Glutamate Induces Calcium Influx by NMDA Receptor Activation in Purified Retinal Ganglion Cells Cultured from Neonatal and Adult Rats

Abstract

Abstract: : Purpose: Excessive or prolonged exposure to glutamate causes an elevation of intracellular calcium levels that can ultimately trigger neuronal death. However, a consensus has not been reached on the pathways that mediate glutamate-dependent increases of calcium in mammalian retinal ganglion cells (RGCs). One possible explanation for the conflicting evidence is that the expression of glutamate receptors in neonatal rats (primarily used for in vitro experiments) is different from that in adult rats (primarily used for in vivo experiments). Therefore, the purpose of this work was to assess the relative contribution of NMDA and kainate/AMPA glutamate receptors to the glutamate-induced calcium influx in purified RGC cultures obtained from both neonatal and adult rats. Methods: Purified RGC cultures from either neonatal (7-8 days old) or adult (5-8 weeks old) rats were generated from papain-dissociated retinas using a 2-step panning procedure with the antibody Thy1.1. The RGCs were maintained in serum-free Neurobasal-A medium with neurotrophic factors and forskolin. The isolated RGCs were transferred to Mg²⁺-free HBSS for calcium imaging, and loaded with the ratiometric calcium indicator dye Fura-2. Changes in the Fura-2 fluorescence ratio, indicative of changes in intracellular Ca²⁺, were monitored with a cooled CCD camera during exposure to glutamate or related agonists both in the presence and absence of selective NMDA and kainate/AMPA receptor antagonists. Results: The NMDA receptor antagonist APV almost completely abolished the calcium influx in both neonatal and adult RGCs exposed to 10 µM glutamate and 10 µM glycine. The glutamate/glycine-induced increase in calcium influx was reduced 80.2% by 25 µM APV and 94.5% by 100 µM APV in neonatal rat RGCs, and was reduced 87.5% by 25 µM APV in RGCs purified from adult rats. The kainate/AMPA receptor antagonists DNQX, CNQX, and NBQX were much less effective at inhibiting the calcium influx. In addition, the calcium influx induced by low doses of glutamate (10 µM) increased with co-administration of glycine (10 µM), and was dramatically attenuated by Mg²⁺ (0.8 mM), consistent with NMDA receptor involvement. Conclusions: The calcium influx induced by a low dose of glutamate appears to be primarily mediated by NMDA receptors in cultured RGCs purified from either neonatal or adult rats. The extracellular levels of magnesium and glycine must be considered when performing glutamate excitotoxicity assays in vitro to ensure NMDA receptor activation is possible.

Keywords: excitatory amino acid receptors • retinal culture • calcium