Download Glycine Binding Sites of Presynaptic NMDA Receptors May

J Neurophysiol 97: 817– 823, 2007.
First published November 8, 2006; doi:10.1152/jn.00980.2006.
Glycine Binding Sites of Presynaptic NMDA Receptors May Tonically
Regulate Glutamate Release in the Rat Visual Cortex
Yan-Hai Li and Tai-Zhen Han
Department of Physiology, School of Medicine, Xi’an Jiaotong University, Zhuque Dajie 205, Xi’an, Shannxi, People’s Republic of China
Submitted 13 September 2006; accepted in final form 4 November 2006
INTRODUCTION
Johnson and Ascher (1987) first demonstrated that, in the
vertebrate CNS, exogenously applied glycine potentiates the
response of N-methyl-D-aspartate receptors (NMDA-Rs) to
NMDA. Binding of glycine to the glycine binding site of
NMDA-Rs is a prerequirement for NMDA-R activation by
glutamate (Kleckner and Dingledine 1988). Thus glycine and
glutamate are co-agonists for NMDA-Rs.
It is generally assumed that the glycine binding site is
constantly saturated due to high concentrations of glycine in
cerebrospinal fluid (CSF) (Ferraro and Hare 1985). However,
glycine in the synaptic cleft is subject to a powerful uptake
system (i.e., glycine transporter 1 and 2, GlyT1 and GlyT2)
(Bergeron et al. 1998; Betz et al. 2006; Chen et al. 2003;
Supplisson and Bergman 1997). In addition, NMDA-R isoforms exhibit different affinities to glycine (Kew et al. 1998;
Kutsuwada et al. 1992). A surge of in vivo and in vitro studies
have suggested that saturation of the glycine binding varies
among different brain areas. Some studies have shown that the
application of exogenous glycine increases NMDA-R responses (Ahmadi et al. 2003; Baptista and Varanda 2005;
Berger et al. 1998; Czepita et al. 1996), whereas others have
found no modulatory effect (Fletcher et al. 1989; Obrenovitch
et al. 1997).
D-serine, which is a potent agonist of the glycine binding site
(Kleckner and Dingledine 1988; Martineau et al. 2006; Schell
et al. 1995) but is not taken up by glycine transporters (SupAddress for reprint requests and other correspondence: T.-Z. Han, Dept. of
Physiology, School of Medicine, Xi’an Jiaotong University, Zhuque Dajie 205,
Xi’an, Shaanxi 710061, PR China (E-mail: htzhen@@mail.xjtu.edu.cn).
www.jn.org
plisson and Bergman 1997), has been shown to potentiate
NMDA synaptic currents (Baptista and Varanda 2005; Mothet
et al. 2000). The ability of glycine or D-serine to potentiate
NMDA-R responses clearly indicates that the glycine binding
site of NMDA-Rs is not saturated.
To date, a number of studies have shown that the NMDA-Rs
are present on the presynaptic membrane in addition to the
postsynaptic area. Presynaptic NMDA-Rs have been found to
enhance neurotransmitter release in different regions of the
CNS including the spinal cord, cerebellum, entorhinal cortex,
and neocortex (Bardoni et al. 2004; Berretta and Jones 1996;
DeBiasi et al. 1996; Glitsch and Marty 1999; MacDermott et
al. 1999; Yang et al. 2006). They may also mediate synaptic
plasticity (i.e., LTP and LTD) (Casado et al. 2002; Duguid and
Sjostrom 2006; Sjostrom et al. 2003). Immunocytochemical
studies have demonstrated that presynaptic NR1 and NR2Bcontaining NMDA-Rs are present in rat visual cortex (Aoki et
al. 1994). Moreover, presynaptic NMDA-Rs enhance neurotransmitter release in the visual cortical layer V neurons as
proven by application of APV, a competitive antagonist of
these receptors (Sjostrom et al. 2003). However, whether the
glycine binding site of presynaptic NMDA-Rs may modulate
glutamatergic neurotransmission in CNS is still uncertain.
