Dynamic regulation of D-serine release in the vertebrate retina

Abstract

Key points: Activation of NMDA receptors (NMDARs) is essential for encoding visual stimuli into signals for the brain, although their over-activation can cause cell death. The recruitment of NMDARs is important for encoding light intensity in retinal ganglion cells. D-serine binding is essential for proper activation of NMDARs, although its role in signal processing and the mechanisms that underlie its availability are not well understood. In these light-evoked experiments, the addition of exogenous D-serine had a large effect on low contrast and low intensity NMDAR responses that decreased as the intensity was increased. The degradation of endogenous D-serine decreased the responses more at higher intensities. The results provide compelling evidence favouring a new interpretation of NMDAR recruitment in which light-evoked D-serine release serves an important regulatory control over the recruitment of NMDARs.

Abstract: The present study aimed to investigate the functional properties of NMDA receptor coagonist release and to specifically evaluate whether light-evoked release mechanisms contribute to the availability of the coagonist D-serine. Two different methods were involved in our approach: (i) whole-cell recordings from identified retinal ganglion cells in the tiger salamander were used to study light adaptation with positive and negative contrast stimuli over a range of ± 1 log unit against a steady background illumination and (ii) the mechanisms for intensity encoding to a range of light intensities covering 6 log10 units were investigated. This latter study employed extracellular recordings of the proximal negative field potential, pharmacologically manipulated to generate a pure NMDA mediated response. For the adaptation study, we examined the light-evoked responses under control conditions, followed by light stimuli presented in the presence of D-serine, followed by light stimulation in the presence of dichlorokynurenic acid to block the coagonist site of NMDA receptors. For the brightness encoding studies, we examined the action of D-serine on each intensity used and then applied the enzyme D-serine deaminase to remove significant levels of D-serine. These studies provided new insights into the mechanisms that regulate coagonist availability in the vertebrate retina. Our results strongly support the idea that light-evoked coagonist release, a major component of which is D-serine, is needed to provide the full range of coagonist availability for optimal activation of NMDA receptors.

Figure 1. Contrast series

The two retinal ganglion cells (red traces; OFF-centre cell upper trace, ON-centre cell lower trace) were stimulated with a 2 s light stimulus, 250 μm in diameter at different contrast levels (0.03, 0.07, 0.15, 0.3, 0.5, and 1.0 log unit) against a steady background light of 9 lux. For each magnitude of the contrast displayed, the negative contrast (darker bar) precedes the positive contrast (lighter bar) stimulus. The first 5 s of each stimulus is displayed (of a 20 s interstimulus interval); the positive 1.0 log unit contrast is not shown. Arrows indicate the onset of the first stimulus because this low contrast step is difficult to discern from the background.

Figure 2. Coagonist sensitivity to D-serine decreases as contrast is enhanced

Both positive (A) and negative (B) contrast stimuli were acquired during each experimental run. A, whole-cell voltage-clamp recording (V_hold = −62 mV) of an ON–OFF retinal ganglion cell recorded from an eyecup preparation in response to a 2 s light stimulus (black bar) consisting of a one (1.0) log unit increase in illumination over the background in control Ringer solution (black trace), d-serine (100 μm, blue trace) and DCK (30 μm, red trace). ON and OFF responses to positive contrast steps of 0.07, 0.3 and 1.0 log units are shown below with an expanded timescale. B, same cell as in (A) but showing the results for negative contrast light steps. C, summary data from eight cells plotting the mean ± SEM charge integrated over 2 s for ON responses and 2 s for OFF responses. Control responses were normalized to 100, represented by a horizontal dashed line. d-serine (100 μm, blue dots) and DCK (30 μm, red triangles) to contrast steps of ±0.03, ±0.07, ±0.15, ±0.3, ±0.5 and ±1.0 log unit. ON and OFF responses from each cell were combined for the same intensity level before averaging.

Figure 3. Increasing the stimulus spot diameter does not change the sensitivity to DCK or d-serine

Whole-cell voltage-clamp recording (V_hold = −58 mV) of a sustained ON retinal ganglion cell in an eyecup preparation responding to a 50 μm (A), 250 μm (B) and 1200 μm (C) light stimulus 2 s in duration in control Ringer solution (black trace), d-serine (100 μm, blue trace) and DCK (30 μm, red trace). Scale bar under the trace in B applies to traces in A, B and C. D, summary data from eight cells plotting the mean ± SEM charge  for responses studied for the range of diameters indicated. Recording in control Ringer solution was standardized to 100% as indicated by the dashed line. d-serine responses (blue dots) and responses recorded in DCK (red triangles). Light stimuli of 50, 100, 250, 600 and 1200 μm diameter were used to generate this data. ON and OFF responses were combined before calculating the mean. E, a current clamp record from an ON ganglion cell, light stimulus is represented by the black line at bottom.

Figure 4. Flash intensity coding revealed through the PNFP illustrates that sensitivity to d-serine decreases as the flash intensity is increased

The PNFP was recorded over a range of 6 log units using 2 s flashes of light of increasing intensity; the interflash interval was 25 s. A, initial intensity series under control conditions (black traces) shown for intensities 1.5, 3.5 and 6.5, followed by an identical series in the presence of 100 μm d-serine (grey traces). B, for each intensity, the control was normalized to 100% (dashed line near bottom of the figure), whereas the response in the presence of d-serine is indicated by the filled circles, including the SEM (n = 6). Inset: range over which the adaption experiments explored the contrast sensitivity shown in Fig.2. C, example of a ‘threshold response’. In the presence of d-serine, a PNFP response was obtained at 0.5 log I when an insignificant response to light was observed under control conditions.

Figure 5. DsdA identifies d-serine as a major player in coagonist release

A, these recordings of the PNFP were generating by ascending flash intensities with a 25 s interval between flashes. The control intensity series (black trace) shows a progressive increase in amplitude. After the control series, DsdA enzyme (10 μg ml⁻¹) was dissolved in the Ringer solution and constantly perfused the retina, during which time the intensity flash series was repeated. The DsdA response traces are shown in red. After obtaining the DsdA response series, the perfusion was changed to one that contained 200 μm glycine to observe recovery of the response (blue traces). B, cumulative normalized responses of the PNFP recorded in DsdA versus control (n = 6).

Figure 6. The maximum PNFP response in DsdA occurs at a lower intensity than in control Ringer solution

V/V_max relationship for control (black squares) and DsdA (grey circles) PNFP responses over a range of 6 log units of flash intensity. Each response amplitude displays the mean ± SEM (n = 6). The data were fit by a Boltzmann curve for the control (black dashed line) and the DsdA responses (grey dashed line; r² for the Boltzmann fit was 99). The main feature of this comparison illustrates how the DsdA responses plateau at 4.5 log I, whereas the control responses continue to increase over the entire intensity range studied.