Cerebral Cortex October 2008;18:2391--2401
doi:10.1093/cercor/bhn008
Advance Access publication February 14, 2008
Bell-Shaped D-Serine Actions on
Hippocampal Long-Term Depression and
Spatial Memory Retrieval
D-Serine, the endogenous coagonist of N-methyl-D-aspartate
receptors (NMDARs), is considered to be an important gliotransmitter, and is essential for the induction of long-term potentiation.
However, less is known about the role of D-serine in another form of
synaptic plasticity, long-term depression (LTD). In this study, we
found that exogenous D-serine regulated LTD in the hippocampal
CA1 region in a ‘‘bell-shaped’’ concentration-dependent manner
through regulating the function of NMDARs in the same manner,
whereas endogenous D-serine was activity-dependently released
and, in turn, contributed to the induction of LTD during lowfrequency stimulation. Furthermore, impairing glial functions with
sodium fluoroacetate (NaFAC) reduced the magnitude of LTD,
which could be restored by exogenous D-serine, indicating that
endogenous D-serine is mainly glia-derived during LTD induction.
More interestingly, similar to the effects on LTD, exogenous
D-serine enhanced spatial memory retrieval in the Morris water
maze in a bell-shaped dose-dependent manner and rescued the
NaFAC-induced impairment of memory retrieval, suggesting links
between LTD and spatial memory retrieval. Our study thus provides
direct evidence of the bell-shaped D-serine actions on hippocampal
LTD and spatial memory retrieval, and underscores the importance
of D-serine in synaptic plasticity, learning, and memory.
Keywords: D-serine, glia, hippocampus, long-term depression, memory
retrieval, NMDA receptor
Introduction
D-Serine is the most abundant D-amino acid in the mammalian
brain. It is not incorporated into proteins, but rather works as
a physiological coagonist of N-methyl-D-aspartate receptors
(NMDARs) (Mothet et al. 2000). The conversion from L-serine
to D-serine by serine racemase (SR) is the only source for
endogenous D-serine in the brain (Wolosker et al. 1999). Thus,
the restricted expression of SR to astrocytes leads to the major
distribution of D-serine in astrocytes (Wolosker et al. 1999; Xia
et al. 2004), although some SR expression and D-serine have
also been found in neurons and microglia (Kartvelishvily et al.
2006; Williams et al. 2006). Furthermore, astrocytic D-serine
can be released by glutamate receptor stimulation through
a Ca2+- and soluble N-ethylmaleimide sensitive fusion protein
attachment protein receptor-dependent exocytotic pathway
(Mothet et al. 2005). This activity-dependent release of
astrocytic D-serine suggests that it acts as a gliotransmitter to
mediate neuron-glia cross-talk and modulates NMDAR-dependent synaptic activity. Indeed, the functions of D-serine parallel
those of NMDARs (Martineau et al. 2006). For example, during
early neuronal development, D-serine released by Bergmann
Ó The Author 2008. Published by Oxford University Press. All rights reserved.
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Zhi Zhang1,2, Neng Gong1,2, Wei Wang3, Lin Xu4 and
Tian-Le Xu1,2
1
Institute of Neuroscience and State Key Laboratory of
Neuroscience, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai 200031, China, 2School
of Life Sciences, University of Science and Technology of China,
Hefei 230027, China, 3Jiangsu Center for Drug Screening, China
Pharmaceutical University, Nanjing 210038, China and
4
Laboratory of Learning and Memory, Kunming Institute of
Zoology, Chinese Academy of Sciences, Kunming 650223,
China
glia seems to be essential in the NMDAR-dependent migration
of granule cells (Kim et al. 2005). On the other hand, in models
of neurotoxicity, D-serine is a key coagonist required for the
cell death caused by NMDAR overactivation (Katsuki et al.
2004; Shleper et al. 2005). Furthermore, several studies on
astrocytic D-serine focus on the astrocyte-dependent modulation of synaptic plasticity. In both neuronal cultures and slices,
NMDAR-dependent long-term potentiation (LTP) is determined
by the concentration of extracellular D-serine, which depends
on the glial environment surrounding neurons (Yang et al. 2003;
Mothet et al. 2006; Panatier et al. 2006). Several studies suggest
that D-serine is also involved in the induction of another type of
synaptic plasticity, long-term depression (LTD) (Panatier et al.
2006; Duffy et al. 2007). However, the mechanism underlying
D-serine modulation of LTD remains unknown.
Synaptic plasticity has been proposed as a cellular mechanism
of learning and memory in the brain, but most studies focus on
the link between LTP and memory (Lynch 2004). Compared
with LTP, the study of LTD has been relatively limited. One
possible reason may be that the magnitude of LTD decreases
with age and LTD is difficult to induce in adult hippocampal
slices (Bear and Abraham 1996). More recently, hippocampal
LTD has been shown to increase in stressful conditions (Xu et al.
1997). In addition, in the nucleus accumbens, LTD has been
related to the expression of specific patterns of behavior, such as
behavioral sensitization (Brebner et al. 2005). This suggests
a more complex role for LTD in learning and memory
(Braunewell and Manahan-Vaughan 2001). Furthermore, LTD
changes synaptic strength which, in turn, regulates the
induction of LTP, thereby contributing indirectly to information
storage (Braunewell and Manahan-Vaughan 2001). Although LTP
and LTD have many processes in common, LTD is not the
mechanistic inverse of LTP. Thus, understanding the role of
D-serine in LTD induction also has importance, perhaps equal to
that of LTP. In this study, we found that exogenous D-serine
regulates NMDAR-dependent LTD in the hippocampal CA1
region in a bell-shaped concentration-dependent manner,
through regulating the function of NMDARs in the same manner,
whereas endogenous astrocytic D-serine is released in an
activity-dependent manner that modulates the induction of
LTD. Behavioral experiments further showed that D-serine plays
a crucial role in spatial memory retrieval. Our findings provide
direct evidence of the bell-shaped D-serine actions on hippocampal LTD and spatial memory retrieval, and suggest the
existence of links between LTD and spatial memory retrieval.
