Supporting InformationLe Bail et al. 10.1073/pnas.1416668112SI Materials and MethodsMaterials. 2,3-Dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxa-line-2,3-dione (NBQX), D-2-amino-5-phosphonovalerate (D-AP5),picrotoxin, strychnine hydrochloride, and Ro25-6981 maleate(Ro25-6981) were purchased from Tocris Bioscience and Abcam.N-[3-(4′-Fluorophenyl)-3-(4′-phenylphenoxy)propyl]sarcosine hydro-chloride (ALX5407) was purchased from Sigma, sodium fluo-roacetate (FAC) from Chem Service, and 5-chloro-benzo[d]isoxazol-3-ol (CBIO) from Maybridge. ZnCl2 purchased fromSigma was used in artificial cerebrospinal fluid (ACSF) con-taining tricine (10 mM) with the relation [zinc]free = [zinc]applied/200 (1, 2). It shall be noted that zinc, as well as Ro25-6981, arepartial antagonists that inhibit ∼70–80% and ∼90% of the currentflowing through GluN2A-NMDARs (GluN1/GluN1/GluN2A/GluN2A) and GluN2B-NMDARs (GluN1/GluN1/GluN2B/GluN2B), respectively (1). All pharmacological agents were bathapplied from appropriate stock solutions stored at −20 °C.

Enzymes. Recombinant wild-type Rhodotorula gracilis D-aminoacid oxidase (referred to as RgDAAO; EC 1.4.3.3) and re-combinant H244K variant of Bacillus subtilis glycine oxidase(referred to as BsGO; EC 1.4.3.19) were overexpressed inEscherichia coli cells, purified, and used as previously described(3, 4). The final RgDAAO and BsGO preparations had a specificactivity of 75 U/mg protein on D-serine as substrate and 2.5 U/mgprotein on glycine as substrate, respectively. Hippocampal sliceswere perfused with RgDAAO (0.2 U/mL) or BsGO (0.2 U/mL)for at least 45 min.

Electrophysiology. Long-term potentiation (LTP) of synaptictransmission was induced by a test stimulus applied every 15 sin normal ACSF for CA1 analysis or in ACSF supplemented with50 μM bicuculline methiodide (Tocris) for DG analysis. Bicu-culline was necessary to shut down the powerful inhibition oc-curring in the DG, which counteracts the induction of synapticplasticity (5). In all cases, the intensity was adjusted to obtaina fEPSP with a baseline slope of 0.1 V/s. The averaged slope ofthree fEPSPs was measured for 15 min before the delivery ofa high-frequency stimulation protocol consisting in a 100-Hzconditioning stimulus (1-s duration at the test intensity). Testingwith a single pulse was then resumed for 60 min to determine thelevel of LTP.Serine racemase knockout mice (SR−/−) and littermate wild-

type mice (SR+/+) were used for electrophysiological recordings.Mice were bred in house and in groups of six in polycarbonatecages and maintained on a 12:12-h light/dark cycle in a temper-ature (22 °C)- and humidity-controlled room. Animals were

given access to food and water ad libitum. Brains from 2- to3-mo-old mice were extracted and dissected as previouslydescribed. Briefly, recordings of NMDAR-dependent field ex-citatory postsynaptic potentials (NMDA-fEPSPs) were per-formed from coronal hippocampal slices (350 μM) at SC–CA1synapses as described herein for rats.

High-Performance Liquid Chromatography Analysis. After extractionof soluble fraction with water-saturated ether from tissue samples,fractions were neutralized with NaOH before precolumn de-rivatization with o-phthaldialdehyde/N-acetyl-L-cysteine in bo-rate buffer (0.1 M; pH 10.4). Diastereoisomer derivatives werethen resolved on an Adsorbosphere XL C8 300A 5 mm (Alltech;4.6 × 250 mm) reversed-phase column in isocratic conditions(0.1 M sodium acetate buffer, pH 6.2, 1% tetrahydrofuran,1 mL/min flow rate). A washing step in 0.1 M sodium acetatebuffer, 3% tetrahydrofuran, and 47% acetonitrile was performedafter every single run. Identification and quantification of theD- and L-serine and glycine was based on retention times andpeak areas, compared with those associated with external stand-ards. Standards were prepared as 10 mM stock solution in Milli-Qwater. Calibration curves were built by injecting increasing amountof standards (2–200 pM).

