Multiple risk pathways for schizophrenia converge in serine racemase knockout mice, a mouse model of NMDA receptor hypofunction

Abstract

Schizophrenia is characterized by reduced hippocampal volume, decreased dendritic spine density, altered neuroplasticity signaling pathways, and cognitive deficits associated with impaired hippocampal function. We sought to determine whether this diverse pathology could be linked to NMDA receptor (NMDAR) hypofunction, and thus used the serine racemase-null mutant mouse (SR(-/-)), which has less than 10% of normal brain D-serine, an NMDAR coagonist. We found that D-serine was necessary for the maintenance of long-term potentiation in the adult hippocampal dentate gyrus and for full NMDAR activity on granule cells. SR(-/-) mice had reduced dendritic spines and hippocampal volume. These morphological changes were paralleled by diminished BDNF/Akt/mammalian target of rapamycin (mTOR) signaling and impaired performance on a trace-conditioning memory task. Chronic D-serine treatment normalized the electrophysiological, neurochemical, and cognitive deficits in SR(-/-) mice. These results demonstrate that NMDAR hypofunction can reproduce the numerous hippocampal deficits associated with schizophrenia, which can be reversed by chronic peripheral D-serine treatment.

Keywords: CREB; MeCP2; glycogen synthase 3 kinase; miR-132.

Fig. 1.

NMDAR mEPSCs in DG granule cells are smaller and faster in SR^−/− mice. (A) Examples of mEPSCs recorded from a DG granule cell at −70 mV in the absence of Mg2⁺ (Left), in the presence of 10 μM d-serine (Center), and in the presence of 50 μM d-AP5 (Right). (B) Averaged mEPSCs (100–300 traces) recorded under control conditions (black), in the presence of d-serine (red), and in the presence of d-AP5 (blue) in slices from WT (Left) and SR^−/− mice (Right). The amplitude of NMDAR mEPSCs were measured 10 ms after the peak (dashed line) of AMPAR currents in the presence of d-AP5. (C) Averaged amplitudes of NMDAR mEPSCs under different conditions. Data are from four WT (n = 6 cells) and four SR^−/− (n = 10 cells) mice. (D) Averaged amplitudes of AMPAR mEPSCs in the presence of d-AP5 from three WT mice (n = 5 cells) and three SR^−/− mice (n = 7 cells). (E) Superimposed averaged mEPSCs recorded from WT (black trace) and SR^−/− mice (gray trace) under control conditions (Left) or in the presence of d-serine (Right). To ease the comparison of decay times, mEPSCs were scaled to the same peak amplitude. (F) Averaged decay times of mEPSCs. Data from four WT (n = 6 cells) and four SR^−/− mice (n = 10 cells). (G) Averaged decay times of mEPSCs in the presence of d-AP5 from three WT (n = 5 cells) and three SR^−/− (n = 7 cells) mice. Asterisk (*) indicates significant difference from the WT group (P < 0.05). All values represent the mean ± SEM.

Fig. 2.

LTP is suppressed at the mPP-DG synapses in SR^−/− mice. (A) Averaged fEPSPs (10 traces) evoked at the mPP-DG synapses by presynaptic stimuli of increasing intensity in slices from WT (Left) and SR^−/− mice (Right). (B) Synaptic input-output curves obtained in slices from six WT (n = 10 slices) and five SR^−/− (n = 11 slices) mice. (C) Examples of fEPSPs at the mPP-DG synapses evoked with paired stimuli (interstimulus intervals 50-ms) in slices from WT (Left) and SR^−/− mice (Right). (D) Averaged paired-pulse ratio values in slices from five WT (n = 11 slices) and five SR^−/− (n = 18 slices) mice at interstimulus intervals 50, 150, and 300 ms. Paired-pulse ratio was calculated as the ratio of the rising slope of the second fEPSP to the first fEPSP. (E and F) Summary of LTP experiments from five WT mice (n = 8 slices) and four SR^−/− mice (n = 8 slices). LTP was induced by a 1-s train of 100 Hz stimulation. Insets in E and F are the averages of: 1, 40 fEPSPs recorded before; 2, 20 fEPSPs recorded 40 min after the induction (at arrow) of LTP in slices from WT and SR^−/− mice. Asterisk (*) indicates significant difference from the WT group (P < 0.05). All values represent the mean ± SEM.

Fig. 3.

Reduced dendritic spine density in the hippocampus of SR^−/− mice is accompanied by altered miR-132 expression. (A) Hippocampal volume was estimated by the Cavaleri method on Nissl-stained coronal brain sections (n = 13–15/genotype). (B) Apical dendritic spines on a Golgi-stained granule (100x) cell in a WT (Left) and a SR^−/− mouse (Right) (Magnification: 100×). (C) Spine density was compared between WT (n = 5) and SR^−/− (n = 5) animals. Spine density on dentate granule neurons (three to six neurons per animal) was reduced on apical dendrites in SR^−/− mice. Spine density is expressed as the number of spines per 10 μm of dendrite. (D) Relative expression of the primary (pri), precursor (pre), and mature miR-132 in WT (n = 7–11) and SR^−/− (n = 7–11) mice were measured by qPCR. Data are expressed as geometric means ± SEM of individual expression values normalized to the appropriate reference gene (pri: GAPDH; pre: miR-16; mature miR-132/212: snoRNA-202) using the comparative 2^−ΔΔCt method. Asterisk (*) indicates significant difference from the WT group (P < 0.05). All values represent the mean ± SEM.

Fig. 4.