Materials and Methods
The care and use of animals in these experiments followed the
guidelines of, and the protocols were approved by, the Institutional
Animals Care and Use Committee of the Institute of Neuroscience,
Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences.
Hippocampal Slice Preparation
Hippocampal slices were prepared from postnatal day 13--18 male
Wistar rats. In brief, rats were decapitated and brains were quickly
removed and placed in ice-cold, oxygenated (95% O2/5% CO2) artificial
cerebrospinal fluid (ACSF). Coronal hippocampal slices (400 lm thick)
were cut with a vibratome (Leica ATF-1000, Leica Instruments Ltd,
Wetzlar, Germany) and transferred into a submersion-type holding
chamber containing oxygenated ACSF at 35 °C for 1 h. Then a single
slice was gently transferred into the recording chamber and held
submerged between 2 nylon nets and maintained at room temperature
(23--25 °C), where they were perfused throughout the experiment
with oxygenated ACSF at a flow rate of 3--5 mL/min. The same ACSF
was used in cutting, incubation and recording, and contained (in mM):
NaCl, 120; KCl, 2.5; NaHCO3, 26; NaH2PO4, 1.25; CaCl2, 2.0; MgSO4, 2.0;
and D-glucose, 10.
Electrophysiological Recordings
Extracellular recordings were made in the CA1 region of the
hippocampus at room temperature (23--25 °C). A bipolar platinumiridium stimulating electrode was placed among the Schaffer collateral
axons to elicit field excitatory postsynaptic potentials (fEPSPs), which
were recorded from the CA1 stratum radiatum using a glass microelectrode (1--3 MX) filled with 3 M NaCl. The recording electrodes
were pulled from borosilicate glass tubing (1.5 mm outer diameter, 0.84
mm inner diameter; World Precision Instruments, Inc., Sarasota, FL)
with a Brown-Flaming micropipette puller (P-97; Sutter Instruments
Co., Novato, CA). Stimuli (0.1 ms in duration) were delivered every 30 s.
Stable basal responses were obtained for at least 10 min. The stimulus
intensity during baseline recording was adjusted to evoke approximately 50% of the maximum response. LTD was induced by lowfrequency stimulation (LFS; 900 pulses, 1 Hz). Stimulus intensity during
LFS was adjusted to evoke approximately 50% of the maximum
response unless otherwise stated. In some experiments, the stimulus
intensity during LFS was adjusted to evoke approximately 30% or 80%
of the maximum response. To record NMDAR-mediated fEPSP, stimulus
intensity was adjusted to evoke approximately 50% of the maximum
response and then 10 lM 6-cyano-7-nitroquinoxaline-2,3-dione was
added to block a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid
receptor-mediated component. Field potentials were amplified by an
Axonpatch-200B amplifier (Axon Instruments, Foster City, CA) and
filtered at 5K Hz. Data were acquired and analyzed using a Digidata1322A interface and Clampfit 9.0 software (Axon Instruments). The
magnitude of LTD was determined 30 min after LFS and expressed as
percentage reduction in fEPSP slope relative to baseline.
Enzyme-Linked Immunosorbent Assay
The sample solution of about 1 mL was collected from the recording
chamber during baseline recording and again during LFS. The
concentrations of D-serine in samples were assayed by competitive
enzyme-linked immunosorbent assay (ELISA). To prepare antigen, 80
mg bovine serum albumin (BSA) and 4 mg D-serine were dissolved in 4
mL of phosphate-buffered saline (PBS) and then 2 mL of glutaraldehyde
(0.02 M) was gently added. The mixed solution was dialyzed in 500 mL
of PBS, which was replaced 4 times every 6--12 h, then used as stock
solution for coating and stored at –60 °C. Stock solution (200 mL) was
diluted 1:50 with 10 mL of PBS and then coated into 96-well plates (100
lL for each well) for 16 h at 4 °C. Then the coated solution was
removed and each well was washed 3 times with PBS. The 96-well
plates were dried and stored at 4 °C. Before use, each well in the plate
underwent blocking by BSA for 1 h at room temperature and then was
washed twice and dried. The samples or standard were mixed with
anti-D-serine antibody (1:1500) (Sigma, St Louis, MO), and then shaken
for 60 min at 37 °C. Then this mixed solution was added to the 96-well
plate. After 90 min of shaking at room temperature, the plate was
washed 5 times with PBS and dried. The plate was then incubated with
secondary rabbit antibody conjugated to horseradish peroxidase (HRP)
(1:1000) at room temperature for 90 min. After 5 washes with PBS, HRP
2392 Bell-Shaped D-Serine Actions on LTD and Memory
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Zhang et al.
substrate was added to produce a color reaction. The absorbance was
read in a plate-reading spectrophotometer and the concentrations of
D-serine in samples were determined by comparison with the standard
curve (Fig. 3C). To avoid the system error, we reproduced the standard
curve before each measurement.
