D-Serine regulates cerebellar LTD and motor coordination through the δ2 glutamate receptor

Journal name:
Nature Neuroscience
Volume:
14,
Pages:
603–611
Year published:
DOI:
doi:10.1038/nn.2791
Received
Accepted
Published online

Abstract

D-Serine (D-Ser) is an endogenous co-agonist for NMDA receptors and regulates neurotransmission and synaptic plasticity in the forebrain. D-Ser is also found in the cerebellum during the early postnatal period. Although D-Ser binds to the δ2 glutamate receptor (GluD2, Grid2) in vitro, its physiological significance has remained unclear. Here we show that D-Ser serves as an endogenous ligand for GluD2 to regulate long-term depression (LTD) at synapses between parallel fibers and Purkinje cells in the immature cerebellum. D-Ser was released mainly from Bergmann glia after the burst stimulation of parallel fibers in immature, but not mature, cerebellum. D-Ser rapidly induced endocytosis of AMPA receptors and mutually occluded LTD in wild-type, but not Grid2-null, Purkinje cells. Moreover, mice expressing mutant GluD2 in which the binding site for D-Ser was disrupted showed impaired LTD and motor dyscoordination during development. These results indicate that glial D-Ser regulates synaptic plasticity and cerebellar functions by interacting with GluD2.

At a glance

Figures

  1. D-Ser causes a decrease in PF-EPSCs through GluD2.
    Figure 1: D-Ser causes a decrease in PF-EPSCs through GluD2.

    (a) Representative data showing PF-EPSC reduction induced by application of exogenous D-Ser (200 μM; 10 min during time 0–10 min) in a cerebellar slice from immature wild-type (WT) mouse. The insets show PF-EPSCs observed just before (1) and 30 min after (2) the application of D-Ser. (b) Averaged data of D-Ser–mediated PF-EPSC rundown recorded from immature wild-type Purkinje cells in the absence (–) or presence (+) of NMDA receptor blockers (100 μM D-AP5 plus 25 μM MK801) in the extracellular solution. (c,d) Averaged data showing effect of D-Ser on PF-EPSC reduction in immature cerebellar slices from Grid2-null (c), Grid2-null TgWT (open circles in d) or Grid2-null TgR/K (filled circles in d) mice. During the recordings, NMDA receptor blockers were continuously perfused. Insets in bd indicate the representative PF-EPSCs just before (black traces) and 30 min after (gray traces) the application of D-Ser in each condition. (e) Dose-response curve for D-Ser–mediated PF-EPSC reduction in wild-type cerebellar slices in the presence of NMDA receptor blockers. Each PF-EPSC amplitude was normalized with that before D-Ser application (for 1 min) and the mean EPSC amplitudes 25−30 min after D-Ser application were plotted against [D-Ser]. We estimated EC50 using the sigmoidal curve fitting methods corresponding to logistical function. The number of experiments is shown in parentheses. P values were obtained using Mann-Whitney's U test. Data represent means ± s.e.m.

  2. D-Ser induces AMPA receptor endocytosis by binding to GluD2.
    Figure 2: D-Ser induces AMPA receptor endocytosis by binding to GluD2.

    (a) D-Ser–mediated reduction in PF-EPSCs was blocked by AP2-specific binding peptide (pep-ΔA849-Q853; 500 μM) but not by the control peptide (pep-K844A; 500 μM) in patch pipettes. Inset traces show representative PF-EPSCs observed just before (black traces) and 30 min after (gray traces) the application of D-Ser (200 μM) for each condition. (b) Representative images showing the cell-surface expression of virally introduced HA-GluA2 (surface HA-GluA2; left) on wild-type Purkinje cell dendrites in the absence (–; top) and presence (+; bottom) of treatment with D-Ser (1 mM for 30 min). After the staining of cell surface HA-GluA2 with anti-HA antibodies and treatment with detergents, all HA-GluA2 was stained using anti-HA antibodies (total HA-GluA2; right). Each Purkinje cell dendrite was identified by the staining of a Purkinje cell-positive marker, calbindin (middle). (c,d) Continuous PF-EPSC recordings obtained by the application of CJ-stim (30 × [parallel fiber stimulation plus Purkinje cell depolarization] at 1 Hz; arrow) followed by exogenous D-Ser (200 μM) (c) or the application of exogenous D-Ser followed by CJ-stim (d) in immature wild-type cerebellar slices. The second conditioning stimulation was added 30 min after the first conditioning stimulation. Insets show representative PF-EPSCs just before the first conditioning stimulation (1) and just before (2) and 30 min after (3) the second conditioning stimulation. The P value was obtained using Mann-Whitney's U test. Data represent means ± s.e.m.

