NMDA receptor subunits have different roles in NMDA-induced neurotoxicity in the retina

To investigate the expression of NMDAR subunits in RGCs, we used a single-cell reverse transcriptase polymerase chain reaction (RT-PCR) method. After dissociation of the retina into single cells, RGCs can no longer be identified by their morphology. We therefore used dissociated retina from B6.Cg-TgN(Thy1-CFP)23Jrs/J transgenic mice (thy1-CFP mice), which express cyan fluorescent protein (CFP) in most RGCs [17]. We first confirmed that the CFP-containing cells in the thy1-CFP mouse retina were RGCs by immunostaining with Brn3, a neurochemical marker for RGCs [18]. CFP expression colocalized with Brn3 immunoreactivity in most somata in the ganglion cell layer (GCL) (Figure 1A-C). A single CFP-expressing cell was picked with a glass capillary from the dissociation mix and transferred to the reaction tube (Figure 1D, E), and was further identified as RGC by expression of Brn3 (Figure 1F). Typical results of single-cell RT-PCR on isolated RGCs are shown in Figure 1F. GluN1 and GluN2A–D could be amplified together with an internal control (β-actin) from a single RGC, as well as from whole retina. In our samples of 4 isolated RGCs, two cells express GluN1/GluN2A/GluN2B/GluN2C/GluN2D, whereas the other two cells express GluN1/GluN2A/GluN2B/GluN2D. These results indicate the presence of GluN1 and all GluN2 subunits (GluN2A–D) in the mouse RGCs.

We used mice lacking any one of the four GluN2 subunits to determine the distinct roles of these GluN2 subunits in NMDA-induced RGC death. Mice lacking GluN2A, GluN2C, and GluN2D are viable [19‐21], whereas GluN2B-deficient mice die shortly after birth [22]. We therefore generated conditional GluN2B KO mice, in which GluN2B was ablated in retinal neurons containing RGCs. For this purpose, we crossed GluN2B^f/f[23] mice with c-kit-Cre mice [24] (GluN2B^f/f/c-kit-Cre). In c-kit-Cre mice crossed with ROSA-tdTomato reporter mice [25] (ROSA-tdTomato/c-kit-Cre), tdTomato-expressing cells were localized in the GCL and inner nuclear layer (INL) and most calretinin immunoreactive cells (RGCs and amacrine cells) contained tdTomato, suggesting that Cre recombinase is expressed in RGCs and cells in the INL, including amacrine cells, in c-kit-Cre mice (Figure 2A). Immunohistochemical analysis revealed that GluN2B protein expression was eliminated in RGCs and cells in the INL in GluN2B^f/f/c-kit-Cre mice (Figure 2B). Western blot analysis showed that GluN2A, GluN2C, and GluN2D proteins were completely eliminated from mutant mice lacking GluN2A, GluN2C, and GluN2D, respectively (Figure 2C, E, F). In GluN2B^f/f/c-kit-Cre mice, GluN2B expression level in the retina was significantly lower than in control mice (Figure 2D).

We next investigated whether the absence of GluN2 subunits affects the anatomical organization of the retina by histological analyses. Hematoxylin and eosin staining revealed the retinae of GluN2A, GluN2B^f/f/c-kit-Cre, GluN2C, and GluN2D mutant mice to be normally organized, consisting of several different cell layers (Figure 3A). The thickness of the inner retinal layer (IRL) in all mutant strains was normal compared with wild-type (WT) mice (Figure 3B). As previous studies showed that ablation of GluN1 increased cell death in the developing somatosensory thalamus [26], we counted cell numbers in the GCL. The cell number in the GCL of GluN2B^f/f/c-kit-Cre mice was significantly lower than that of WT mice at 5 weeks, whereas cell number in the GCL of the other mutant strains was comparable to that of control mice at 5 weeks (Figure 3C). These results suggest that GluN2B subunit plays a survival role for RGCs during retinal development, but the other GluN2 subunits (GluN2A, GluN2C and GluN2D) are not involved in retinal development and survival in RGCs.