Behavioral Testing
The Morris water maze consisted of a circular pool (250 cm diameter,
60 cm deep) filled with water at 24--26 °C to a depth of 20 cm. The
water surface was covered with floating black resin beads. Yellow
curtains were drawn around the pool (50 cm from the pool periphery)
and contained distinctive visual marks that served as distal cues. Before
training, a 180 s free swim trial without the platform was run. For
training, a submerged (1.5 cm below the surface of the water, invisible
to the animal) Perspex platform (13 3 13 cm) was fixed in the center of
a quadrant so that the animal had to learn the location of the platform,
which was the only escape from the water. Adult Wistar rats (150--200 g)
were given 8 trials with starting positions that were equally distributed
around the perimeter of the maze in an acquisition session, and for 3
days were allowed to learn the position of the platform. A trial was
terminated when the rat had climbed onto the escape platform or when
120 s had elapsed. Each rat was allowed to stay on the platform for 30 s.
The probe trial was performed on the fourth day. The platform was
removed and the rat behavior was recorded for 120 s. Swimming paths
for training session and probe trial were monitored using an automatic
tracking system. This system was used to record the swimming trace
and calculate the time spent in each quadrant. In the drug-treated
groups, sodium fluoroacetate (NaFAC) was intraperitoneally injected
6 h before, whereas D-serine and 7-chlorokynurenic acid (7-ClKY) was
injected 3 h before the memory retrieval task.
All drugs and chemicals in these experiments were purchased from
Sigma. In the experiments on slices, for bath application, the stock
solutions of drugs were diluted into the bathing medium and the given
concentration could be reached in the recording chamber after 3--5
min; for ‘‘rapid’’ application, we directly replaced the ACSF flowing into
the chamber by ACSF containing drugs with a given concentration at
the entrance of the recording chamber, so that the drugs at a given
concentration could reach the slices immediately. All the data are
shown as the mean ± SEM. Comparisons of 2 groups were made by
Student’s t-test. All statistical analysis was performed using Origin 7.0
(OriginLab, Northampton, MA).
Results
Exogenous D-Serine Regulates NMDAR-Dependent LTD in
a Bell-Shaped Concentration-Dependent Manner
To define the role of D-serine in LTD, we first examined the
effects of exogenous D-serine on LFS-induced LTD. LFS (900
pulses, 1 Hz) with the 50% stimulus intensity induced an
obvious LTD in the hippocampal CA1 region (Fig. 1A,B). With
bath application of exogenous D-serine, we found that 5 lM
significantly enhanced the magnitude of LTD from 19.3 ± 3.7%
(n = 5) to 58.3 ± 5.6% (n = 6; P < 0.01), whereas 100 lM had no
significant effect—the LTD magnitude was 21.6 ± 4.1% (n = 6;
P > 0.05) (Fig. 1A,B). To further determine the exact
concentration--effect relationship of D-serine on LTD, we
measured its effects at various concentrations (3, 5, 10, 50,
and 100 lM). As shown in Figure 1D, exogenous D-serine
regulated LTD in a bell-shaped concentration-dependent
manner with the effect reaching maximum at 5 lM. The
NMDAR antagonist D-AP5 (50 lM) blocked LTD both in the
absence and presence of D-serine (data not shown). In addition,
the specific NMDAR glycine site antagonist 7-ClKY (50 lM)
(Kemp et al. 1988) blocked LTD both in the absence (0.1 ±
7.5%, n = 5) and presence of 5 lM D-serine (0.5 ± 5.8%, n = 5) and
100 lM D-serine (1.5 ± 5.8%, n = 5) (Fig. 1C), indicating that the
Figure 1. Exogenous D-serine regulates NMDAR-dependent LTD in a bell-shaped concentration-dependent manner. (A) Representative traces before LFS (trace 1) and 30 min
after LFS (trace 2) during control, 5 lM D-serine and 100 lM D-serine. (B) D-Serine at 5 lM (filled circles; control, open circles) but not 100 lM (filled triangles) significantly
increased the magnitude of LTD. LTD was induced by LFS with 50% stimulus intensity. (C) The NMDAR glycine site antagonist 7-ClKY (50 lM) blocked LTD both in the absence
and presence of D-serine (5 and 100 lM). (D) Concentration--effect relationship between D-serine and LTD. The concentration of exogenous D-serine ranged from 3 to 100 lM.
For each concentration, n 5 6. (E) Concentration--effect relationship between glycine and LTD. The concentration of exogenous glycine ranged from 5 to 100 lM. For each
concentration, n 5 5, *P \ 0.05, **P \ 0.01.
glycine site of NMDAR is required for inducing LTD whether
or not exogenous D-serine was present. To ascertain that
a NMDAR-dependent mechanism is involved in D-serine
action, we further examined the effect of glycine. Similar
to that of D-serine, glycine modulated LTD in a bell-shaped
concentration-dependent manner with the effect reaching
maximum at 10 lM (Fig. 1E). These results indicate that
D-serine exerts its action on LTD through regulating NMDARs.