In this study, we examined whether the glycine binding site of
presynaptic NMDA-Rs regulates glutamate release in the rat
visual cortex. Our results show that the glycine binding site on the
postsynaptic NMDA-Rs is not saturated and that glycine binding
sites at presynaptic NMDA-Rs regulate glutamate release in the
layer II/III pyramidal neurons of the rat visual cortex.
METHODS
Slice preparation
Visual cortical slices were prepared from Sprague–Dawley rats
aged 13–15 days. All animals were housed in a standard environment
on a 12/12-h light/dark cycle with light on at 07:00, and they were
allowed ad libitum access to water and food. The use and care of
animals in this study follow the guidelines of the Xi’an Jiaotong
University Animal Research Advisory Committee. Rats were initially
anesthetized with ether and then immersed in ice-cold water, with the
nose exposed, for 3 min to reduce brain temperature. Immediately
after decapitation, the brain was placed in an ice-cold artificial
cerebrospinal fluid (ACSF) bubbled with 95% O2-5% CO2 (pH 7.4).
The ACSF consisted of (in mM) 124 NaCl, 5 KCl, 1.2 KH2PO4, 1.3
MgSO4, 2.4 CaCl2, 26 NaHCO3, and 10 glucose, and it had an
osmolality of 305–310 mosM/kg H2O. A block of tissue containing
the primary visual cortex was cut into 350-␮m-thick slices with a
vibratome (Campden Instruments, London, UK). Slices were transThe costs of publication of this article were defrayed in part by the payment
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0022-3077/07 $8.00 Copyright © 2007 The American Physiological Society
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Li Y-H, Han T-Z. Glycine binding sites of presynaptic NMDA
receptors may tonically regulate glutamate release in the rat visual
cortex. J Neurophysiol 97: 817– 823, 2007. First published November
8, 2006; doi:10.1152/jn.00980.2006. In the CNS, activation of Nmethyl-D-aspartate receptor (NMDA-R) glycine binding sites is a
prerequisite for activation of postsynaptic NMDA-Rs by the excitatory neurotransmitter glutamate. Here we provide electrophysiological
evidence that the glycine binding sites of presynaptic NMDA-Rs
regulate glutamate release in layer II/III pyramidal neurons of the rat
visual cortex. Specifically, our results reveal that the frequency of
miniature excitatory postsynaptic currents is significantly reduced by
7-chloro-kynurenic acid (7-Cl KYNA), a NMDA-R glycine binding
site antagonist, and glycine or D-serine reverses this effect. Similar
results are obtained when the open-channel NMDA receptor blocker,
MK-801, is included in the recording pipette. Our data indicate that
the glycine binding site of postsynaptic NMDA-Rs is not saturated.
Moreover, they suggest that presynaptic NMDA-Rs are located in
layer II/III pyramidal neurons of the rat visual cortex and that the
glycine binding site of presynaptic NMDA-Rs tonically regulates
glutamate release.
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Y.-H. LI AND T.-Z. HAN
ferred to an incubating chamber containing ACSF equilibrated with
carbogen (95% O2-5% CO2) and incubated for ⱖ1.5 h at room
temperature (20°C) prior to electrophysiological recording.
Recordings
Data analysis
Miniature excitatory postsynaptic currents (mEPSCs) were detected
and analyzed with MiniAnalysis software (Version 6.0, Synaptosoft)
using a threshold-crossing criterion. Although the threshold level
varied from neuron to neuron, it was always the same before and after
drug application (Bradaia and Trouslard 2002). Detection criteria also
included rise time ⬍3 ms (Sjostrom et al. 2003), which may eliminate
the purely NMDA-R-mediated mEPSCs because the average 10 –90%
rise time of purely NMDA-R-mediated mEPSC events is 8 ⫾ 2 ms
(O’Brien et al. 1997). This means that our data do not include the
silent synapses. Multiple peak events were discarded. Averaged
mEPSCs were obtained by aligning events on the rising phase. Events
occurring during 120 s were averaged and analyzed in every group. To
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RESULTS
Glycine or D-serine potentiates NMDA-R–mediated mEPSCs
Whole cell mEPSCs recordings were performed on 58 pyramidal neurons in layer II/III of the visual cortical slices. Spontaneous mEPSCs in the presence of TTX (0.5 ␮M), strychnine (1
␮M), and picrotoxin (100 ␮M) were abolished by application of
D-APV (50 ␮M) and CNQX (10 ␮M) in the Mg-free ACSF (data
not shown). This indicates that the mEPSCs were composed of
both NMDA and non-NMDA glutamatergic components (Berger
et al. 1998; O’Brien et al. 1997). Application of exogenous
glycine (100 ␮M) enhanced the NMDA-R-mediated mEPSCs,
and this effect was completely blocked by D-APV (50 ␮M; Fig. 1,
A and B). This enhancement of NMDA-R-mediated mEPSCs or
increased NMDA charge transfer (fC), is shown in Fig. 1C.
