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

Abstract

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.

Fig 1

Serine racemase (SR) distribution in the tiger salamander retina shows a prominent staining pattern in retinal Müller cells. An antibody to SR (green) labeled Müller cells in this cross-section of salamander retina (upper left). SR label can also be appreciated in the photoreceptor layer. A three-dimensional reconstruction of the staining from the SR antibody (red) from a z-series in this same section of retina (upper right). This label is severely curtailed in an adjacent retinal section when the antibody was preadsorbed with peptide (lower left). A western blot of protein extracted from salamander at different concentrations labeled with the SR antibody displayed a single band at approximately 37 kDa, the anticipated weight of the enzyme.

Fig 2

Blocking serine racemase (SR) decreased N-methyl-d-aspartate currents in retinal ganglion cells. (a) A whole-cell voltage-clamp recording (V_Hold = −67 mV) from a retinal ganglion cell in an eyecup preparation showed the response to a 2 s light stimulus in control Ringer, which was enhanced by the addition of d-serine (100 μM), and blocked by 5,7-dichlorokynurenic acid (5,7-DCK, 30 μM). When the SR antagonist, phenazine ethosulfate (Phz-ES; 10 μM, thick bar) was added, the response was diminished in amplitude similar to that observed in 5,7-DCK. This reduced response could still be potentiated by the addition of d-serine (DS). Phz (100 μM) did not alter the response to a significant degree. (b) Summary data from seven cells showing reduced On and Off responses when Phz-ES was added to the bathing medium and an unaltered potentiation by DS and the lack of change in the presence of Phz. (c) Summary data from seven cells showing a small, nonsignificant additional reduction in inward current when 5,7-DCK was added to Phz-ES. (d) Time course of the block by the Phz-ES fit with a Boltzman. Whole cell recording (WCR, ■) and proximal negative field potential (PNFP,●) measurements were averaged from responses at 20 s intervals (error bars not shown). Transit time of enzyme from reservoir subtracted before plotting. LS: l-serine; L-E PSP: light-evoked post-synaptic potential (*P < 0.01 compared with control).

Fig 3

(a) Extracellular recording of the proximal negative field potential (PNFP). Application of phenazine-ethosulfate (Phz-ES) decreased the amplitude of the PNFP. An example set of traces shows a decrease in PNFP amplitude after bath application of Phz-ES (light gray trace) when compared with the control cocktail response (black trace). The addition of exogenous d-serine (DS) to the Phz-ES bathing media (gray trace) increased the response beyond that of the original control. (b) Cumulative results showed a significant decrease of 26.8 ± 2.6% in the PNFP in the presence of Phz-ES and a significant increase of 9.1 ± 1.1% after addition of DS (both compared with control, n = 6). (c) Shows the change in measured levels of DS for the intact retina exposed for 10 min to Phz-ES, which produced an approximate 50% decline in DS levels. (d) Shows that l-serine levels measured from the same retinas were not significantly changed.