Immunofluorescence Staining and Confocal Microscopy Analysis.Immunostainings of hippocampus were performed as describedpreviously (6, 7) on 30-μm sections of 2- to 3-mo-old male rats.Immunofluorescence signals for the glial-specific marker glialfibrillary acid protein (GFAP) (secondary antibody coupled toAlexa 488), for the neuronal nuclei marker NeuN (secondaryantibody coupled to Alexa 633), together with DAPI stainingswere analyzed using a laser-scanning confocal microscope (LeicaTCS SP2; Leica Microsystems). The confocal images were ac-quired using the Leica TCS software with a sequential mode toavoid interference between each channel. Moreover, emissionwindows were fixed for each fluorophore in conditions where nosignal is detected from the other fluorophore. Stack images weretaken and a Z projection was formed.

RgDAAO Activity Assay. The effect of CBIO and D-AP5 onRgDAAO activity was determined by an oxygen consumptionassay. Measurements were performed in the presence of 1 and5 μM CBIO and 50 μM D-AP5, by using ACSF added of 28 mMD-alanine as a substrate. One unit corresponds to the amount ofenzyme that converts 1 μmol of substrate per minute at 25 °C (8).The results are reported as relative activity in the presence of theinhibitors with respect to controls.

1. Paoletti P, Ascher P, Neyton J (1997) High-affinity zinc inhibition of NMDA NR1-NR2Areceptors. J Neurosci 17(15):5711–5725.

2. Papouin T, et al. (2012) Synaptic and extrasynaptic NMDA receptors are gated bydifferent endogenous coagonists. Cell 150(3):633–646.

3. Fantinato S, Pollegioni L, Pilone MS (2001) Engineering, expression and purification ofa His-tagged chimeric D-amino acid oxidase from Rhodotorula gracilis Enz. MicrobTechnol 29(6-7):407–412.

4. Job V, Molla G, Pilone MS, Pollegioni L (2002) Overexpression of a recombinant wild-type and His-tagged Bacillus subtilis glycine oxidase in Escherichia coli. Eur J Biochem269(5):1456–1463.

5. Arima-Yoshida F, Watabe AM, Manabe T (2011) The mechanisms of the strong in-hibitory modulation of long-term potentiation in the rat dentate gyrus. Eur J Neurosci33(9):1637–1646.

6. Puyal J, Martineau M, Mothet JP, Nicolas MT, Raymond J (2006) Changes in D-serinelevels and localization during postnatal development of the rat vestibular nuclei. JComp Neurol 497(4):610–621.

7. Fossat P, et al. (2012) Glial D-serine gates NMDA receptors at excitatory synapses inprefrontal cortex. Cereb Cortex 22(3):595–606.

8. Pollegioni L, Piubelli L, Sacchi S, Pilone MS, Molla G (2007) Physiological functions ofD-amino acid oxidases: From yeast to humans. Cell Mol Life Sci 64(11):1373–1394.

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Fig. S1. Expression of serine racemase (SR) during postnatal development and in adult. Proteins (30 μg per lane) from whole hippocampal (H), CA1 anddentate gyrus (DG) lysates obtained from P10 and P90 rats were submitted to SDS/PAGE (12%) electrophoresis. (A) Immunoblots for SR (37 kDa) and actin(42 kDa) were performed using specific antibodies and chemiluminescence revelation. (B) Optical density was analyzed using ImageJ software, and results wereplotted. Normalized SR/actin intensity ratio shows no changes in the expression of SR between brain areas and during development. Histograms representmean ± SEM of nine independent experiments.