To determine which GluN2 subtypes are involved in NMDA-induced RGC death in the retina, we examined the effect of intraocular injection of NMDA on retinal cell death in GluN2 KO and WT mice. First, to examine the acute injury of NMDA, TUNEL analysis was performed on the retinas of WT and GluN2 mutants at 1 day after NMDA treatment. A number of TUNEL-positive cells were observed in the GCL and INL in both WT and GluN2 mutant strains after NMDA injection (Figure 4A), but the percentage of TUNEL-positive cells in the GCL of GluN2B^f/f/c-kit-Cre and GluN2D^−/− mice was significantly lower than that in WT mice (Figure 4B). Following NMDA injection, the number of RGCs and the thickness of IRL decreased from days 1 to 7, with no further decrease being observed from days 7 to 14 [27, 28]. To examine the chronic injury of NMDA, morphological changes were measured 7 days after NMDA or phosphate-buffered saline (PBS) injection. Intraocular administration of NMDA induced cell death in the GCL in both WT and GluN2 mutant mice (Figure 4C), but the percentage of surviving cells in the GCL was significantly larger in GluN2B^f/f/c-kit-Cre and GluN2D^−/− mice than in WT mice (Figure 4D). Additionally, the thickness of IRL was significantly larger in GluN2B^f/f/c-kit-Cre mice than in WT mice (Figure 4E). Taken together, these results suggest that GluN2B and GluN2D were involved in NMDA-induced RGC death.

We have reported that the neuroprotective role of apolipoprotein E-containing lipoproteins in glaucomatous retinal degeneration in GLAST KO mice is mediated through promoting interaction between low density lipoprotein receptor-related protein 1 (LRP-1) and the GluN2B subunit [29]. Recently, we have also demonstrated that Dock3 overexpression prevented retinal cell death in GLAST KO mice by promoting GluN2B degradation [28]. To determine whether GluN2B is involved in RGC degeneration in GLAST-deficient mice, we evaluated the effect of a specific GluN2B antagonist, HON0001, on RGC degeneration in GLAST KO mice. As shown in Figure 5, the number of cells in GLAST KO mice subjected to HON0001 (10 mg/kg) treatment (281 ± 26) was significantly greater than that in GLAST KO mice not subjected to HON0001 treatment (203 ± 10). These results suggest that GluN2B is involved in RGC loss in GLAST KO mice.

B6.Cg-TgN(Thy1-CFP)23Jrs/J transgenic mice (thy1-CFP mice) and c-kit-Cre transgenic mice have been described previously [17, 24]. c-kit-Cre transgenic mice were bred with ROSA-tdTomato mice [25] to examine Cre activity. c-kit-Cre mice were bred with GluN2B^flox/flox (GluN2B ^f/f ) mice [23] to generate GluN2B conditional knockout mice (GluN2B ^f/f /c-kit-Cre). The homozygous GluN2A KO (GluN2A ^−/− ) [19], GluN2C KO mice (GluN2C ^−/− ) [20] and GluN2D KO mice (GluN2D ^−/− ) [21] were obtained by crossing heterozygous GluN2A^+/−, GluN2C^+/− and GluN2D^+/− mice, respectively. GLAST KO mice have been described previously [39, 40]. In all experiments, age matched WT and GluN2B^f/f littermate controls were used. All mice were of the C57BL/6 J genetic background, and all animal procedures were approved by the Animal Committee of Tokyo Medical and Dental University (0130166C).

5 week old Thy1-CFP mice were deeply anesthetized by diethyl ether and retinas were dissociated by using the Papain Dissociation System (Worthington Biochemical Corporation) at 37°C for 30 min. Single-CFP-expressing cell was aspirated by glass microcapillaries and placed into the PCR-tube containing 10 μl of resuspention buffer. Single-cell RT-PCR was performed using the SuperScript III CellsDirect cDNA Synthesis System (Invitrogen). Total RNA (5 μg) from whole retina were used to synthesize first-strand cDNA by using SuperScript III First-Strand Synthesis System (Invitrogen). The retina cDNA served as positive controls. The following primers were used for cDNA detection: GluN2A FWD: 5′ GTG TGC GAC CTC ATG TCC G 3′; REV: 5′ GCC TCT TGG TCC GTA TCA TCT C 3′; GluN2B FWD: 5′ CAG CAA AGC TCG TTC CCA AAA 3′; REV: 5′ GTC AGT CTC GTT CAT GGC TAC 3′; GluN2C FWD: ATC CCC GAC GGC TGA GA 3′; REV: 5′ TTC CTA GTC CAA GCA CAA AAC G 3′; GluN2D FWD: 5′ TGT GTG GGT GAT GAT GTT CGT 3′; REV: 5′ CCA CAG GAC TGA GGT ACT CAA AGA 3′; GluN1 FWD: 5′ GCC GAT TTA AGG TGA ACA GC 3′; REV: 5′ AAT TGT GCT TCT CCA TGT GC 3′; Brn3 FWD: 5′ GCA GTC TCC ACT TGG TGC TTA CTC 3′; REV: 5′ TTC CCC CTA CAA ACA AAC CTC C 3′; β-actin FWD: 5′ ATA TCG CTG CGC TGG TCG TC 3′; REV: 5′ TCA CTT ACC TGG TGC CTA GGG 3′. The thermal cycler conditions were 5 min at 94°C and then 40 cycles of 30 s at 94°C, 30 s at 60°C, and 30 s at 72°C, followed by 7 min at 72°C.

