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. 2003 Dec 9;100(25):15194-9.
doi: 10.1073/pnas.2431073100. Epub 2003 Nov 24.

Contribution of astrocytes to hippocampal long-term potentiation through release of D-serine

Yunlei Yang  1 , Wooping Ge, Yiren Chen, Zhijun Zhang, Wanhua Shen, Chienping Wu, Muming Poo, Shumin Duan
Affiliations

Contribution of astrocytes to hippocampal long-term potentiation through release of D-serine

Yunlei Yang et al. Proc Natl Acad Sci U S A. .

Abstract

Repetitive correlated activation of pre- and postsynaptic neurons induced long-term potentiation (LTP) of synaptic transmission among hippocampal neurons grown on a layer of astrocytes (mixed cultures) but not among neurons cultured in glial conditioned medium. Supplement of D-serine, an agonist for the glycine-binding site of N-methyl-D-aspartate (NMDA) receptors, enhanced NMDA receptor activation and enabled LTP induction in glial conditioned medium cultures. The induction of LTP in both mixed cultures and hippocampal slices was suppressed by NMDA receptor antagonists, glycine-binding-site blockers of NMDA receptors, or an enzyme that degrades endogenous D-serine. By providing extracellular D-serine that facilitates activation of NMDA receptors, astrocytes thus play a key role in long-term synaptic plasticity.