Charge transfers under control conditions and in response to
glycine treatment were 622 ⫾ 111 and 795 ⫾ 104 fC, respectively
(28% increase, P ⬍ 0.007, n ⫽ 8). These results indicate that the
glycine binding site on synaptically active NMDA-Rs is not
saturated. However, application of glycine (100 ␮M) did not alter
the frequency or the peak amplitude of the mEPSCs (Fig. 1, D and
E). Application of D-APV (50 ␮M) decreased the frequency
(control, 2.82 ⫾ 0.5 Hz vs. treated, 1.50 ⫾ 0.5 Hz, P ⬍ 0.001, n ⫽
8) but not the peak amplitude of the mEPSCs (control, 46.9 ⫾ 4.4
pA vs. treated, 46.0 ⫾ 4.6 pA, P ⬎ 0.18, n ⫽ 8). This result
suggests that glycine binding site on presynaptic NMDA-Rs is
saturated and the NMDA-Rs may tonically regulate glutamate
release.
D-serine, another agonist at the glycine-site of NMDA-Rs, is
not transported by glycine transporters (Supplisson and Bergman 1997). Therefore we examined the effect of D-serine on
NMDA-mediated mEPSCs to verify the results obtained with
glycine. Application of exogenous D-serine (100 ␮M) also
increased the NMDA-R-mediated component of mEPSCs,
which was abolished by D-APV (50 ␮M; Fig. 2, A and B).
NMDA-mediated charge transfer increased from 631 ⫾ 83 fC
in the control to 960 ⫾ 186 fC in D-serine-treated neurons
(52% increase, P ⬍ 0.005, n ⫽ 9, Fig. 2C). Thus D-serine had
a greater effect on the glycine binding site of NMDA-Rs than
glycine. However, D-serine did not alter the frequency or the
peak amplitude of the mEPSCs (Fig. 2, D and E). Application
of D-APV reduced the frequency (control, 2.27 ⫾ 0.6 Hz vs.
treated, 0.98 ⫾ 0.3, P ⬍ 0.001, n ⫽ 9) but not the peak
amplitude (control, 40.3 ⫾ 3.6 pA vs. treated, 39.4 ⫾ 3.5 pA,
P ⬎ 0.21, n ⫽ 9) of the mEPSCs. These results verified that
D-serine has the same effect as glycine.