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Fig. S2. Impaired NMDAR-dependent long-term potentiation in CA1 and DG by RgDAAO or BsGO is reversed by supplementation with exogenous coagonistsglycine and D-serine, respectively. Time course of LTP induced at SC–CA1 (A) and mPP–DG (B) synapses by 1 × 100-Hz conditioning stimulation (arrow) in slicessupplemented with the NMDAR antagonist D-AP5 (80 μM; n = 10 and 11; white circles), in slices pretreated with RgDAAO and supplemented with glycine(500 μM; n = 11; brown circles), and in slices pretreated with BsGO and supplemented with D-serine (100 μM; n = 10 and 8; green squares).

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Fig. S3. Expression pattern of GluN2A and GluN2B vs. GluN1 between brain areas and during postnatal development. Signal density was analyzed usingImageJ software, and results were plotted. Normalized GluN2A/GluN1 (A) and GluN2B/GluN1 (B) intensity ratios show opposite evolution in whole hippo-campus (H) and the CA1 area during postnatal development (P10 vs. P90). Histograms represent mean ± SEM of seven independent experiments. **P < 0.01using one-way ANOVA.

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Fig. S4. The potentiating effect of CBIO on NMDA-fEPSPs at mature SC–CA1 synapses are mediated by D-serine and by activation of GluN2A-containingNMDARs. (A) The potentiating effect of CBIO (1 μM) (2) on NMDA-fEPSPs is largely compromised in rat hippocampal slices treated with RgDAAO (1) to depleteendogenous D-serine (125.5 ± 15.4%; P = 0.1724; n = 4). (B) Blocking the GluN1/GluN2A-NMDARs with zinc (zinc pre: 250 nM) (2) decreased NMDA-fEPSPs (1)(37.67 ± 9.8% of decrease; P = 0.0143; n = 4) as already reported (Figs. 3 and 5) and prevented the potentiating effect of CBIO (3). (C) The activity of RgDAAOmeasured by the oxygen consumption assay in the presence of CBIO (1 and 5 μM) added to ACSF shows that CBIO is not a substrate, as well as it does not inhibitthe activity of this enzymatic scavenger. (D) CBIO (5 μM) potentiates NMDA-fEPSPs in wild-type mice (SR+/+: 183.5 ± 19.2%; P = 0.0065; n = 6) but not inSR-deficient littermates (SR−/−: 117.2 ± 9.6%; P = 0.0752; n = 7). Note that the effect of CBIO in SR+/+ mice is fully comparable to the one observed in rats (Fig. 7).Data represent mean ± SEM. *P < 0.05, **P < 0.01.

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Table S1. Antibodies used for Western blot analysis and immunostainings

Antibody Dilution Supplier Catalog no./ref.

PrimarySer racemase (rabbit polyclonal) 1:500 Customized Ref. 1GluN1 (goat polyclonal) 1:500 Santa Cruz sc-1467GluN2A (rabbit polyclonal) 1:1,000 Millipore AB1555PGluN2B (rabbit polyclonal) 1:750 Millipore AB15362Actin (ascites) 1:500 Developmental Studies Hybridoma

Bank, University of IowaJLA 20S

GFAP (rabbit polyclonal) 1:1,000 DAKO Z0334NeuN (mouse monoclonal) 1:1,000 AbCys VMA377

SecondaryGoat purified anti-rabbit (HRP) 1:4,000 Abliance BI2407Mouse purified anti-goat (HRP) 1:4,000 Abliance B12403Rabbit purified anti-mouse (HRP) 1:4,000 Abliance BI2413LAlexa Fluor 633 goat anti-mouse 1:2,000 Molecular Probes A21052Alexa Fluor 488 goat anti-rabbit 1:2,000 Molecular Probes A11008DAPI 1:1,000 Molecular Probes D21490

1. Kartvelishvily E, Shleper M, Balan L, Dumin E, Wolosker H (2006) Neuron-derived d-serine release provides a novel means to activate N-methyl-d-aspartate receptors. J Biol Chem281(20):14151–14162.

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