Exogenous D-Serine Regulates NMDAR--fEPSP in a
Bell-Shaped Concentration-Dependent Manner
To establish the critical role of NMDARs underlying the bellshaped D-serine action on LTD, we directly examined the
effects of exogenous D-serine on NMDAR-mediated fEPSPs. As
shown in Figure 2A--C,G, exogenous D-serine at 5 lM but not
100 lM significantly increased the amplitude of NMDAR--fEPSP
(5 lM, 133.6 ± 7.6% normalized to control, n = 8, P < 0.01; 100
lM, 101.8 ± 2.5% normalized to control, n = 7, P > 0.05), which
could be blocked by the NMDAR glycine site antagonist 7-ClKY
(50 lM). The amplitudes of NMDAR--fEPSPs were measured 20
min after D-serine application, when the NMDAR--fEPSPs
reached stable value. Interestingly, following the application
of 100 lM D-serine, a transient increase of the NMDAR--fEPSP
amplitude was observed (Fig. 2C). We attributed this phenomenon to the time lag for local D-serine to reach the maximal
concentration. To test this hypothesis, we altered the method
of drug application in order to speed up D-serine to reach
a high level (see Methods). Under such conditions, however,
we still observed first an increase, then a decrease of NMDAR-fEPSP. (Fig. 2D,G). To make sure that this modulation is
dependent on the NMDAR glycine site, we examined glycine
with the same rapid application method as we used for
applying D-serine. Consistently, we found that glycine at low
concentration (10 lM) caused a persistent increase of NMDA
response, whereas glycine at high concentration also induced
a first increase, and then a decrease of NMDAR--fEPSP (10 lM,
125.9 ± 8.5% normalized to control, n = 5, P < 0.01; 100 lM,
100.2 ± 10.4% normalized to control, n = 5, P > 0.05) (Fig. 2E--G).
These results together suggest that exogenous D-serine also
modulates NMDAR response in a bell-shaped concentrationdependent manner through a NMDAR glycine site-dependent
mechanism.
Cerebral Cortex October 2008, V 18 N 10 2393
Figure 2. Exogenous D-serine regulates NMDAR--fEPSP in a bell-shaped concentration-dependent manner. (A) Representative traces showing that bath application of D-serine at
5 lM but not 100 lM increased the amplitude of NMDAR--fEPSP, which was abolished by 7-ClKY (50 lM). (B and C) Time courses showing the effects of exogenous D-serine on
NMDAR--fEPSPs and their antagonism by 7-CIKY. (D) Time courses showing the effects of rapid D-serine (100 lM) application on NMDAR--fEPSPs. (E and F) Time courses
showing the effects of rapid glycine (10 and 100 lM) application on NMDAR--fEPSPs. (G) Statistical results showing the modulation of NMDAR--fEPSP by D-serine and glycine
with different application methods. For each group, n 5 5--8, **P \ 0.01. NS represents no statistical significance. The data were obtained 20 min after D-serine application,
when the NMDAR--fEPSPs reached stable value.
Endogenous D-Serine is Activity-Dependently Released
during LTD Induction
Because exogenous D-serine regulated LTD in a concentrationdependent manner, an important question was whether
endogenous D-serine also participates in the induction and
regulation of LTD, similar to its well-known function in LTP
(Yang et al. 2003; Mothet et al. 2006; Panatier et al. 2006). To
address this question, we first determined whether endoge2394 Bell-Shaped D-Serine Actions on LTD and Memory
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Zhang et al.
nous D-serine release occurred during the induction of LTD.
Regarding the question of whether D-serine release was
activity-dependent, we choose 3 different stimulus intensities
to mimic different degrees of neuronal activity. As shown in
Figure 3A, LFS at 3 different stimulus intensities, which were
adjusted to evoke 30%, 50%, and 80% of the maximum
response, induced LTD with different magnitudes (30% group,
11.5 ± 2.7%, n = 6; 50% group, 19.3 ± 3.7%, n = 5; 80% group,
43.8 ± 5.2%, n = 5). Furthermore, all these LTD induced by
different stimulus intensities were blocked by the NMDAR
glycine site antagonist 7-ClKY (Fig. 3B), indicating a NMDAR
glycine site-dependent mechanism underlying the differences
among the LTD induced by different intensities. By collecting
the solutions during baseline and LFS, we assessed the
concentration of extracellular D-serine in recording chamber
by ELISA assay and the concentrations of D-serine in samples
were determined by comparison with the standard curve
(Fig. 3C). The specificity of the assay for D-serine was validated
by testing 2 other amino acids c-amino butyric acid (GABA) and
glycine, and samples treated with a specific enzyme that
degrades D-serine, D-amino acid oxidase (DAAO, 0.1 units/mL,
from porcine kidney, Sigma) (Table 1). In all 3 groups with
different stimulus intensities, the concentrations of D-serine
during LFS were significantly higher than those during baseline
activity (30% group, 5.69 ± 0.39 lM, baseline, 2.78 ± 0.19 lM,
n = 8, P < 0.01; 50% group, 10.46 ± 0.67 lM, baseline, 2.70 ±
0.26 lM, n = 8, P < 0.01; 80% group, 14.80 ± 0.71 lM, baseline,
3.08 ± 0.27 lM, n = 8, P < 0.01) (Fig. 3D). Furthermore, the
extracellular D-serine levels during LFS at higher stimulus
intensities were significantly higher (50% compared with 30%,
P < 0.01; 80% compared with 50%, P < 0.05) (Fig. 3D). Besides
the stimulus intensity, the extracellular D-serine also depended
on the stimulus frequency. High-frequency stimulation (HFS)
with 100 Hz/1 s could induce a robust LTP (data not shown).
During HFS with 50% intensity, the concentration of D-serine in
recording chamber was significantly higher than those during
baseline activity (HFS, 18.45 ± 1.68 lM, baseline, 3.15 ± 0.93 lM,
n = 8, P < 0.01) and LFS with 50% intensity (HFS compared
with 50% LFS, n = 8, P < 0.01). These results suggest that
D-serine is activity-dependently released during the induction
of LTD, and this may contribute to the observed differences in
LTD magnitudes with various LFS intensities.
