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W.H. Baldridge, T.M. Maillet, A.T. E. Hartwick; NMDA– and Kainate–Induced Calcium Dynamics in Rat Retinal Ganglion Cells . Invest. Ophthalmol. Vis. Sci. 2004;45(13):5376.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The application of either NMDA or kainate has been shown to increase intracellular calcium concentration in retinal ganglion cells (RGCs). Both NMDA and kainate can depolarize RGCs, but the contribution of voltage–gated calcium channels (VGCCs) to NMDA– or kainate–mediated calcium influx is uncertain. The purpose of this work was to characterize and compare the kainate and NMDA–induced calcium dynamics in rat RGCs, using both intact retinas and purified RGC cultures. Methods: Dextran–conjugated calcium indicator dye (Fura–2) was injected into flat–mounted adult rat retinas, and following incubation for 4–8 hrs, the dye was retrogradely transported to RGC somata. Purified RGC cultures from neonatal rats were generated using a 2–step panning procedure with the antibody Thy1.1, and the isolated RGCs were loaded with Fura–2 AM. All calcium imaging experiments were done in Mg–free Hank's solution. Changes in the Fura–2 fluorescence ratio, indicative of changes of free intracellular calcium concentration, were monitored with a cooled CCD camera during exposure to NMDA or kainate both in the presence and absence of selective NMDA and kainate/AMPA receptor antagonists (APV and NBQX, respectively) and a cocktail of VGCC blockers (verapamil plus ω–conotoxin). Results: In RGCs in the intact retina preparation, the calcium influx induced by 200 µM NMDA was abolished by APV but NBQX had no effect (n=8). Conversely, the calcium influx induced by 50 µM kainate was blocked completely by NBQX but was not affected by APV (n=8). Similarly, in the cultured RGCs, NMDA and kainate (both at 200 µM; n=10 for each) caused an increase in intracellular calcium that could be blocked by the appropriate antagonist. The verapamil/ω–conotoxin mixture reduced the kainate–induced calcium influx in the isolated RGCs by 61 ± 6 % (n=7), but did not have a significant effect on NMDA–induced calcium influx (n=8). Conclusions: These results confirm that both NMDA and kainate/AMPA receptor activation can lead to increases in RGC calcium levels but do so through separate pathways, as kainate–induced calcium influx in the intact retina was not due to the activation of NMDA receptors by endogenously released glutamate (and similarly, kainate/AMPA receptor activation did not contribute to NMDA–induced calcium influx). These glutamate agonists influence RGC calcium dynamics via different mechanisms, as VGCC activation accounts for the majority of the calcium influx induced by kainate, but contributes little to the influx induced by NMDA.
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