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Abstract
Physiological [Mg 2+ ] produces a voltage-dependent channel block of NMDA receptors (NMDAR). Removal of Mg 2+ from the extracellular environment therefore induces spontaneous Ca 2+ oscillations in neurons mediated by activation of NMDAR and L-type Ca 2+ channels. In an effort to develop a physiologically relevant model of NMDAR-mediated Ca 2+ signaling, we studied the mechanism of Ca 2+ oscillations in murine neocortical neurons under physiological [Mg 2+ ]. AMPA receptors (AMPAR) regulated the frequency of Ca 2+ oscillations, which were independent of L-type Ca 2+ channels and partially mediated by NMDAR. Activation of metabotropic glutamate receptors and release of Ca 2+ from endoplasmic reticulum were critical for the generation of Ca 2+ oscillations. We further studied whether Ca 2+ -permeable AMPAR (Ca-AMPAR) contribute to the Ca 2+ oscillations. Ca-AMPAR exhibit characteristic permeability to Co 2+ , not seen among other divalent cation-permeable channels. Based on this conductance property, we developed a fluorometric method using dye calcein whose fluorescence is quenched by Co 2+ , while unaffected by intracellular Ca 2+ levels. AMPAR did not produce measurable Co 2+ influx in the basal state. Activation of AMPAR by bath application of AMPA or kainate induced Co 2+ influx in neocortical neurons, which was blocked by an AMPAR antagonist. These results suggest that Ca-AMPAR may not be activated during basal synaptic activity and only mediate Ca 2+ influx when activated by an excitatory stimulus. We further studied the effect of brevetoxin (PbTx), potent allosteric enhancers of voltage-gated sodium channel (VGSC) function associated with "Red Tide" blooms, on Ca 2+ dynamics and downstream signaling in neocortical neurons. Depending on the extent of VGSC activation, PbTx-2 treatment produced distinct Ca 2+ responses resulting in distinct temporal pattern of extracellular signal-regulated kinases 1/2 (ERK1/2) activation. Differential modulation of ERK1/2 by PbTx-2 may be explained by the activation of synaptic versus extrasynaptic NMDAR. An elevation of intracellular Na + and Src kinase-mediated tyrosine phosphorylation of NMDAR is known to increase NMDAR function. Inhibition of Src kinase reduced PbTx-2 induced Ca 2+ influx. PbTx-2 treatment also increased tyrosine phosphorylation of NR2B subunit. In addition, PbTx-2 augmented NMDAR-mediated Ca 2+ influx and ERK2 activation. These results suggest that brevetoxin upregulates NMDAR signaling in neocortical neurons which may be mediated by coincidence of an elevation of intracellular [Na + ] and Src kinase activation.