Endogenous D-Serine Contributes to the Modulation of
LTD
The concentration of endogenous D-serine varied with LFS
intensity. Thus, we supposed that exogenous D-serine may
exert different effects on LTD induced by different stimulus
intensities, due to variations in the levels of endogenously
released D-serine during LFS. Indeed, we found that the
Figure 3. Endogenous D-serine is activity-dependently released. (A) LTD induced by LFS with 3 different stimulus intensities adjusted to evoke 30% (triangles), 50% (circles), and
80% (squares) of the maximum response. (B) 7-ClKY (50 lM) abolished LTD induced by 3 different stimulus intensities. (C) The standard curve in the ELISA assay for D-serine. In
the concentration range from 3 lm to 30 mM, the OD value showed a linear relationship to the D-serine concentration (R 5 0.98074; P \ 0.0001). Each point was averaged
from 3 results. (D) Levels of D-serine during LFS with 3 different stimulus intensities as indicated. HFS (100 Hz/1 s) was also compared. At all stimulus protocols, the levels of Dserine during stimulation were much higher than those during baseline recording. For each group, n 5 8, **P \ 0.01. D-Serine level also depended on stimulus intensity (n 5 8,
50% LFS compared with 30% LFS, ##P \ 0.01; 80% LFS compared with 50% LFS, #P \ 0.05) and stimulus frequency (n 5 8, HFS compared with 50% LFS, ##P \ 0.01).
Table 1
The specificity of the ELISA assay for D-serine was tested by using ACSF, 20 lM glycine, 20 lM GABA, as well as 0.1 units/mL DAAO (n 5 5 for each group)
Group
ACSF
Glycine (20 lM)
GABA (20 lM)
50% baseline
50% LFS
50% DAAO
50% LFS DAAO
OD value
[D-Serine] (lM)
0.299 ± 0.014
0.003 ± 0.003
0.275 ± 0.005
0.003 ± 0.001
0.285 ± 0.004
0.001 ± 0.000
0.204 ± 0.001
6.508 ± 0.323
0.195 ± 0.001
17.693 ± 0.623
0.234 ± 0.005
0.367 ± 0.162
0.241 ± 0.006
0.298 ± 0.231
Cerebral Cortex October 2008, V 18 N 10 2395
concentration--effect relationship of D-serine on LTD differed
with stimulus intensity. As shown in Figure 1A,B, 5 lM D-serine
greatly increased the magnitude of 50% stimulus-induced LTD,
whereas 100 lM had no significant effect. However, in 30%
stimulus-induced LTD (11.5 ± 2.7%, n = 6), 5 lM exogenous
D-serine had little effect on LTD (18.0 ± 5.1%, n = 5, P > 0.05
compared with 30% control) but 100 lM significantly
enhanced the LTD magnitude (42.6 ± 4.6%, n = 7, P < 0.01
compared with 30% control) (Fig. 4A,C), whereas in 80%
stimulus-induced LTD (43.8 ± 5.2%, n = 5), 5 lM D-serine had
no significant effect (37.5 ± 5.0%, n = 5, P > 0.05 compared
with 80% control) but 100 lM significantly reduced the LTD
magnitude (25.0 ± 4.8%, n = 8, P < 0.05 compared with 80%
control) (Fig. 4B,C). These results indicate that endogenous
D-serine released during LFS participates in the induction of
LTD. Thus stimulus intensity and exogenous D-serine concurrently modulated LTD, suggesting that endogenous D-serine
also contributes to the bell-shaped modulation of LTD.
To directly examine the role of endogenous D-serine in LTD,
we used DAAO, a specific enzyme that degrades D-serine.
Similar to its effect on NMDAR-dependent LTP induction (Yang
et al. 2003), bath application of DAAO for more than 45 min
(0.1 units/mL) significantly reduced the magnitude of LTD induced by both 50% stimulus (control group, 19.3 ± 3.7%, n = 5;
DAAO group, –0.2 ± 4.6%, n = 8) and 80% stimulus (control
group, 43.8 ± 5.2%, n = 5; DAAO group, –1.9 ± 7.8%, n = 6)
(Fig. 5A,B). This impairment of LTD was rescued by exogenous
application of 100 lM D-serine (28.4 ± 4.2%, n = 8) (Fig. 5C).
Similarly, an inhibitor of glycine transporter 1 (GlyT1),
sarcosine (1 mM), was able to rescue the impairment of LTD
by DAAO (22.1 ± 5.8%, n = 9) (Fig. 5C), indicating that
increasing glycine overcomes the loss of D-serine and restores
NMDAR activation. Therefore, the effect of DAAO was due to
the specific depletion of D-serine but not glycine. Taken
together, these results indicate that endogenous D-serine
participates in the induction of LTD.
Astrocytes Contribute to the Release of D-Serine during
LTD Induction
Because endogenous D-serine can be released in an activitydependent manner and, in turn, contributes to the induction of
LTD, we attempted to identify the source of endogenous
D-serine. It is well known that D-serine is a gliotransmitter and
astrocyte-derived D-serine is essential for the induction of LTP
(Yang et al. 2003; Mothet et al. 2006). Thus, we first determined
whether astrocytes also contribute to endogenous D-serine
release during LTD induction. We used NaFAC (3 mM for 1 h),
the glia-specific metabolic inhibitor (Swanson and Graham
1994) to ‘‘interfere’’ with glial functions in slices and found that
LTD was significantly inhibited (3.8 ± 5.2%, n = 4) (Fig. 6A).
