mGlu1 receptors and endocannabinoids: neuroprotection studies in an in vitro model of cerebral ischemia

Antagonists of mGlu1 receptors attenuate CA1 pyramidal cell death in models of ischemia by a mechanism that involves the release of GABA [1]. Stimulation of mGlu1 postsynaptic receptors in CA1 pyramidal cells promote the release of endogenous cannabinoids [2], that may act as retrograde transmitters to suppress the output of GABA via activation of CB1 receptors located on interneuron presynaptic terminals [3]. The effects of synthetic and endogenous CBs were evaluated in rat organotypic hippocampal slices exposed to 20 min oxygen-glucose deprivation (OGD). When present in the incubation medium, the synthetic CB agonists WIN 55212-2 and CP 55940 (1-30 µM) and the CB1 agonist ACEA exacerbated CA1 injury induced by OGD, whereas the CB1 receptor antagonists AM 251 and LY 320135 were neuroprotective with maximal activity at 1 µM. The endocannabinoid 2-arachidonoylglycerol (2-AG) reduced OGD injury in hippocampal slices at 0.1-1 µM, whereas anandamide (AEA) was neurotoxic at the same concentrations. The effects of WIN 55212-2, AEA and 2-AG in slices were all dependent on the activation of CB1 but not CB2 receptors, except for the toxic effects of AEA that were also dependent on vanilloid TRPV1 receptors. In the same model of rat organotypic hippocampal slices we investigated the cross-talk between mGlu1 and CB1 receptors. WIN 55212-2, but not AM251, significantly reverted the neuroprotective effects of LY367385 and 3-MATIDA in 30 min of OGD. The mGlu1/5 agonist DHPG (100 µM) exacerbated CA1 injury induced by 20 min OGD, but this effect was not synergic with that of WIN 55212-2. AM251, but not WIN 55212-2, was able to revert the neurotoxic effects of the mGlu1/5 agonist DHPG. We then determined by LC-MS/MS the CA1 contents of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in control and OGD-treated hippocampal slices. AEA, and to a lesser extent 2-AG, increased significantly immediately after OGD and returned to basal levels 3 h later; LY367385 was able to prevent the increase induced by OGD. Our results suggest that endocannabinoids and mGlu1 receptors may play a cooperative role in the mechanisms of post-ischemic neuronal death.