Glycine or D-serine reverses the inhibitory effects of 7-Cl
KYNA on the frequency of NMDA-R–mediated mEPSCs
To determine whether the glycine binding site of the
NMDA-Rs may be involved in the modulation of glutamate
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For recording, slices were individually transferred to a recording
chamber where they were perfused (2.5–3 ml/min) with oxygenated
Mg-free ACSF at 31 ⫾ 0.5°C. The temperature of the recording
chamber was continuously monitored and controlled by a custommade temperature controller. The slices were placed on an upright
infra-red video microscope with differential interference contrast
(DIC) optics (OLYMPUS BX51WI), which was mounted on a
Gibraltar X–Y table. Slices were observed through a ⫻40 waterimmersion objective using an infrared-sensitive camera (DAGE-MTI,
IR-1000). Layer II/III pyramidal neurons of the visual cortex were
visually selected (Mason and Larkman 1990). Patch-clamp recordings
were performed in the whole cell configuration. Unpolished and
uncoated patch pipettes (1.5 mm/1.1 mm; Sutter Instruments, Novato,
CA) with a resistance of 4 – 6 M⍀ were pulled using a horizontal
puller (model P-97, Sutter Instruments, Novato, CA). The pipette
solution contained (in mM) 130 cesium methanesulfonate, 5 NaCl, 5
QX-314, 2 MgCl2, 0.1 CaCl2, 1 EGTA, 10 HEPES, 2 Na2ATP, and
0.25 Na3GTP (pH 7.3–7.4, adjusted with CsOH, 280 –290 mosM/kg
H2O). To block postsynaptic NMDA-Rs, the following pipette solution was used (in mM): 124 cesium methanesulfonate, 5 QX-314, 2
MgCl2, 10 BAPTA, 1 (⫹)-MK-801, 10 HEPES, 2 Na2ATP, and 0.25
Na3GTP (pH 7.3–7.4, adjusted with CsOH, 280 –290 mosM/kg H2O).
To facilitate the blockade of the open channel blocker MK-801,
neurons were depolarized to ⫺10 mV for 10 s at intervals during a
10-min period after breakthrough to whole cell access, and this
interval might be enough for MK-801 to block all the channels
according to Yang’s work (Yang et al. 2006). Currents were recorded
at a holding potential of ⫺60 mV using an Axopatch 700B amplifier
(Axon Instruments, USA) and digitized using a data-acquisition board
(Digidata 1322A) operated by pCLAMP 9.2 software (Axon Instruments). They were filtered at 2 kHz and digitized at 10 kHz. Compensation for the series resistance was not employed. Statistical
comparisons were performed only when series resistance was ⬍20
M⍀ and did not change by ⬎10%.
Glycine, D-serine, strychnine, picrotoxin, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), D-2-amino-5-phosphonovalerate (D-APV),
(⫹)-MK-801, QX-314, BAPTA, and 7-chloro-kynurenic acid (7-Cl
KYNA) were purchased from Sigma. Tetrodotoxin (TTX) was obtained from Hebei Province Marine Science and Technolog, China.
TTX (0.5 ␮M), strychnine (1 ␮M) and picrotoxin (100 ␮M) were
added to Mg-free ACSF to suppress the sodium currents, glycinergic
and GABAergic transmission, respectively. Mg-free ACSF was obtained by omitting MgSO4 (1.3 mM) from the ACSF without compensation for the loss of osmolarity or for the amount of divalent ions.
All drugs were applied at known concentrations by changing the
perfusion line.
quantify the NMDA component of mEPSCs, we integrated the average mEPSCs waveforms occurring over a 90-ms time period, which
began 10 ms after the start of the mEPSCs (Berger et al. 1998). This
integral was used as an index of NMDA-R-mediated charge transfer.
Comparison of mEPSC distributions was performed using the Kolmogorov-Smirnov test, and values of P ⬍ 0.01 were accepted as
significant (Berretta and Jones 1996). Group means were compared
using a paired Student’s t-test, and P ⬍ 0.05 was considered significant. All data are expressed as means ⫾ SE.
GLYCINE TONICALLY REGULATES GLUTAMATE RELEASE
819
release, we employed 7-Cl KYNA, an antagonist of the glycine
binding site of the NMDA-Rs. As shown in Fig. 3D, inclusion
of 7-Cl KYNA (20 ␮M) in the extracellular solution blocked
the postsynaptic NMDA component and reduced the frequency
of the mEPSCs (control, 1.9 ⫾ 0.7 Hz vs. treated, 1.1 ⫾ 0.4
Hz, P ⬍ 0.001, n ⫽ 10). Combined treatment with glycine (100
␮M) and 7-Cl KYNA reversed this effect (control, 1.9 ⫾ 0.7
Hz vs. treated, 1.8 ⫾ 0.6 Hz, P ⬎ 0.08, n ⫽ 10) and recovered
the postsynaptic NMDA component (Fig. 3, B and D). As
shown in Fig. 3C, the peak amplitude of the mEPSCs was not
influenced by 7-Cl KYNA or glycine (control, 41.0 ⫾ 3.3 pA;
7-Cl KYNA, 39.6 ⫾ 4.0 pA; 7-Cl KYNA ⫹ glycine, 40.4 ⫾
3.9 pA, n ⫽ 10).