Exogenous application of D-serine reversed this impairment in
a concentration-dependent manner (Fig. 6B,C). D-Serine at 100
lM had the maximal effect, restoring the LTD to 29.8 ± 4.2%
(n = 6, P < 0.01 compared with NaFAC group), and this effect
was blocked by 50 lM 7-ClKY (8.5 ± 4.5%, n = 4, Fig. 6B). These
results suggest that astrocyte-derived D-serine contributes to
LTD, as it does to LTP (Yang et al. 2003; Panatier et al. 2006).
Furthermore, we found that increasing the stimulus intensity to
80% could also partially reverse the NaFAC-induced impairment of LTD (19.9 ± 3.5%, n = 5) (Fig. 6D). This rescue effect
was blocked by the treatment of DAAO (0.1 units/mL) (-1.1 ±
2396 Bell-Shaped D-Serine Actions on LTD and Memory
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Zhang et al.
Figure 4. Stimulus intensity and exogenous D-serine concurrently modulates LTD.
(A and B) The effects of exogenous D-serine depended on stimulus intensity. In 30%
stimulus-induced LTD (A), 5 lM exogenous D-serine (filled circles; control, open
circles) had little effect, but 100 lM D-serine (filled triangles) significantly enhanced
the LTD magnitude; whereas in 80% stimulus-induced LTD (B), 5 lM D-serine had no
effect but 100 lM D-serine significantly reduced LTD magnitude. (C) The statistical
results showing the different effects of exogenous D-serine (5 and 100 lM) on the
magnitude of LTD induced by 30%, 50%, and 80% stimuli. For each group, n 5 5--8,
*P \ 0.05, **P \ 0.01. NS represents no statistical significance.
5.3%, n = 5) (Fig. 6D), suggesting that either NaFAC only
partially impairs glial function or there is also a neuron-derived
source of D-serine.
and Collingridge 1993; Bear and Malenka 1994). Thus, we
further examined the effects of D-serine on spatial memory
retrieval by using the Morris water maze. In the probe trail after
3 days training, rats showed significantly greater preference for
the target quadrant (the quadrant with platform during training
session) than the opposite quadrant (the quadrant opposite
platform), represented by the time spent in the quadrant
(Control group: target, 69.76 ± 4.0 s; opposite, 27.35 ± 3.54 s;
n = 10, P < 0.05) (Fig. 7A). Intraperitoneal injection of D-serine
at various concentrations did not change the preference
(100 mg/kg group: target, 64.76 ± 4.80 s, opposite, 38.16 ±
3.33 s, n = 10, P < 0.05; 1000 mg/kg group: target, 90.02 ±
3.20 s, opposite, 18.60 ± 2.83 s, n = 10, P < 0.05; 3000 mg/kg
group: target, 65.53 ± 6.27 s, opposite, 32.37 ± 7.10 s, n = 10,
P < 0.05). However, D-serine improved memory retrieval only
at an intermediate dose of 1000 mg/kg, when more time was
spent in the target quadrant (1000 mg/kg group compared
with control group without D-serine injection, n = 10, P < 0.05)
(Fig. 7A,B), which was in parallel with the bell-shaped actions
of D-serine on LTD induction (Fig. 1D). This effect was
completely abolished by coinjection of 7-ClKY (15 mg/kg)
(Fig. 7A), suggesting that D-serine exerts its effects through the
glycine site of NMDARs. Interestingly, 7-ClKY also blocked the
rat’s preference for the target quadrant (Target, 35.70 ± 6.82 s;
opposite, 29.98 ± 6.52 s; n = 8, P > 0.05) (Fig. 7A), suggesting
that endogenous D-serine derived from glial cells may play
a role in establishing the target preference, as it does in LTD
induction (Fig. 1C). Consistent with this assumption, intraperitoneal injection of 3 mg/kg NaFAC impaired memory retrieval
by abolishing the preference for the target quadrant (control
group: target, 66.30 ± 10.54 s, opposite, 13.18 ± 6.52 s, n = 7,
P < 0.05; NaFAC group: target, 41.81 ± 5.02 s, opposite, 32.77 ±
7.49 s, n = 7, P > 0.05), which could be restored by coinjection
of 1000 mg/kg D-serine (target, 60.21 ± 3.62 s; opposite, 28.24 ±
5.27 s; n = 7; P < 0.05) (Fig. 7C,D). Again, coinjection of 7-ClKY
(15 mg/kg) diminished the rescue effects of D-serine on the
re-establishment of the target preference (target, 32.97 ± 6.53 s;
opposite, 36.47 ± 5.42 s; n = 7; P > 0.05) (Fig. 7C). Together,
these results suggest that both exogenous and endogenous
D-serine, contributes to spatial memory retrieval in the same
manner as it affects LTD.
Discussion
Figure 5. Endogenous D-serine contributes to the induction of LTD. (A and B) DAAO
(0.1 units/mL) abolished the LTD induced by 50% (A) and 80% stimulus intensity (B).
(C) The DAAO-induced impairment of LTD was rescued by exogenous application of
100 lM D-serine or the GlyT1 inhibitor sarcosine (1 mM) (filled circles, DAAO; filled
triangles, DAAO þ 100 lM D-serine; open triangles, DAAO þ sarcosine).