  3. Activity-dependent increase in extracellular [D-Ser] in immature wild-type cerebellar slices.
    Figure 3: Activity-dependent increase in extracellular [D-Ser] in immature wild-type cerebellar slices.

    (ah) Representative 2D-HPLC spectrograms measured from extracellular solutions superfusing the cerebellar slices without parallel fiber stimulation (no stim; a,f), with parallel fiber stimulation in the absence (PF burst; b,g) or presence of NaFAC (NaFAC + PF burst; c) or NASP (NASP + PF burst; d) treatment, and with kainate treatment (100 μM for 5 min; e,h). The spectrograms in ae and fh are from immature and mature wild-type cerebellar slices, respectively. Filled and open circles represent signal peaks for D- and L-Ser, respectively. (i) Averaged data showing extracellular [D-Ser] after each stimulation in immature (top; n = 17 slices from 12 mice each) and mature (bottom; n = 14 slices from 7 mice each) slices. [D-Ser] was calculated from the fluorescence intensity of the signal peak for D-Ser. nd, not determined. *P < 0.05 (ANOVA with Dunnett's test). Data represent means ± s.e.m.

  4. Endogenous D-Ser is released mainly from Bergmann glia and regulates LTD in the developing cerebellum.
    Figure 4: Endogenous D-Ser is released mainly from Bergmann glia and regulates LTD in the developing cerebellum.

    (a) Diagram showing the CJ-stim protocol (30 × [10 cycles of parallel fiber stimuli at 50 Hz plus Purkinje cell depolarization from −60 mV to +20 mV] at 1 Hz; top) and the CJ-stim–derived Ca2+ spike responses (bottom). All responses are overlaid as gray traces and the representative one is shown as a black trace. (b,c) Averaged data showing cerebellar LTD from immature (b) or mature (c) wild-type cerebellar slices in the absence (–) or presence (+) of DAAO (0.125 U ml−1, preincubation for at least 60 min and perfusion during recordings). (d,e) Averaged data for cerebellar LTD from immature (d) or mature (e) wild-type cerebellar slices in the absence (control) or presence of NaFAC (3 mM, preincubation for at least 90 min) or NASP (100 μM, perfusion during recordings) treatment. (f) Immunohistochemical images showing the adenoviral (AV) expression of GFP (green) and a glial marker, 3-phosphoglycerate dehydrogenase (3-PGDH; red), in a cerebellar slice from an immature (P13) wild-type mouse. (g) Averaged data for cerebellar LTD from immature wild-type Purkinje cells whose neighboring Bergmann glia were infected with AV-GFP or AV-GFP-TeNT. CJ-stim was applied at 0 min (arrow). Insets (be,g) show PF-EPSC traces just before (black) and 30 min after (gray) CJ-stim in each condition. P values were obtained using Mann-Whitney's U test in b,c,g and ANOVA in d,e. Data represent means ± s.e.m.

  5. Cerebellar LTD is enhanced by D-Ser binding to GluD2 in the developing cerebellum.
    Figure 5: Cerebellar LTD is enhanced by D-Ser binding to GluD2 in the developing cerebellum.

    (a,b) Results of cerebellar LTD in the absence (control) or presence of treatment with NMDA receptor blockers (100 μM D-AP5 plus 25 μM MK801, perfusion during recording) or NMDA receptor blockers plus DAAO (0.125 U ml−1, preincubation for at least 60 min and perfusion during recordings) in immature (a) and mature (b) wild-type cerebellar slices. (c) Averaged data of LTD recordings from mature Purkinje cells in Dao+/+ and Dao−/− cerebellar slices treated with or without DAAO (0.125 U ml−1, preincubation for at least 60 min and perfusion during recordings). (d,e) Averaged data showing cerebellar LTD in immature (d) and mature (e) Purkinje cells from Grid2-null TgWT or Grid2-null TgR/K cerebellar slices. NMDA receptor blockers were constantly added to the extracellular solution during the recordings in ce. CJ-stim was applied at 0 min (arrow). Insets show representative PF-EPSCs just before (black traces) and 30 min after (gray traces) CJ-stim for each condition. P values were obtained using Mann-Whitney's U test. Data represent means ± s.e.m.