As shown in Fig. 4, D-serine (100 ␮M) also reversed the
inhibitory effects of 7-Cl KYNA (20 ␮M) on the frequency of
NMDA-mediated mEPSCs (control, 1.69 ⫾ 0.4 Hz; 7-Cl
KYNA, 1.1 ⫾ 0.2 Hz; 7-Cl KYNA ⫹ D-serine, 1.65 ⫾ 0.3 Hz,
P ⬍ 0.001, n ⫽ 12). The peak amplitude of the mEPSCs was
unaffected by treatment either with 7-Cl KYNA only or along
with D-serine (control, 40 ⫾ 3.8 pA; 7-Cl KYNA, 38.6 ⫾ 5.1
pA; 7-Cl KYNA ⫹ D-serine, 39.0 ⫾ 4.4 pA; n ⫽ 12; Fig. 4C).
These results suggest that the glycine binding site is involved
in the modulation of the glutamate release.
Glycine or D-serine reverses the inhibitory effects of 7-Cl
KYNA in the presence of MK-801
It is possible that 7-Cl KYNA reduced the frequency of the
mEPSCs by acting on the glycine binding site of presynaptic
NMDA-Rs or postsynaptic NMDA-Rs. In the latter case,
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presynaptic neurotransmitter release could be affected through
retrograde messengers. To test these possibilities, we included
the open-channel NMDA-R blocker, dizocilpine maleate (MK801; 1 mM), in the recording pipette as described by Berretta
and Jones (1996). Blockade of the postsynaptic NMDA-Rs
with MK-801 completely abolished the NMDA component of
mEPSCs (Fig. 5). Inclusion of 7-Cl KYNA (20 ␮M) under
these conditions reduced the frequency of the mEPSCs. Moreover, this effect could be reversed by the addition of glycine
(100 ␮M; control, 2.6 ⫾ 0.5 Hz; 7-Cl KYNA, 1.7 ⫾ 0.3 Hz;
7-Cl KYNA ⫹ glycine, 2.6 ⫾ 0.6 Hz; P ⬍ 0.001, n ⫽ 10; Fig.
5C). As before, the peak amplitude of the mEPSCs was not
affected by treatment with 7-Cl KYNA and glycine. (control,
34.3 ⫾ 1.5 pA; 7-Cl KYNA, 34.4 ⫾ 1.2 pA; 7-Cl KYNA ⫹
glycine, 34.5 ⫾ 1.7 pA; n ⫽ 10).
As shown in Fig. 6C, D-serine (100 ␮M) also reversed the
effects of the 7-Cl KYNA on mEPSC frequency in the presence of MK-801 (control, 2.8 ⫾ 0.7 Hz; 7-Cl KYNA, 1.6 ⫾ 0.5
Hz; 7-Cl KYNA ⫹ D-serine, 2.8 ⫾ 0.6 Hz; P ⬍ 0.001; n ⫽ 9).
The peak amplitude of the mEPSCs was not affected by these
treatments (control, 32.8 ⫾ 3.9 pA; 7-Cl KYNA, 32.4 ⫾ 4.1
pA; 7-Cl KYNA ⫹ D-serine, 33.1 ⫾ 4.2 pA; n ⫽ 9; Fig. 6B).
These results suggest that presynaptic, but not postsynaptic,
NMDA-R glycine binding sites tonically regulate glutamate
release in the visual cortex.
Additional experiments showed that there were no effects of
glycine, D-serine or 7-Cl KYNA on the frequency and amplitude of mEPSCs in the presence of D-APV, which blocked both
pre- and postsynaptic NMDARs (data showing in Supplemental materials).