D-Serine Contributes to Spatial Memory Retrieval
It is widely believed that long-term synaptic plasticity serves as
a cellular mechanism underlying learning and memory (Bliss
As the most common coagonist of NMDARs, it is not surprising
that exogenous D-serine can regulate hippocampal LTD
through modulating the functions of NMDARs. However, we
found that exogenous D-serine modulated LTD in a bell-shaped
concentration-dependent manner. The maximal LTD was only
achieved at an intermediate concentration of D-serine. Interestingly, in the Morris water maze, we found that D-serine
enhanced spatial memory retrieval in a similar profile to its
effects on LTD. The mechanisms underlying D-serine’s bellshaped actions are currently unclear. There are several possible
explanations for this phenomenon. The most obvious one is
that the dose-dependence may be accounted for by the
Bienenstock--Cooper--Munro (BCM) theory (Bienenstock et al.
1982), which incorporates a bell-shaped curve for LTD
induction. In the BCM model, different stimulus intensities
reflect different Ca2+ levels in postsynaptic neurons (Nishiyama
+
et al. 2000). The induction of LTD needs a suitable level of Ca2
influx to activate the specific downstream pathways, a level
Cerebral Cortex October 2008, V 18 N 10 2397
Figure 6. Astrocytes play a key role. (A) Slices incubated in NaFAC (3 mM) for 1 h had significantly impaired LTD (open circles, control; filled circles, NaFAC). (B) The impairment
was rescued by 100 lM exogenous D-serine, which was diminished by 50 lM 7-ClKY (filled circles, NaFAC; filled triangles, NaFAC þ 100 lM D-serine; open triangles, NaFAC þ
100 lM D-serine þ 7-ClKY). (C) Exogenous application of D-serine prevented the NaFAC-induced impairment in a concentration-dependent manner. For each data point, n 5 5--6,
*P \ 0.05, **P \ 0.01. Note that D-serine at 100 lM had the maximal effect. (D) Increasing the stimulus intensity to 80% partially rescued the NaFAC-induced impairment of
LTD, an effect that was prevented by DAAO (filled circles, NaFAC þ 50% stimulus; filled squares, NaFAC þ 80% stimulus; open squares, NaFAC þ 80% stimulus þ DAAO).
lower than that for LTP (Nishiyama et al. 2000; Franks and
Sejnowski 2002). Thus, only a certain degree of NMDAR
activation is needed for LTD induction. In the case of LTD
induced by a 50% stimulus, it is possible that exogenous
D-serine at 5 lM increases the magnitude of LTD, whereas at
100 lM D-serine may decrease LTD during the transition from
LTD to LTP according to the BCM theory. However, our results
showed that exogenous D-serine modulated NMDAR-mediated
fEPSPs also in a bell-shaped manner, indicating that D-serine at
high concentrations does not cause more activation of
NMDARs. Therefore, the bell-shaped D-serine action on LTD
may not represent a high [Ca2+] shift to inducing LTP. We noted
that glycine and D-serine at high concentrations can prime
NMDAR internalization through their specific interactions with
the glycine-binding site (Nong et al. 2003). Interestingly,
D-serine and glycine at high concentrations modulated NMDAR
response in a biphasic mode, with first an increase and then
a decrease (Fig. 2). The result suggests that the activation of
NMDARs is required for the secondary decline of NMDAR
response, which is consistent with the notion that a prebinding
of D-serine at high concentration is needed for the NMDAR
internalization (Nong et al. 2003). The secondary reduction of
NMDAR response might be counteracted by D-serine enhancement of the residual uninternalized NMDARs, leading to
a sustained NMDAR response comparable to the value observed
in control conditions without D-serine treatment. Accordingly,
it is possible that D-serine--induced NMDAR internalization
could account for its bell-shaped actions on LTD and memory
retrieval. Alternatively, a recent study suggests that D-serine
specifically regulated NMDA-NR2B receptor-dependent hippocampal LTD (Duffy et al. 2007), consistent with the newly
2398 Bell-Shaped D-Serine Actions on LTD and Memory
d
Zhang et al.
proposed ‘‘NMDAR NR2A/2B subtype’’ theory underlying
bidirectional synaptic plasticity (Liu et al. 2004; Fox et al.
2006). Regardless of its underlying mechanisms, the bellshaped concentration--effect relationship between D-serine and
LTD is likely to be important, by providing a novel mechanism
for activity-dependent homeostatic regulation of synaptic
plasticity.
Astrocytes sense changes in neuronal activity and, in turn,
regulate synaptic function by releasing a variety of neuroactive
substances known as gliotransmitters (Haydon 2001; Fields and
Stevens-Graham 2002; Volterra and Meldolesi 2005). Similar to
glutamate and adenosine triphosphate, D-serine has been
suggested to be a gliotransmitter, and there is evidence that
astrocytic D-serine is essential for the induction of LTP (Yang
et al. 2003; Mothet et al. 2006; Panatier et al. 2006). Our data
indicates that endogenous D-serine is released in an activitydependent manner during the induction of LTD. The magnitudes of LTD induced by different stimulus intensities
paralleled measurements of D-serine released in slices. Although there could be other mechanisms underlying differing
magnitudes of LTD, such as different numbers of synapses
recruited and depressed, our results raise the possibility that
release of endogenous D-serine regulates the induction of LTD.
