D-serine and serine racemase are present in the vertebrate retina and contribute to the physiological activation of NMDA receptors

Abstract

d-serine has been proposed as an endogenous modulator of N-methyl-d-aspartate (NMDA) receptors in many brain regions, but its presence and function in the vertebrate retina have not been characterized. We have detected d-serine and its synthesizing enzyme, serine racemase, in the retinas of several vertebrate species, including salamanders, rats, and mice and have localized both constituents to Müller cells and astrocytes, the two major glial cell types in the retina. Physiological studies in rats and salamanders demonstrated that, in retinal ganglion cells, d-serine can enhance excitatory currents elicited by the application of NMDA, as well as the NMDA receptor component of light-evoked synaptic responses. Application of d-amino acid oxidase, which degrades d-serine, reduced the magnitude of NMDA receptor-mediated currents, raising the possibility that endogenous d-serine serves as a ligand for setting the sensitivity of NMDA receptors under physiological conditions. These observations raise exciting new questions about the role of glial cells in regulating the excitability of neurons through release of d-serine.

Fig. 1.

(A) Confocal image of the vitreal surface of a rat retina, showing an astrocyte immunoreactive for d-serine. (B–E) Sections of rat retina. Retinal section doubly immunostained by using the anti-d-serine antibody preabsorbed with l-serine conjugated to BSA (B, green in overlay in D) and glutamine synthetase (C, red in D). d-serine immunoreactivity extends throughout the depth of the retina, from the ILM to the ELM. Prominent staining is seen in the Müller cell endfeet that form the ILM, in the plexiform layers, and in cell bodies in the INL, where Müller cell somata are located. (E) Retinal section doubly immunostained with anti-glutamine synthetase (red) and anti-d-serine that had been preincubated with both d- and l-serine conjugated to BSA (green). Compare this section with that shown in D. (F) d-serine immunoreactivity in isolated Müller cells of tiger salamander. (G–J) Section of mudpuppy retina doubly immunostained for d-serine (G, green in overlays in I and J) and glutamine synthetase (H, red in I and J). (I) Note that the thick proximal processes of Müller cells, one of which is indicated by the arrow, appear yellow in this overlay, indicating colocalization of the two labels. The region within the white box in I is shown at higher magnification in J. (K–O) Mouse retinal wholemount doubly immunostained for serine racemase (green) and glutamine synthetase (red). (K and L) Each panel represents the superimposition of six confocal images collected at 0.2-μm intervals in a plane parallel to the retinal layers. (K) In the IPL, the proximal stalks of Müller cells, seen in cross section, stain for both serine racemase and glutamine synthetase; arrows indicate two such stalks. (L) Processes of Müller cells in the inner nuclear layer also stain for both antigens; examples are indicated by arrows. (M–O) “Virtual section” reconstructed from a stack of optical sections collected parallel to the retinal layers. Both serine racemase (M, green in overlay in O) and glutamine synthetase (N, red in O) are found throughout the depth of the mouse retina. (P) Western blot. Soluble proteins from rat brain and retina were separated by SDS/PAGE under reducing conditions, transferred to poly(vinylidene difluoride) (PVDF) membranes, and probed with anti-serine racemase. Positions of molecular mass markers are shown to the left of the blot. OPL, outer plexiform layer; GCL, ganglion cell layer. Calibration bars = 10 μm, A, J, and L (applies to K and L); 15 μm, O (applies to M–O); 20 μm, E (applies to B–E), F, and I (applies to G–I).

Fig. 2.

A summary of HPLC analysis of N-tert-butyloxycarbonyl-l-cysteine o-phthaldialdehyde-derived amino acids extracted from the rat (left) and tiger salamander (right) retinas. Twelve retinas from each species were used, and three retinas were pooled for each HPLC determination. When expressed as nmol/mg protein, values for the rat were 2.0 ± 0.023, whereas those of the salamander were 1.1 ± 0.012.

Fig. 3.

WCRs from ganglion cells in the isolated, perfused rat retina. (A)(Top) Pressure-ejection of NMDA (1 mM) from an extracellular pipette evoked an inward current (holding potential -61 mV) in Mg²⁺-free Ringer's solution. The response to NMDA was enhanced when d-serine (100 μM) was added to the bathing medium (Middle), and recovery to the control amplitude was observed after removing d-serine (Bottom). The average response in d-serine was 131.8 ± 7.1% of the control response (left bars in D). (B) Pressure-ejection of NMDA evoked an inward current (Top) that was reduced after several minutes of exposure to d-AAO (100 μg/ml, Middle). The response partially recovered after washout of d-AAO (Bottom). In 10 recordings, the average response in d-AAO was 62.0 ± 4.1% of the control response (right bars in D). (C) d-serine evoked a small inward current accompanied by a large increase in noise; the application of the NMDA antagonist AP7 induced a small net outward current associated with a significant reduction in membrane noise and virtually eliminated the response to d-serine (dashed line at zero current). The action of AP7 was reversible. (D) Summary of electrophysiological results. RTC, return to control solution.

Fig. 4.

Light-evoked responses in the tiger salamander retina. (A) WCR from a sustained ON ganglion cell. In a Mg²⁺-free control Ringer's solution, presentation of a 130-μm wide bar of light evoked a relatively sustained inward current that was substantially enhanced when d-serine (100 μM) was added to the bathing medium; when 100 μM AP7 was added to the d-serine solution, the light-evoked current was reduced to a value approximately equal to the control. Numbers indicate the average inward current at -65 mV before the light stimulus. The average response in d-serine was 133.6 ± 7.5% (n = 10) of the control response; the average response in d-serine plus AP7 was 83 ± 10.4% (n = 9) of the control response (left bars in D). (B) Light-evoked PNR, an extracellularly recorded response of the inner retina (negative up). A perfusion medium containing picrotoxin (100 μM) and NBQX (100 μM) enhanced the PNR above that in Mg²⁺-free Ringer's solution (not shown); the enhanced PNR was reversibly reduced when d-AP7 was added to the bathing medium. d-AP7 reduced the light response to 42.9 ± 15.0% (n = 15) of the response in picrotoxin/NBQX (“control mixture,” middle bars in D). (C) d-AAO reduced the PNR in picrotoxin/NBQX; the response recovered after washout of d-AAO. d-AAO reduced the light response to 69 ± 9% (n = 10) of the response in picrotoxin/NBQX (right bars in D). (D) Summary of electrophysiological results; RTCC, return to “control mixture.”