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Figures

Fig. 1.
Fig. 1.
Comparing synaptic potentiation induced by correlated pre- and postsynaptic activities in two different culture preparations. (A and B) Immunostaining with anti-MAP2 (neuronal marker, red) and anti-GFAP (astrocyte marker, green) showing images of mixed (A) and GCM (B) cultures, respectively. (C and D) Correlated pre- and postsynaptic activation induced LTP in mixed cultures (C) but not in GCM cultures (D). (Upper) Phase-contrast images showing dual patch recordings from neurons in mixed (Left) and GCM (Right) cultures. The amplitude of EPSCs was normalized by the mean amplitude observed during the control period (t = -10 to 0 min). Data represent averages from all experiments (n = 10 and 9 for C and D, respectively). Arrows mark the time of repetitive correlated activation (1 Hz, 80 s), with presynaptic stimulation (1 ms, +100 mV) preceded postsynaptic stimulation (1 ms, 2 nA) by 5 ms. Traces depict samples of averaged EPSCs from one experiment during the control period (I) and at 20–30 min after correlated stimulation (II). (Scales, 20 μm for photo images; 200 pA and 20 ms for recording traces.)
Fig. 2.
Fig. 2.
Dependence of LTP induction in mixed cultures on NMDAR activation, the glycine-binding site, and d-serine. (A–C) LTP induction in mixed cultures in the presence of glycine-binding site antagonist DCKA (10 μM, A), d-serine-degrading enzyme DAAO (0.1 units/ml, B), and d-serine (10 μM) in addition to DAAO (0.1 units/ml, C). Data were normalized in the same manner as that shown in Fig. 1C. (D) Summary of results from all experiments, including data shown in A–C. Other experiments include treatments with NMDAR antagonist APV (25 μM), glycine-binding site antagonist 7-CLKY (10 μM), glycine-binding site agonist d-serine (10 μM), and heat-inactivated DAAO (0.1 units/ml; heated at 90°C for 5 min). The enhancement of EPSCs was measured by the mean amplitude of EPSCs at 20–30 min after correlated stimulation, normalized in each neuron by the mean EPSC amplitude observed during the control period (-10 to 0 min). The number associated with each column refers to the number of neurons tested in each condition. Asterisks indicate significant difference from the control group (not treated with any drug, P < 0.001, t test).
Fig. 3.
Fig. 3.
d-serine enabled the induction of LTP in GCM cultures. (A) No LTP induction was induced in GCM cultures under control conditions. (B) Supplement of d-serine (10μM) enabled the induction of LTP in GCM cultures. (C) The enabling effect of d-serine was abolished by NMDAR antagonist APV. (D) Summary of results from all experiments, including data shown in A–C and experiments in which l-serine (10 μM) was used instead of d-serine. Asterisks indicate significant difference from the control group (P < 0.001, t test).
Fig. 4.
Fig. 4.
Comparison of miniature synaptic currents in three different culture preparations. (A) Continuous traces depict membrane currents and spontaneous mEPSCs recorded from postsynaptic neurons in three types of cultures (see Materials and Methods). Extracellular Mg2+ was omitted in the recording medium. (Scales, 25 pA and 1 s.) Traces (Right) depict samples of mEPSCs (average of 10 events) observed in the experiment depicted (Left). (Scales, 10 pA and 20 ms.) (B and C) Summary of the average frequency and amplitude of mEPSCs observed in three different cultures (n = 10 for each group). Asterisks indicate significant difference from the data of mixed cultures (*, P < 0.05; **, P < 0.01; ***, P < 0.001, t test.)
Fig. 5.
Fig. 5.
Analysis of NMDAR-mediated EPSCs and mEPSCs in mixed and GCM cultures. (A) Samples of EPSCs (average of 5 ∼ 10 events) recorded in the mixed and GCM cultures before (control) and during the perfusion with APV (25 μM) or DCKA (10 μM) or d-serine (10 μM), and after washout of d-serine. Note the significant increase in the NMDAR component (APV-sensitive slow component) in the GCM culture after perfusion with d-serine. (Scales, 100 pA and 50 ms.) (B) Samples of mEPSCs (average of 22 events) recorded in the mixed cultures before (control) during the perfusion with APV or DAAO and after washout of DAAO. (Scales, 5 pA and 20 ms.) (C) Ratio of the NMDAR and AMPAR components of EPSCs obtained from data similar to those shown in A, before and after perfusion with d-serine. (D) Ratio of the NMDAR and AMPAR components of mEPSCs obtained from experiments in mixed cultures, similar to those shown in B, before and after treatment with DAAO. Asterisks indicate significant differences between the two groups (*, P < 0.01; **, P < 0.001, t test).
Fig. 6.
Fig. 6.
d-serine synthesis and release in different cultures. (A) Free d-serine level in the supernatant of three different cultures was assayed by chemiluminescence and HPLC methods. There is significant difference between astrocyte culture and GCM culture (P < 0.01), between astrocyte culture and mixed culture (P < 0.001), and between mixed culture and GCM culture (P < 0.001). (B) Treatment of cultures with tetrodotoxin and CNQX significantly decreased d-serine in the supernatant of mixed cultures but not in the supernatant of pure astrocyte cultures. (C) d-serine in the supernatant from different cultures under control, pretreatment of l-serine for 48 h, or 48-h pretreatment of l-serine plus 10-min exposure to glutamate. Data were normalized with cell content (protein level) of cultures. (D) d-Serine measured from cell lysates from different cultures with or without 48-h pretreatment with l-serine. d-serine was measured by chemiluminescence assay in B–D. Results were from four independent experiments in each group for A–D. Asterisks in B–D indicate significant differences from other groups of the same culture preparation (**, P < 0.01; ***, P < 0.001, t test).
Fig. 7.
Fig. 7.
The involvement of endogenous d-serine in the induction of LTP in hippocampal slices. (A) Summary of all experiments in control slices showing the induction of LTP in CA1 pyramidal neurons by TBS (see Materials and Methods). (B–E) Under the same stimulation conditions as in A, LTP induction was significantly inhibited by perfusing the slice with NMDA receptor antagonists, APV and MK801 (B), antagonist of glycine-binding site DCKA (C), and d-serine degrading enzyme DAAO (D). The blockade of LTP induction by DAAO was rescued by perfusing d-serine (100 μM) in addition to DAAO (E). Data shown are averaged values of EPSP slope normalized by the mean EPSP slope observed during the control period (-10 to 0 min) in each experiment. The arrow marks the time of the TBS. (F) Summary of results from all experiments shown in A–E. Data represent the averaged slope of EPSP at 35–45 min after TBS, normalized in each neuron by its mean EPSP slope observed during the control period. Numbers associated with each column refer to the number of neurons tested in each condition. Asterisks indicate data significantly different from the data obtained in control slices (*, P < 0.001, t test).
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