First, with both 50% and 80% LFS stimulus intensities, degradation of endogenous D-serine by DAAO could block the induction
of LTD (Fig. 5). Second, stimulus intensity and exogenous
D-serine cooperatively modulated LTD (Fig. 4C). Third, exogenous D-serine restored LTD in slices treated with the astrocytic
metabolic inhibitor NaFAC, and did so in a concentrationdependent manner. Finally, we found that increased stimulus
intensity during LFS compromised NaFAC-induced impairment
Figure 7. D-Serine contributes to spatial memory retrieval. (A and B) Intraperitoneal injection of D-serine improved spatial memory retrieval in a bell-shaped dose-dependent
manner. Time spent in the target and opposite quadrant was shown in (A). Rats showed significant preference for the target quadrant following learning training. For each group,
n 5 8--10, #P \ 0.05, compared the time spent in target quadrant with that in opposite quadrant. D-Serine significantly increased the time spent in the target quadrant only at
the dose of 1000 mg/kg (n 5 10, *P \ 0.05, compared with control group without D-serine injection). Coinjection of 15 mg/kg 7-ClKY with 1000 mg/kg D-serine completely
abolished the effect of D-serine (n 5 8). (B) Representative swimming traces during the probe test. (C and D) Intraperitoneal injection of 3 mg/kg NaFAC impaired memory
retrieval by abolishing the preference for the target quadrant (n 5 7, #P \ 0.05), which could be restored by coinjection of 1000 mg/kg D-serine (n 5 7; #P \ 0.05). Coinjection
of 15 mg/kg 7-ClKY prevented the rescuing effect of D-serine on the re-establishment of the target preference (n 5 7). NS represents no statistical significance.
of LTD, and DAAO diminished this rescuing effect, consistent
with an elevated D-serine release with increased stimulus
intensity.
Incomplete blockade of LTD by astrocytic poisoning could
be due to incomplete depletion of glial D-serine, or indicate
additional neuronal release of D-serine (Kartvelishvily et al.
2006). It is reasonable to speculate that a suitable level of
endogenous D-serine is needed for LTD induction. Besides
D-serine, glycine is also a ligand for the glycine-binding site
(Thomson et al. 1989). Both D-serine (Yang et al. 2003; Mothet
et al. 2006; Panatier et al. 2006) and glycine (Martina et al. 2004;
Zhang et al. 2007) have been implicated in regulating LTP
induction. In our study, we found that, after DAAO treatment,
application of glycine transporter GlyT1 inhibitor, sarcosine,
was effective in restoring DAAO-impaired LTD, indicating the
involvement of glycine site in NMDAR in LTD. Together, our
findings support the idea that endogenous D-serine is a key
coagonist of NMDAR in the induction of LTD.
In parallel with the bell-shaped relationship between
D-serine and LTD, we found that intraperitoneal injection of
D-serine also affected spatial memory retrieval in a bell-shaped
dose-dependent manner. Furthermore, this aspect of memory
was also impaired by NaFAC and restored by systematic
D-serine injection. Synaptic plasticity, including both LTP and
LTD at excitatory synapses, has been proposed as a cellular
substrate of both information processing and memory storage
(Bliss and Collingridge 1993; Bear and Malenka 1994).
However, the exact roles of LTP and LTD in memory are far
from clarified. Although some previous studies suggest that the
balance between LTP and LTD in different pathways may
determine the expression of specific patterns of behavior
(Brebner et al. 2005; Goto and Grace 2005), most studies
mainly focus on the correlation between LTP and memory. The
observed correlation between LTD and memory retrieval in the
present study, although not implying a causal relation, suggests
that LTD may also be critical for learning and memory retrieval.
Interestingly, we noted that another study also showed
a specific D-serine action on LTD, which was accompanied by
the change of spatial reversal learning behavior (Duffy et al.
2007). Although we have no idea about the relationship
between these 2 tasks (memory retrieval and reverse learning)
due to the different experimental systems, these results
together suggest that D-serine and LTD may be involved in
some specific patterns of behavior. A previous study has shown
that intraperitoneal injection of D-serine at a dose of 10 mmol/kg
(1000 mg/kg) causes a substantial rise in hippocampal
D-serine (from about 0.2 to 0.35 mM) (Hashimoto and Chiba
2004). This happens to be the dose that was effective in our
water maze memory retrieval testing (Fig. 7A,B). We have no
way of knowing what brain regions are influenced by actions of
Cerebral Cortex October 2008, V 18 N 10 2399
D-serine on memory retrieval following systemic injection. Our
data are reminiscent of D-serine treatment of diseases related
to the hypofunction of NMDARs such as schizophrenia
(Hashimoto et al. 2003) and Alzheimer’s disease (Hashimoto
et al. 2004). The relationship between impaired LTD and these
psychiatric disorders awaits further exploration.
Funding
National Natural Science Foundation of China (No. 30621062); the
National Basic Research Program of China (No. 2006CB500803);
and the Knowledge Innovation Project from the Chinese
Academy of Sciences (KSCX2-YW-R-35).
Note Added in Proof
Note: Added in Proof—While this paper was under review,
Duffy et al. (Duffy S, Labrie V and Roder JC. 2007. D-Serine
augments NMDA-NR2B receptor-dependent hippocampal longterm depression and spatial reversal learning. Neuropsychopharmacology. Forthcoming) reported similar D-serine effects
on NMDA receptor-dependent hippocampal LTD.
Notes
We thank Dr. Patric K. Stanton for helpful comments, Bin Zhao for
assistance with ELISA, Yue-Xiong Yang for assistance with behavioral
test, Xue-Hua Sun and Ming-Hu Cui for assistance with slice
preparation. Conflict of Interest : None declared.
Address correspondence to Tian-Le Xu, PhD, Institute of Neuroscience and State Key Laboratory of Neuroscience, Shanghai Institutes of
Biological Sciences, The Chinese Academy of Sciences, 320 Yue-yang
Road, Shanghai 200031, China. Email: [email protected].
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