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FIG. 1. Glycine enhances N-methyl-D-aspartate receptor
(NMDA-R)-mediated miniature excitatory postsynaptic
currents (mEPSCs). A: traces of mEPSCs recorded from the
same neuron (holding potential ⫽ ⫺60 mV) under control
conditions (top), after the addition of glycine (middle), and
after addition of D-APV (bottom). The NMDA component
of mEPSCs was increased by glycine and decreased by
D-APV. B: average mEPSCs from the same neuron shown
in A. C: NMDA charge transfer occurring between 10 and
100 ms after mEPSC onset was increased in the presence of
100 ␮M glycine (P ⬍ 0.007, n ⫽ 8). D: pooled data from
8 neurons shows the peak amplitudes of the mEPSCs were
not altered by treatment with glycine and D-APV. E: glycine (100 ␮M) did not alter the frequency of the mEPSCs.
However, application of D-APV (50 ␮M) decreased the
frequency (P ⬍ 0.001, n ⫽ 8).
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Y.-H. LI AND T.-Z. HAN
DISCUSSION
In the present study, we have used a whole cell recording
technique to examine the effects of glycine and D-serine on the
spontaneous synaptic activities in layer II/III pyramidal neurons of the rat visual cortex. All data were obtained in the
presence of TTX (0.5 ␮M), strychnine (1 ␮M), and picrotoxin
(100 ␮M) in the Mg-free ACSF and at a holding potential of
⫺60 mV. Under these conditions, the spontaneous spikes as
well as glycinergic and GABAergic synaptic activities could be
excluded, and the NMDA-R- mediated mEPSCs could be
FIG. 3. Glycine reverses the inhibitory effect of
7-chloro-kynurenic acid (7-Cl KYNA) on NMDA-R-mediated mEPSCs. A: traces of mEPSCs recorded from the
same neuron (holding potential ⫽ ⫺60 mV) under control
conditions (top) and in the presence of only 20 ␮M 7-Cl
KYNA (middle) and along with 100 ␮M glycine (bottom).
B: average mEPSCs from the same neuron shown in A. C:
pooled data from 10 neurons shows the peak amplitudes of
the mEPSCs were not altered by treatment with 7-Cl
KYNA and glycine. D: frequency of mEPSCs was reduced
by 7-Cl KYNA (P ⬍ 0.001, n ⫽ 10), and this effect was
reversed by addition of glycine (P ⬎ 0.08, n ⫽ 10).
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FIG. 2. D-serine enhances the NMDA-R-mediated
mEPSCs. A: traces of mEPSCs recorded from the same
neuron (holding potential ⫽ ⫺60 mV) under control
conditions (top), after the addition of D-serine (middle),
and after addition of D-APV (bottom). The NMDA
component of mEPSCs was increased by D-serine and
decreased by D-APV. B: average mEPSCs from the
same neuron shown in A. C: NMDA charge transfer
occurring between 10 and 100 ms after mEPSC onset
was increased in the presence of D-serine (P ⬍ 0.005,
n ⫽ 9). D: pooled data from nine neurons shows the
peak amplitudes of the mEPSCs were not altered by
treatment with D-serine and D-APV. E: D-serine (100
␮M) did not alter the frequency of the mEPSCs. However, application of D-APV (50 ␮M) decreased the
frequency (P ⬍ 0.001, n ⫽ 9).
GLYCINE TONICALLY REGULATES GLUTAMATE RELEASE
821
facilitated. The finding that mEPSCs were abolished by the
presence of D-APV (50 ␮M) and CNQX (10 ␮M) in extracellular fluid indicates that the mEPSCs were composed of both
NMDA and non-NMDA glutamatergic components. On the
other hand, mEPSCs were made up of only non-NMDA
glutamatergic components when MK-801 was included in the
recording pipette.
As described in the preceding text, application of exogenous
glycine potentiated NMDA-R-mediated mEPSCs. Glycine
may achieve this effect through at least three mechanisms.
