Modulation of ionotropic glutamate receptors in retinal neurons by the amino acid D-serine

Abstract

D-Serine is regarded as an obligatory co-agonist required for the activation of NMDA-type glutamate receptors (NMDARs). In the retina D-serine and a second NMDAR coagonist, glycine, are present at similar concentration and the cells that produce and release them are in close apposition. This arrangement allows for an abundant supply of coagonists and under certain conditions the NMDAR coagonist binding site could be saturated. There is also evidence suggesting that D-serine can act in an inhibitory manner at AMPA/kainate-type glutamate receptors (GluRs). Glutamate receptor activation can lead to direct and indirect elevation of intracellular calcium (Ca2+) concentration ([Ca2+]i). Therefore, in this thesis, I predominantly used Ca2+ imaging techniques to study the effect of D-serine on GluR activation in the mammalian retina. I first describe a novel method I developed to load retinal cells with Ca2+ indicator dye using electroporation and show that retinas remain viable and responsive following electroporation. This technique was used to explore the excitatory role of D-serine at NMDARs and its potential inhibition of AMPA/kainate receptors using cultured retinal ganglion cells (RGCs) and isolated retina preparations. Using cultured RGCs I demonstrated that D-serine and glycine enhance NMDAR-mediated Ca2+ responses in a concentration-dependent manner and are equally effective as coagonists. In isolated retinas I showed that D-serine application enhanced NMDA-induced responses consistent with sub-saturating endogenous coagonist concentration. Degradation of endogenous D-serine reduced NMDAR-mediated Ca2+ responses supporting the contribution of this coagonist to NMDAR activation in the retina. Using imaging and two different electrophysiological approaches, I found that D-serine reduced AMPA/kainate receptor-mediated responses in cultured RGCs and isolated retinas at concentrations that are saturating at NMDARs. Antagonist experiments suggest that the majority of inhibition is due to D-serine acting on AMPA receptor activity. Degradation of endogenous D-serine enhanced AMPA/kainate-induced responses of some cells in isolated retina suggesting that, under these conditions, D-serine concentration may be sufficient to inhibit AMPA receptor activity. Overall, the work in this thesis illustrates the utility of electroporation as a method to load Ca2+-sensitive fluorescent dyes into retinal cells and highlights the potential role for D-serine as a modulator of ionotropic GluRs in the CNS.