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S. J. Sullivan, M. Esguerra, R. F. Miller; AMPA Receptor Activation Induces D-serine Release in the Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1030.
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NMDA receptors (NMDARs) located on retinal ganglion cells (RGCs) contribute to light evoked responses. Glutamate activation of NMDARs requires a coagonist, either glycine or D-serine. D-serine and its synthesizing enzyme serine racemase are primarily localized to retinal glia, and evidence suggests that D-serine is the primary coagonist for NMDARs. However, little is known about D-serine release in the retina. To overcome some of the difficulties in measuring retinal D-serine, we have utilized a mutant mouse lacking function D-amino acid oxidase (DAO-), which has elevated D-serine, to explore the possibility that D-serine release is induced by AMPA receptor (AMPAR) activation.
D-serine measurement: Amino acids were isolated from the retina and fluorescently tagged with NBDF for laser induced detection. Samples were separated via capillary electrophoresis and a cyclodextrin-containing buffer allowed for separation of enantiomeric amino acids.Calcium imaging: Ca2+ changes in isolated mouse retinas loaded with Fluo-4 were measured using a multiphoton imaging system.
D-serine measurement: DAO- retinas had significantly more tissue and extracellular D-serine when compared to wt controls. AMPA and cyclothiazide together induced D-serine release in both mice but this effect was exaggerated in DAO- mice. In DAO- mice, D-serine release was attenuated by N-acetyl spermine (NAS), a Ca2+ permeable AMPAR antagonist. Elevating extracellular glutamate by blocking glutamate transporters resulted in D-serine release only when cyclothiazide was co-applied and this effect was blocked by an AMPAR antagonist.Calcium imaging: AMPA and cyclothiazide treatment of whole-mount retinas elevated Ca2+ in in glial cells and this response was significantly reduced by NAS.
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