Retinas were quickly removed and homogenized in 100 μl of cold lysis buffer (50 mM Tris–HCl, 1% Nonidet P-40, 5 mM EDTA, 150 mM NaCl, 0.5% Na-deoxycholate, 1 mM MgCl₂, 1 mM DTT, 1 mM Na₃VO₄, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride (PMSF), and Complete Protease Inhibitor Cocktail [Roche]). Protein concentration was determined by BCA Protein Assay kit (Sigma-Aldrich). Thirty microgram of the protein was loaded per lane. Primary antibodies used were GluN2A (1:500, Covance), GluN2B (1:500) [41], GluN2C (1:100, Invitrogen), GluN2D (1:500) [42], β-actin (1:1000, Santa Cruz). They were then incubated with anti-rabbit, guinea pig or mouse IgG-HRP-conjugated secondary antibody (1:5000, Jackson ImmunoResearch). SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) was used to visualize the immunoreactive proteins.

Sections were prepared as previously described [15]. Frozen retinal sections of 12 μm thickness were incubated using anti-Brn3 (1:50, Santa Cruz), anti-calretinin (1:500, Swant) and anti-GluN2B (1:100) antibodies. For Brn3 and calretinin detection, Cy-3-conjugated donkey anti-goat IgG (1:500, Jackson ImmunoResearch) and goat anti-rabbit IgG Alexa 488 (1:1000, Molecular Probes) were used as secondary antibodies. For GluN2B detection, peroxidase labelled polymer conjugated to goat anti-rabbit IgG (DAKO) was used as secondary antibody. Images were recorded with an LSM-510 META confocal laser microscope (Carl Zeiss).

Eyes from mice at postnatal day 35 (P35) were enucleated and fixed in Davidson’s solution fixative [43], then embedded in paraffin wax. In some experiments, HON0001 (10 mg/kg, a gift from T. Honda at Hoshi University) [16] or saline was injected orally (p.o.) into GLAST KO mice daily from P21 to P35. These mice were sacrificed on P35 and processed for RGC count. Paraffin sections (7 μm thick) were cut though the optic nerve and stained with hematoxylin and eosin (H&E). The number of neurons in the GCL was counted as previously described [15]. The thickness of the IRL (from GCL to INL) was measured at a distance of 0.5 to 1.0 mm from optic disc.

Intravitreal injection of NMDA (Sigma) was conducted as previously described [15]. Briefly, a single 2-μl injection of 20 mM NMDA in 0.1 M PBS (pH 7.4) was administered intravitreally into the right eye of each mouse, the same volume of PBS was administered to the contralateral (left) eye as control. The animals were sacrificed at 1 day or 7 days after injection, and eyes were enucleated for morphometric and TUNEL analysis. Paraffin sections (5 μm thick) were cut though the optic nerve and stained with H&E. The extent of NMDA-induced retinal cell death after 7 days was quantified by counts of neurons in the GCL and the thickness of the IRL. The changes of the number of ganglion cells and thickness of IRL after NMDA injection were expressed as percentages of the control eyes.

At 1 day after the NMDA or PBS injection, TUNEL staining was performed with paraffin sections (5 μm thick) according to the manufacturer’s instructions (Promega). Fluorescence detection was carried out using Alexa-fluor-568-conjugated streptavidin (Molecular Probes). TUNEL-positive cells in the GCL were counted and expressed as percentages of total DAPI stained cells in the GCL.