Preformed vs. on‐demand: Molecular economics of endocannabinoid signalling

Endogenous cannabinoids are powerful retrograde transmitters that modulate synaptic transmission in many areas of the mammalian CNS. Endocannabinoid- mediated retrograde transmission can be induced by postsynaptic calcium influx, by postsynaptic activation of specific Gq/11-coupled metabotropic receptors, or by a combination of such stimuli. Once mobilized, endocannabinoids travel backwards across the synaptic cleft where they inhibit transmitter release via activation of Gi/o-coupled presynaptic type I cannabinoid receptors (CB1Rs). Over the last 10–15 years, an enormous amount has been learned about the nature, diversity, power and therapeutic potential of endocannabinoid-mediated signalling systems. However, over that time, some questions have been more persistent than others have, and as is perhaps often the case, some hard won answers have generated new and pressing questions of their own. A good case in point has to do with the molecular identity of the endogenous cannabinoid that acts as a retrograde messenger in most classic forms of CB1R-mediated synaptic plasticity. At this time, the best available evidence would indicate that the answer is 2-arachidonoylglycerol (2-AG). The strongest data supporting that conclusion comes from a recent series of studies that rely on genetic deletion of diacylglycerol lipase (DAGL), an enzyme necessary for the synthesis of 2-AG. Indeed, three laboratories independently developed DAGLα knockout animals and all observed essentially complete loss of CB1R-mediated short-term synaptic plasticity, collectively across a wide variety of brain areas, including the hippocampus, cerebellum, striatum and prefrontal cortex (Gao et al. 2010; Tanimura et al. 2010; Yoshino et al. 2011). What is surprising about work during this period is that a series of parallel studies relying on acute pharmacological inhibition of DAGL produced a complex set of highly inconsistent results. Thus was raised a new question: If 2-AG is in fact the retrograde messenger required for most classic forms of CB1R-dependent signalling, as is consistently indicated by studies that genetically remove a key synthetic enzyme, why is acute pharmacological inhibition of the same enzyme sometimes so ineffective? In an effort to address this question, several investigators proposed that there may exist a pre-formed pool of 2-AG that is synthesized in a DAGL-dependent fashion, but then stored in neurons for later DAGL independent release (e.g. see Min et al. 2010; Alger & Kim, 2011). While this hypothesis effectively addresses a sharp disparity that exists between the genetic studies and a subset of the pharmacological results, it does not directly address the inconsistencies between various pharmacological studies themselves. Further, it also challenges a long held hypothesis that endogenous cannabinoids are synthesized and released on-demand, a possibility that is much easier to reconcile with their extremely lipophilic and thus membrane permeant nature. Ultimately, for the field to embrace the preformed pool hypothesis more fully, it will probably be necessary to develop a more detailed and mechanistic understanding of exactly how, once synthesized, 2-AG may be stored and selectively mobilized without DAGL. By contrast, to reject the preformed pool hypothesis more fully, it would be helpful to develop a better understanding of why acute application of DAGL inhibitors has failed to block CB1R-dependent signalling in a significant number of studies. In the current issue of The Journal of Physiology, Hashimotodani et al. (2013) weigh in on this issue with a careful study of the effects of the DAGL inhibitor OMDM-188 on CB1R-dependent retrograde signalling in acute slices of hippocampus, striatum and cerebellum, and in cultured hippocampal neurons. In the acute slice preparations, they use a consistent 1 h preincubation with low micromolar concentrations of OMDM-188 and take care to patch neurons close to the surface of the slice. Their results show uniformly excellent inhibition of multiple types of CB1R-dependent phenomena, which interestingly, is accompanied by a significant reduction in basal 2-AG levels. While it might be argued that these data suggest that preformed pools of 2-AG are depleted by spontaneous synaptic activity during the 60 min incubation period, that conclusion is not consistent with subsequent studies in cultured hippocampal neurons. In these experiments, the authors demonstrate that a 2 min exposure to OMDM-188 is sufficient to block over 50% of CB1R-dependent retrograde signalling, while a nearly complete block is obtained within 8 min. These data more directly argue that if preformed pools exist at all, they must be very rapidly depleted and, indeed, they must be depleted in quiescent neurons without a requirement for direct experimental stimulation of endocannabinoid release. Based on these results, the authors argue that poor penetration of highly lipophilic OMDM-188 (and other DAGL inhibitors) into acute slices is in fact likely to be the key issue underlying inconsistent results in previous work. They further suggest a variety of potentially important but previously uncontrolled variables that may have contributed to this issue. These include temperature, degree of penetration into the slice required to reach the relevant site of action, and the extent to which studied phenomena involve saturating concentrations of 2-AG or are rate limited by 2-AG synthesis. Overall, the paper by Hashimotodani et al. (2013) clearly leaves some questions open, perhaps most notably about regulation of basal levels of 2-AG. Further, it does not completely eliminate the hypothesis that preformed pools of 2-AG, particularly readily depleting pools, may exist. However, it does provide several strong arguments suggesting a much more prominent role for on-demand synthesis/release of 2-AG. As such, it represents an important and timely addition to a current debate in the field.