D-serine is an endogenous ligand for the glycine site of the N-methyl-D-aspartate receptor

Abstract

Functional activity of N-methyl-D-aspartate (NMDA) receptors requires both glutamate binding and the binding of an endogenous coagonist that has been presumed to be glycine, although D-serine is a more potent agonist. Localizations of D-serine and it biosynthetic enzyme serine racemase approximate the distribution of NMDA receptors more closely than glycine. We now show that selective degradation of d-serine with D-amino acid oxidase greatly attenuates NMDA receptor-mediated neurotransmission as assessed by using whole-cell patch-clamp recordings or indirectly by using biochemical assays of the sequelae of NMDA receptor-mediated calcium flux. The inhibitory effects of the enzyme are fully reversed by exogenously applied D-serine, which by itself did not potentiate NMDA receptor-mediated synaptic responses. Thus, D-serine is an endogenous modulator of the glycine site of NMDA receptors and fully occupies this site at some functional synapses.

Figure 1

DAAOX diminishes basal and evoked cerebellar NMDA receptor-mediated neurotransmission monitored by NOS activation. The activity of NOS was used as an index of NMDA receptor activity. NOS was assayed by the conversion of [³H]l-arginine (3 μCi/ml in the assay) to [³H]l-citrulline in cerebellar slices from 9- to 11-day-old rats. (A) DAAOX reduces basal NOS activity. The IC₅₀ for DAAOX is ≈10 μg/ml. (B) NMDA augments NOS activity in a concentration-dependent manner. DAAOX (10 μg/ml) diminishes NMDA-evoked NOS activity. When DAAOX is heat-inactivated, it fails to reduce basal or NMDA-evoked NOS activity (data not shown). (C) DAAOX (10 μg/ml) reduces glutamate-evoked NOS activity. Data are the mean ± SEM of three to five independent experiments, with triplicate determinations in each case. The basal activity was 5,300 ± 603 cpm/mg. Statistical comparisons are with respect to the corresponding condition in the absence of DAAOX. #, P < 0.05 and *, P < 0.001; one-way ANOVA with Tukey's multiple comparison test.

Figure 2

DAAOX does not affect either basal or NMDA-evoked NOS activity monitored in mature cerebellum. (A) d-Serine and glycine levels were determined by HPLC in cerebellar slices from immature (9- to 11-day-old) and adult (6-week-old) rats. †, P < 0.0001; t test. (B) Basal and NMDA-evoked NOS activity was determined as in Fig. 1. Data are the means ± SEM of four independent experiments, with triplicate determinations in each case. Statistical comparisons are with respect to the corresponding controls in the absence of NMDA and DAAOX. *, P < 0.001; one-way ANOVA with Tukey's multiple comparison test. NMDA-evoked NOS activity in adult was significantly less than in the immature cerebellum (#, P < 0.05).

Figure 3

d-Serine reverses inhibitory effects of DAAOX on NMDA receptor activation in cerebellar slices. NOS was monitored as in Fig. 1, and cGMP formation was measured by RIA. (A and B) The inhibitory effect of DAAOX on NMDA-evoked NOS activity (A) and cGMP formation (B) was almost wholly reversed by exogenous d-serine. (C) The AMPA/kainate receptor antagonist NBQX does not influence the inhibitory action of DAAOX on NMDA-evoked NOS activity. (D) Effects of 5,7-dichlorokynurenic acid (DCKA), an antagonist of the strychnine-insensitive glycine site, on NMDA-stimulated cGMP formation. Data are the means ± SEM of three to five independent experiments, with triplicate determinations in each case. Some of the SE bars are too small to be seen at this scale. Asterisks indicate a significant difference from NMDA-stimulated values. *, P < 0.001; one-way ANOVA with Tukey's multiple comparison test.

Figure 4

DAAOX reduces spontaneous NMDA receptor-mediated synaptic currents in cultured hippocampal neurons. Whole-cell spontaneous excitatory postsynaptic currents (EPSCs) were recorded in pyramidal neurons of embryonic rat hippocampal in monolayer culture. Inward currents are downward. Triangles indicate artifacts resulting from valve opening for solution changes. (A1 and A2) CPP (50 μM) abolishes the synaptic currents, indicating that they are mediated by NMDA receptors. Five-minute exposure to heated DAAOX (10 μg/ml) fails to affect the synaptic currents (B1 and B2) whereas active 10 μg/ml DAAOX (C1 and C2) reduces the peak synaptic current amplitude by more than 50%. (C3 and C4) d-Serine (100 μM) plus DAAOX fully reversed the inhibitory effect of the enzyme. (D1– D4) Spontaneous AMPA receptor currents, which are blocked by 10 μM NBQX, are not affected by DAAOX application. (E) Summary of results of experiments similar to those of A–C analyzed as described in Results. Numbers of cells for each condition are shown in parentheses. Values are expressed as mean ± SEM of the control values. Statistical significance was evaluated by using ANOVA analysis followed by Scheffé posthoc comparison. (**, P < 0.0001.)

Figure 5

DAAOX inhibition and d-serine reversal of NMDA-evoked currents in cultured hippocampal neurons. Solutions were applied by using a multibarrel fast-perfusion device. Tetrodotoxin (1 μM) was present in the perfusion media. (A1) Representative current evoked by 10 μM NMDA. (A2) Heated DAAOX minimally affects the current amplitude. (A3) DAAOX (10 μg/ml) produces a 75% reduction in the current amplitude that recovers slowly upon termination of the DAAOX perfusion. (A4) d-Serine (100 μM) rapidly overcomes the DAAOX and induces a marked potentiation of the NMDA-evoked current. (B) Summary of data from 18 experiments similar to that of A. Acute application of DAAOX produced a significant decrease in the magnitude of inward current (†, P = 0.0014), whereas heat-inactivated DAAOX application did not alter significantly NMDA-evoked current (not shown). DAAOX-induced inhibition of NMDA current is reversed and the current is enhanced further by d-serine (**, P < 0.0001). Total number of recordings for each condition is indicated in parentheses. Bars indicate mean ± SEM of the maximal response expressed as a percentage of control. Statistical significance was evaluated by using ANOVA analysis followed by Scheffé posthoc comparison.

Figure 6

DAAOX reduces NMDA receptor currents evoked with the micropuffer perfusion technique in cultured hippocampal neurons. To avoid washing away endogenous d-serine, NMDA (100 μM, 1 sec) was applied by gentle pressure ejection from a puffer pipette located ≈50 μm from the cell soma. Perfusion with DAAOX (1–10 μg/ml) attenuates the response to NMDA. The effect of DAAOX is fully reversed by the application of d-serine (100 μM), which also potentiates the NMDA-evoked current response. Recordings were in the presence of 1 μM tetrodotoxin. Values are expressed as mean ± SEM percent change in peak current amplitude with respect to control. Total number of recordings for each condition are shown in parentheses. Representative traces are shown to the right. Statistical significance was evaluated by using ANOVA analysis followed by Scheffé posthoc comparison. #, P < 0.05; **, P < 0.0001.