DHPG and NMDA preconditioning induce ischemic tolerance with differential mechanisms

Ischemic tolerance is a neuroprotective mechanism in which exposure to a mild preconditioning stress results in resistance to a subsequent lethal ischemic insult. Because cerebral ischemia and excitotoxicity involve the activation of glutamate receptors, we investigated whether AMPA responses were modified following preconditioning in rat organotypic hippocampal slices, and the possible underlying mechanisms for these changes. For our study, we used two models of preconditioning by exposing the slices to subtoxic bouts of either DHPG (10 M for 30 min) or NMDA (3 M for 60 min) and then, 24 h later, to excitotoxic conditions (30 min oxygen-glucose deprivation (OGD)) or 10 µM AMPA for 24 h, which leads to selective injury of the CA1 subregion 24 h later. After preconditioning, we observed a significant reduction in CA1 damage following our toxic insults. We first performed whole-cell voltage-clamp recordings in control or preconditioned CA1 pyramidal neurons of organotypic slices. Twenty-four h after exposure to preconditioning doses of DHPG or NMDA, AMPA induced-currents were significantly reduced. We then evaluated whether the expression of the GluR1, NR2A and NR2B subunits and of PSD-95 were modified after our preconditioning stimuli, by using Western blot analysis performed in postsynaptic densities. Our results show that following NMDA, but not DHPG preconditioning, the expression of GluR1 was significantly reduced and this reduction appeared to be associated with the internalization of AMPA receptors on the postsynaptic densities. To clarify the mechanisms whereby DHPG induces tolerance, we investigated the involvement of endocannabinoid system. Exposure of slices to the CB1 antagonist AM251 during preconditioning and the subsequent 24 h period prevented the development of tolerance to AMPA toxicity induced by DHPG but not NMDA. In conclusion, our results show that preconditioning induced by NMDA is associated with internalization of AMPA receptors, whereas preconditioning induced by DHPG involves a possible interplay between CB1 and mGluR1 receptors.