Eric C. Gustafson

Glycine transport accounts for the differential role of glycine vs. d‐serine at NMDA receptor coagonist sites in the salamander retina

In this study, we demonstrate that d‐serine interacts with N‐methyl‐d‐aspartate receptor (NMDAR) coagonist sites of retinal ganglion cells of the tiger salamander retina by showing that exogenous d‐serine overcomes the competitive antagonism of 7‐chlorokynurenic acid for this site. Additionally, we show that exogenous d‐serine was more than 30 times as effective at potentiating NMDAR currents compared with glycine. We thus examined the importance of glycine transport through the application of selective antagonists of the GlyT1 (NFPS) and GlyT2 (ALX‐5670) transport systems, while simultaneously evaluating the degree of occupancy of the NMDAR coagonist binding sites. Analysis was carried out with electrophysiological recordings from the inner retina, including whole‐cell recordings from retinal ganglion cells and extracellular recordings of the proximal negative field potential. Blocking the GlyT2 transport system had no effect on the light‐evoked NMDAR currents or on the sensitivity of these currents to exogenous d‐serine. In contrast, when the GlyT1 system was blocked, the coagonist sites of NMDARs showed full occupancy. These findings clearly establish the importance of the GlyT1 transporter as an essential component for maintaining the coagonist sites of NMDARs in a non‐saturated state. The normal, unsaturated state of the NMDAR coagonist binding sites allows modulation of the NMDAR currents, by release of either d‐serine or glycine. These results are discussed in light of contemporary findings which favor d‐serine over glycine as the major coagonist of the NMDARs found in ganglion cells of the tiger salamander retina.

Light-evoked NMDA receptor-mediated currents are reduced by blocking D-serine synthesis in the salamander retina

Experiments were carried out in the retina of the tiger salamander (Ambystoma tigrinum) to evaluate the importance of D-serine synthesis on light-evoked N-methyl D-aspartate (NMDA) receptor-mediated components of ganglion cells and contributions to the proximal negative field potential. We blocked the synthesis of D-serine through brief exposures of the retina to phenazine ethosulfate and validated the changes in the tissue levels of D-serine using capillary electrophoresis methods to separate and measure the amino acid enantiomers. Ten minute exposures to phenazine ethosulfate decreased D-serine levels in the retina by about 50% and significantly reduced the NMDA receptor contribution to light responses of the inner retina. This is the first report of a linkage between D-serine synthesis and NMDA receptor activity in the vertebrate retina.

Retinal NMDA receptor function and expression are altered in a mouse lacking d-amino acid oxidase

D-serine is present in the vertebrate retina and serves as a coagonist for the N-methyl-D-aspartate (NMDA) receptors of ganglion cells. Although the enzyme D-amino acid oxidase (DAO) has been implicated as a pathway for d-serine degradation, its role in the retina has not been established. In this study, we investigated the role of DAO in regulating D-serine levels using a mutant mouse line deficient in DAO (ddY/DAO(-)) and compared these results with their wild-type counterparts (ddY/DAO(+)). Our results show that DAO is functionally present in the mouse retina and normally serves to reduce the background levels of D-serine. The enzymatic activity of DAO was restricted to the inner plexiform layer as determined by histochemical analysis. Using capillary electrophoresis, we showed that mutant mice had much higher levels of D-serine. Whole cell recordings from identified retinal ganglion cells demonstrated that DAO-deficient animals had light-evoked synaptic activity strongly biased toward a high NMDA-to-AMPA receptor ratio. In contrast, recordings from wild-type ganglion cells showed a more balanced ratio between the two receptor subclasses. Immunostaining for AMPA and NMDA receptors was carried out to compare the two receptor ratios by quantitative immunofluorescence. These studies revealed that the mutant mouse had a significantly higher representation of NMDA receptors compared with the wild-type controls. We conclude that 1) DAO is an important regulatory enzyme and normally functions to reduce D-serine levels in the retina, and 2) D-serine levels play a role in the expression of NMDA receptors and the NMDA-to-AMPA receptor ratio.