BDNF expression and TrkB activation are reduced in the hippocampus of SR^−/− mice. (A) Relative mRNA expression of BDNF obtained from the hippocampus of WT (n = 6; black bars) and SR^−/− (n = 5; white bars) mice was measured by qPCR. Data are expressed as geometric means ± SEM of individual expression values normalized to the housekeeping gene GAPDH using the comparative 2^−ΔΔCt method. (B) Nuclear protein levels of CREB and p-CREB (Ser133) were measured by Western blot in the hippocampus of WT (n = 5–7) and SR^−/− (n = 6–9) mice. (C) Nuclear protein levels of MeCP2 were measured by Western blot in the hippocampus of WT (n = 12) and SR^−/− (n = 10) mice. (D) BNDF protein levels were measured in WT (n = 7) and SR^−/− (n = 6) mice using ELISA. (E) TrkB protein levels were measured by Western blot from WT (n = 13) and SR^−/− (n = 14) mice. (F) Hippocampal lysates from WT (n = 10) and SR^−/− (n = 10) were subjected to immunoprecipitation with rabbit anti-TrkB antibody and immunoblotted with pY99 antibody. Immunoprecipitation of total TrkB was confirmed by immunoblotting with mouse-antiTrkB antibodies. Densitometric analyses are shown for p-TrkB receptor relative to the total TrkB obtained from immunoprecipitation. Asterisk (*) indicates significant differences from the WT group (P < 0.05). All values represent the mean ± SEM.

Fig. 5.

Akt/mTOR signaling is reduced in the hippocampus of SR^−/− mice. Protein levels of Akt/p-Akt (Ser473, Thr308), mTOR/p-mTOR (Ser2448), p70 S6 kinase/p-p70 S6K (Thr389), eEF2/p-eEF2 (Thr56), and 4-E-BP1/p-4E-BP1 (Thr37/46) were measured in the total homogenate (A–E) and synaptoneurosomal fractions (F–J) from the hippocampus of WT (n = 8–12; black bars) and SR^−/− (n = 8–12; white bars) mice. Asterisk (*) indicates significant differences from the WT group (P < 0.05). All values represent the mean ± SEM.

Fig. 6.

Chronic peripheral administration of d-serine elevates serum and normalizes brain d-serine levels in SR^−/− mice. WT and SR^−/− mice received 21 d of either vehicle or d-serine (300 mg/kg day 1, 150 mg/kg days 2–21; s.c.) and were killed 24 h after the last injection. HPLC (HPLC) was used to quantify d-serine levels with representative chromatographs for WT and SR^−/− serum and cortex samples shown. (A) (Left) Amino acid peaks and retention times of a WT serum sample. Glutamate (9 min), l-HCA (9.5 min), l-serine (18 min), and d-serine (19 min) are indicated. (Right) A slight reduction in d-serine in a SR^−/− serum sample compared with WT. (B) (Left) Amino acid peaks and retention times of a WT cortical sample. Glutamate (16.5 min), l-HCA (16 min), l-serine (24 min), and d-serine (27 min) are indicated. (Right) A marked reduction in d-serine in a SR^−/− cortical sample compared with WT. (C and D) d-serine was measured in the serum, hippocampus, and cortex of WT (vehicle; n = 4–5; black bars), SR^−/− mice (vehicle; n = 3–4; white bars), and SR^−/− mice treated with d-serine (n = 2–4; gray bars). Significant one-way ANOVA results were followed up by Newman–Keuls Multiple comparison test. Asterisk (*) indicates significant difference from the WT group (P < 0.05) and a pound sign (#) indicates significant difference from the SR^−/− vehicle group. All values represent the mean ± SEM. (E–G) H&E-stained kidneys (40×) from WT (E; vehicle), SR^−/− (F; vehicle), and SR^−/− mice treated with d-serine (G). Glomeruli (arrows) from d-serine treated mice do not show signs of necrosis. (Scale bars, 50 μm.)

Fig. 7.

Chronic d-serine treatment reverses the electrophysiological, biochemical, and cognitive deficits found in SR^−/− mice. WT and SR^−/− mice received 21 d of either vehicle or d-serine (300 mg/kg day 1, 150 mg/kg days 2–21; s.c.) and were killed 24 h after the last injection. (A–C) Summary of LTP experiments at the mPP-DG synapses from five WT (n = 10 slices; black circles) mice receiving vehicle injections, four SR^−/− mice (n = 9 slices; white circles) receiving vehicle injections, and four SR^−/− (n = 8 slices; gray circles) mice receiving chronic d-serine injections. Insets are the averages of: 1, 40 fEPSPs recorded before; and 2, 20 fEPSPs recorded 40 min after the induction of LTP (at arrow). Significant one-way repeated measures ANOVA during the last 10 min of recording was followed up by Bonferroni’s multiple comparison test. Protein levels of (D) p-CREB and MeCP2, (E) BDNF, (F) Akt/p-Akt (Ser473, Thr308), (G) Akt1/p-Akt1 (Ser473), (H) mTOR/p-mTOR (Ser2448), and (I) GSK3α Ser21/β Ser9 were measured in the hippocampus of WT (n = 12; black bars), SR^−/− (n = 5–6; white bars), and SR^−/− mice treated with d-serine (n = 5–6; gray bars). BDNF values are expressed as pg of BDNF/mg of protein. Significant one-way ANOVA results were followed up by Newman–Keuls Multiple comparison test. (J) Diagram illustrating the trace fear-conditioning paradigm. WT (n = 11), SR^−/− (n = 5), and SR^−/− mice treated with d-serine (n = 5) were subjected to the paradigm during the last 3 d of treatment. Injections were given 3 h before behavioral testing. (K) Representation of the average time animals froze during the first 3 min of being placed in the chamber on day 2. (L) Represents the average freezing of each mouse during the seven, 20-s tone presentations on day 3. Asterisk (*) indicates significant difference from the WT group (P < 0.05) and pound symbol (#) indicates significant difference from the SR^−/− vehicle group. All values represent the mean ± SEM.