D-Serine Modulates Glutamatergic Increases of Intracellular Calcium in Rat Retinal Ganglion Cells

Abstract

Purpose: : It is well documented that glutamatergic activation of NMDA receptors is enhanced by glycine at a separate binding site. Recent studies on retinal ganglion cells (RGCs) have revealed that this co-agonist binding site can also be occupied endogenously by D-serine. Previous work has focused primarily on D-serine modulation of post-synaptic NMDA currents. Given the significant permeability of NMDA channels to calcium we have investigated the effect of D-serine on glutamatergic-induced intracellular calcium [Ca²⁺]_i increases in RGCs in vitro and in situ.

Methods: : Calcium imaging of immunopanned isolated RGCs (P6-8 Long Evans rats) and RGCs in adult whole mount retinas was performed. Isolated RGCs were loaded with fura-2 AM calcium indicator dye and RGCs in the whole mount retinas were loaded with fura dextran. All experiments were performed in Mg²⁺-free solutions.

Results: : Isolated RGC [Ca²⁺]_i increases produced by application of 10 µM glutamate were enhanced by co-exposure to D-serine in a dose dependant manner. D-Serine concentrations ≥1 µM increased the glutamate-induced [Ca²⁺]_i response (mean ± sd; 1 µM, 118 ± 84% increase; 10 µM, 282 ± 273%; 100 µM, 425 ± 490%). The peak effect of D-serine was seen at 100 µM. At 10 µM, glycine or D-serine enhanced the 10 µM glutamate-induced calcium response to similar levels (379 ± 231% and 474 ± 422%, respectively). This effect was partially blocked by the NMDA co-agonist binding site antagonist 1-aminocyclobutane-1-carboxylic acid (ACBC, 100 µM). In whole mount retinas NMDA-induced (200 µM) [Ca²⁺]_i increases showed no change with the co-application of 100 µM glycine or D-serine. In other experiments endogenous D-serine was degraded by a 10 min exposure to D-amino acid oxidase (400 µg/ml). Subsequent NMDA-induced [Ca²⁺]_i increases were significantly reduced to 65 ± 21% in most RGCs. The calcium response to NMDA was recovered by addition of 500 µM D-serine to 103 ± 35% of the original NMDA-induced response.

Conclusions: : At 10 µM, D-serine and glycine are equally effective as co-agonists of glutamate-induced increases in [Ca²⁺]_i in vitro. In situ, D-serine did not augment the NMDA-induced [Ca²⁺]_i increase possibly due to saturation of the NMDA receptor co-agonist site. Consistent with this hypothesis, enzymatic degradation of D-serine resulted in a reduction of the NMDA-induced [Ca²⁺]_i increase that was recovered by exogenous D-serine application.

Keywords: ganglion cells • calcium • excitatory neurotransmitters