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Eric C. Gustafson, Stephane Marinesco, Robert F. Miller; Measuring Release Of The NMDA Receptor Agonist D-serine In The Retina Using An Enzymatic Biosensor. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1946.
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© ARVO (1962-2015); The Authors (2016-present)
Excitatory input to retinal ganglion cells activates both AMPA and NMDA-type glutamate receptors. In addition to glutamate, NMDA receptors require a second agonist for channel opening. Previously, our lab showed that the co-agonist D-serine is essential for activation of retinal ganglion cell NMDA receptors. Still, the mechanisms that regulate D-serine, including those of release and of uptake, are poorly understood. To investigate these processes we have utilized a microelectrode biosensor that detects D-serine in the low µM to nM levels.
Enzymatic microelectrodes were constructed by inserting a 25 µm diameter 90% platinum/ 10% iridium wire into a pulled glass capillary. The 150 µm of wire protruding from the glass was covered in a layer of m-phenylenediamine then in the enzyme D-amino acid oxidase (DAO), for D-serine sensitivity, or in BSA for control electrodes. Recordings of current were made with a VA-10 amplifier (NPI Electronics).We manipulated microelectrodes onto the surface of the retina in a salamander retina-eyecup preparation. Changes in electrode current were monitored in response to bath applied pharmaceutical agents. Electrode calibration was done at the end of experiments by applying known concentrations of D-serine to the preparation.
Biosensor currents showed a linear relationship with D-serine standards from 100 nM to 100 µM. Bath application of the D-serine degrading enzyme D-serine deaminase (DsdA) produced a current equivalent to that produced by 1.9 ± 0.2 µM of D-serine standard, suggesting an approximate value for the background level of D-serine.AMPA receptor mediated D-serine release was examined by applying AMPA (50µM) and cyclothiazide (50µM). The biosensor response reached a peak within 30 seconds of drug application. The resulting current corresponded to a D-serine level of 5.9 ± 2.2 µM.AMPA induced D-serine release was substantially reduced in the presence of DsdA to 1.6 ± 0.1 µM.
Using a D-serine biosensor, we directly measured D-serine levels from a perfused retinal eyecup preparation. The resting D-serine level, recorded at the retinal surface, was about 2 µM--well below the level of saturation for the coagonist site. AMPA receptor stimulation caused the release of D-serine and resulted in a three-fold increase in D-serine at the surface of the retina. This level, while not a saturating dose, is likely to represent only a fraction of the concentration present near the sights of release and near retinal NMDA receptors of the inner plexiform layer. Future refinement of the microelectrodes should allow us to access D-serine levels at different layers within the retina and to more effectively study release mechanisms and localization.
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