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M. Esguerra, K.B. O'Brien, M.T. Bowser, R.F. Miller; Rapid Measurements of D-serine Release in the Intact Retina by Online Microdialysis and Capillary Electrophoresis . Invest. Ophthalmol. Vis. Sci. 2003;44(13):5137.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: D-serine release by glial cells has been proposed as an endogenous modulator of NMDA receptor activation in the central nervous system. D-serine and its synthesizing enzyme serine racemase have been detected in retinal Muller cells, and D-serine modulates light-evoked responses in isolated retina. However, the mechanisms of D-serine release have not been studied in intact CNS tissues. We describe a highly sensitive electrophoretic method for rapid, online analysis of D-serine and other neurotransmitters in the extracellular space of intact retinas. This method was used to test the hypothesis that AMPA receptors on retinal glia modulate release of D-serine (Schell et al, 1994). Methods: Isolated tiger salamander retinas (A. tigrinum) are maintained in a 5 µL continuous perfusion chamber. The effluent is sampled by a microdialysis probe, and dialysate immediately analyzed on-line by capillary electrophoresis with laser-induced fluorescence (LIF) detection in a custom sheath-flow cell. Primary amines are derivatized online with o-phthaldialdehyde for LIF, and D- and L- enantiomers separated by complexing with hydroxypropyl-γ-cyclodextrin. Separations are complete in 10 seconds, allowing rapid analysis of changes in the extracellular space. Retinas were exposed to varying concentrations of AMPA receptor agonists and modulators, and the resulting changes in extracellular D- and L-amino acids analyzed. Results: Stimulation of retinas with kainate (up to 100 µM) in the presence of cyclothiazide (50 µM) led to dose-dependent, transient increases in extracellular D-serine concentration (2- to 7-fold). Increases in GABA, glutamate, and taurine were also reliably detected with this method. Similar results were obtained after stimulation with 60 mM K+. Addition of cobalt (1mM) modulated these responses differentially, suggesting that both synaptic and non-synaptic mechanisms participate in their generation. Conclusions: 1) AMPA receptor activation in retina leads to release of D-serine into the extracellular space. This effect requires addition of cyclothiazide, suggesting that AMPA receptors in this preparation are tonically desensitized by standing levels of glutamate. 2) Microdialysis/CE is an effective method for directly measuring amino acids and neurotransmitters in the extracellular environment of the intact retina in response to physiological stimuli.
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