A lthough bacteria and many invertebrate species use both Dand L-enantiomers of amino acids for cellular functions, it was generally believed that higher organisms had a more restricted stereospecificity and were confined to the use of L-amino acids. Thus, the D-amino acids detected in vertebrates were generally assumed to come from ingested material or intestinal flora (1). Indeed, the enzyme D-amino acid oxidase (D-AAO), which degrades many D-amino acids, is found in vertebrate tissues, but it was assumed to exist for degrading D-amino acids from external sources. However, in the past decade, it has become clear that some D-amino (6) proposed that D-serine released by glia plays a regulatory role as a necessary coagonist for the glutamate activation of NMDA receptors. Thus, an expanded model for glutamate synapses emerged in which perisynaptic glial processes respond to glutamate released from the presynaptic neuron by releasing D-serine, which, in turn, facilitates the activation of NMDA receptors on the postsynaptic cell. The functional importance of D-serine was further supported by the more recent discovery that the enzyme serine racemase, which converts L-serine to D-serine, is localized to astrocytes in the nervous system (7). The discovery of this mechanism adds to the accumulating evidence that glial cells dynamically influence neuronal activity.Although D-serine and serine racemase have been found in many regions of the brain, no study has yet been carried out in the vertebrate retina, a tissue that offers several advantages for studying the actions of D-serine. The retinal network is a well-characterized region of the central nervous system that can be studied in an intact preparation, permitting the use of light stimulation to provide natural activation of the neural circuitry and test more directly the physiological significance of D-serine. Virtually all retinal ganglion cells have AMPA and NMDA receptors, which both contribute to light-evoked excitation (8-12), and NMDA receptor currents can be enhanced through pharmacological techniques and studied in relative isolation from AMPA receptor contributions (13).In the present study, we have used immunohistochemistry, immunoblotting, analytical chemistry (HPLC), and electrophysiology to evaluate the presence of D-serine and serine racemase in the retinas of several vertebrate species. Our findings indicate that D-serine and serine racemase are present in the retina and seem to be localized to Müller glial cells and astrocytes. In addition, we have demonstrated that exogenous D-serine can enhance NMDA receptor responses and modulate light-evoked activity in retinal ganglion cells. The enhancement of NMDA receptor function seems to involve both tonic and phasic components of glutamatergic input. When the D-serine-degrading enzyme D-AAO (14) is added to the bathing medium, currents mediated by NMDA receptors are reduced, suggesting that these responses depend on endogenous D-serine as a coagonist for the glutamate activation of NMDA receptors. Method...
We have combined electrophysiology and chemical separation and measurement techniques with capillary electrophoresis (CE) to evaluate the role of endogenous d-serine as an NMDA receptor (NMDAR) coagonist in the salamander retina. Electrophysiological experiments were carried out using whole cell recordings from retinal ganglion cells and extracellular recordings of the proximal negative response (PNR), while bath applying two D-serine degrading enzymes, including d-amino acid oxidase (DAAO) and D-serine deaminase (DsdA). The addition of either enzyme resulted in a significant and rapid decline in the light-evoked responses observed in ganglion cell and PNR recordings. The addition of exogenous D-serine in the presence of the enzymes restored the light-evoked responses to the control or supracontrol amplitudes. Heat-inactivated enzymes had no effect on the light responses and blocking NMDARs with AP7 eliminated the suppressive influence of the enzymes as well as the response enhancement normally associated with exogenous d-serine application. CE was used to separate amino acid racemates and to study the selectivity of DAAO and DsdA against D-serine and glycine. Both enzymes showed high selectivity for D-serine without significant effects on glycine. Our results strongly support the concept that endogenous D-serine plays an essential role as a coagonist for NMDARs, allowing them to contribute to the light-evoked responses of retinal ganglion cells. Furthermore under our experimental conditions, these coagonist sites are not saturated so that modulation of NMDAR sensitivity can be achieved with further modulaton of d-serine.
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