  6. Disruption of D-Ser binding to GluD2 impaired motor coordination and learning in developing mice.
    Figure 6: Disruption of D-Ser binding to GluD2 impaired motor coordination and learning in developing mice.

    (a,c,e,g,i) Continuous measurements of the rotor-rod test performed over 5 d (Day 1 to 5) in immature (a,g) or mature (c,e,i) Grid2-null TgWT and Grid2-null TgR/K mice. Daily sessions consisted of six trials and the retention time on the rotating rod was measured (maximum score, 120 s in a,c,e and 300 s in g,i). The rotating speeds were set at 5 r.p.m. in a,c, 20 r.p.m. in e and 4−40 r.p.m. in g,i. P values were obtained using two-way repeated measure ANOVA. (b,d,f,h,j) Averaged data of a,c,e,g and i, respectively. The results were averaged every day (6 trials) for each condition. ***P < 0.001, **P < 0.01, *P < 0.05 (ANOVA with Bonferroni's correction for multiple comparisons). Data represent means ± s.e.m.

  7. D-Ser conveys signals for LTD through the cytoplasmic C-terminal tails of GluD2 independent of its channel function but dependent on PKC activities in immature Purkinje cells.
    Figure 7: D-Ser conveys signals for LTD through the cytoplasmic C-terminal tails of GluD2 independent of its channel function but dependent on PKC activities in immature Purkinje cells.

    (a) Immunohistochemical images showing Sindbis viral expression of GFP (green) and GluD2 (red) in a cerebellar slice from an immature (P13) Grid2-null mouse. (b) Rescue of impaired cerebellar LTD in Grid2-null Purkinje cells by the viral expression of GFP plus GluD2WT (GluD2WT) or GluD2V/R (GluD2V/R), but not GFP alone (vector). (c,d) LTD in the developing cerebellum requires the C-terminal tails of GluD2. Averaged data of cerebellar LTD (c) and PF-EPSC changes induced by exogenous D-Ser (200 μM during 0−10 min, d) in immature Grid2-null TgΔCT7 mice. (e,f) LTD in the developing cerebellum requires PKC activities in Purkinje cells. Averaged data of cerebellar LTD (e) and PF-EPSC changes induced by exogenous D-Ser (200 μM during 0−10 min, f) in immature wild-type Purkinje cells loaded with a PKC inhibitory peptide (PKC[19–36]; 500 μM) or a control peptide (PKC[19–36]-R27E; 500 μM) in the patch pipette. In experiments shown in bd and f NMDA receptor blockers were constantly added to the extracellular solution. CJ-stim was applied at 0 min (arrows). Insets show representative PF-EPSCs observed just before (black traces) and 30 min after (gray traces) CJ-stim (b,c,e) or D-Ser application (d,f). P values were obtained using ANOVA in b and Mann-Whitney's U test in e,f.

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Author information

Affiliations

  1. Department of Physiology, School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan.

    • Wataru Kakegawa,
    • Shinji Matsuda,
    • Keiko Matsuda,
    • Kazuhisa Kohda,
    • Kyoichi Emi,
    • Junko Motohashi &
    • Michisuke Yuzaki
  2. Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Kawaguchi, Saitama, Japan.

    • Wataru Kakegawa,
    • Shinji Matsuda,
    • Keiko Matsuda,
    • Kazuhisa Kohda,
    • Kyoichi Emi,
    • Junko Motohashi &
    • Michisuke Yuzaki
  3. Graduate School of Pharmaceutical Science, Kyushu University, Higashi-ku, Fukuoka, Japan.

    • Yurika Miyoshi,
    • Kenji Hamase &
    • Kiyoshi Zaitsu
  4. Center for Medical Science, International University of Health and Welfare Graduate School, Ohtawara, Tochigi, Japan.

    • Ryuichi Konno

Contributions

W.K. designed the experiments, performed the electrophysiological, immunohistochemical and behavioral studies, analyzed the data, and wrote the manuscript. Y.M., K.H. and K.Z. performed 2D-HPLC analysis. S.M. and K.M. performed cell surface staining. K.K. prepared the recombinant viruses and performed the biochemical analysis. K.E. supported behavioral experiments. J.M. performed biochemical assays and maintained mouse lines. R.K. provided the Dao−/− mouse. M.Y. supervised the project, designed the experiments and wrote the manuscript.

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The authors declare no competing financial interests.

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