Roscovitine triggers excitotoxicity in cultured granule neurons by enhancing glutamate release

Cerebellar granule neurons are highly susceptible to injury in vivo and in vitro, and primary cultures are widely used to characterize relevant receptors and signaling pathways. However, there are problems associated with their use. In particular, cultures are typically grown in medium supplemented with elevated KCl levels because it improves survival, but accumulating evidence indicates that this causes profound neuroadaptations. For example, growth in elevated KCl levels renders neurons electrically silent. Thus, they cannot be used to examine excitotoxicity of synaptic origins. On the other hand, cultures grown in physiological medium are rarely studied because a proportion undergoes apoptosis. Herein, we provide evidence that mature neurons cultured in physiological KCl develop spontaneous action potentials that support survival through N-methyl-D-aspartate (NMDA) receptor-mediated mechanisms. Furthermore, the cdk inhibitor roscovitine enhances the coupling between tetrodotoxin-sensitive action potentials and P/Q-type voltage-dependent calcium channels (VDCCs), thereby converting this survival program to excitotoxicity of synaptic origin. Therefore, roscovitine-triggered necrosis requires spontaneous Na+-based action potentials (tetrodotoxin inhibits, (+/-)-2-amino-4-phosphonobutyric acid enhances), P/Q-type VDCC currents (omega-agatoxin-IVA and omega-conotoxin-MVIIC inhibit, but not omega-conotoxin-GVIA), intact vesicle fusion processes (tetanus toxin inhibits), and transmitter-filled vesicles (concanamycin and bafilomycin inhibit). From a postsynaptic standpoint, roscovitine-mediated excitotoxicity requires the functionally linked activation of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate/kainate (AMPA/KA) and NMDA receptors, which is consistent with evidence that activated AMPA/KA receptors relieve the voltage-dependent Mg2+ block of NMDA receptors, resulting in excitotoxic Ca2+ influx. In the end, NMDA receptor-linked pathways transduce excitotoxicity. On the other hand, L-type VDCC blockers are not protective. Further characterization of this new model is expected to provide important insights about excitotoxicity of synaptic origins and about roscovitine as a selective modulator of this process.