First, glycine might act through presynaptic, strychnine-sensitive glycine receptors to facilitate the release of glutamate, an
action that has been demonstrated in the brain stem (Turecek
and Trussell 2001). In this study, the contribution of such as
mechanism is probably limited because mEPSCs were recorded in the presence of strychnine (1 ␮M) and picrotoxin
(100 ␮M). Second, glycine might act on the NMDA-Rs containing NR3A or NR3B subunits (Chatterton et al. 2002),
which are activated by glycine in the absence of glutamate. The
finding that D-serine, which blocks NMDA-Rs containing
NR3A or NR3B subunits, potentiates NMDA-R–mediated
mEPSCs argues against this possibility. Third, glycine might
act postsynaptically by activating the glycine binding site of
NMDA-Rs. To test this hypothesis, we employed D-serine,
another NMDA-R glycine binding site agonist. The ability of
D-serine to increase the NMDA component provides evidence
that glycine potentiates NMDA-R–mediated mEPSCs via the
NMDA-R glycine binding site. In addition, the non-NMDA
FIG. 5. Glycine reverses the inhibitory effects of
7-Cl KYNA on mEPSC frequency in the presence of
MK-801. A: NMDA-R open channel blocker, MK-801
(1 mM), was applied via the recording pipette, and
mEPSCs were recorded under control conditions (top)
and in the presence of only 20 ␮M 7-Cl KYNA
(middle) and along with 100 ␮M glycine (bottom).
The mEPSC tracings from the same neuron (holding
potential ⫽ ⫺60 mV) are shown. Blockade of
postsynaptic NMDA-Rs via MK-801 completely abolished NMDA-mediated mEPSCs. B: average mEPSCs
shown in A. C: pooled data from 10 neurons shows the
peak amplitudes of the mEPSCs were not significantly
changed under any of these conditions. D: in the
presence of MK-801, the frequency of mEPSCs was
reduced by 7-Cl KYNA, and this effect was reversed
by ddition of glycine (P ⬍ 0.001, n ⫽ 10).
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FIG. 4. D-serine reverses the inhibitory effect of 7-Cl
KYNA on NMDA-R-mediated mEPSCs. A: traces of
mEPSCs recorded from the same neuron (holding potential ⫽ ⫺60 mV) under control conditions (top) and in the
presence of 20 ␮M 7-Cl KYNA (middle) and along with
100 ␮M D-serine (bottom). B: average mEPSCs from the
same neuron shown in A. C: pooled data from 12 neurons
shows the peak amplitudes of the mEPSCs were not
altered by treatment with 7-Cl KYNA and D-serine. D:
frequency of mEPSCs was reduced by 7-Cl KYNA, and
this effect was reversed by addition of D-serine (P ⬍
0.001, n ⫽ 12).
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Y.-H. LI AND T.-Z. HAN
component was not influenced by glycine or D-serine, and the
application of APV or 7-Cl KYNA abolished the NMDA
component of the mEPSCs. Taken together, these results suggest that the glycine binding site on the postsynaptic
NMDA-Rs is not saturated as has been shown in cat visual
cortex (Czepita et al. 1996).
The levels of synaptic glycine are known to be tightly
controlled by glycine transporters. There are two types of
glycine transporters, GlyT1 and GlyT2. GlyT1, which is localized to glia and neurons, is closely associated with the
NMDA-Rs (Cubelos et al. 2005); whereas, GlyT2 is colocalized with strychnine-sensitive glycine receptors (Gomeza et al.
2003). As has been described in the hippocampus (Watanabe et
al. 1992), D-serine was capable of potentiating NMDA synaptic
currents. Here, application of exogenous D-serine increased the
NMDA component by ⬃52%, and glycine increased the
NMDA component of the mEPSCs by⬃ 28%. The greater
effect of D-serine, which is not taken up by glycine transporters
(Supplisson and Bergman 1997), suggests that levels of synaptic glycine are relatively low and are tightly controlled by
glycine transporters.
Our results also show that APV or 7-Cl KYNA reduce the
frequency but not the peak amplitude of mEPSCs, regardless of
the presence of MK-801 in the recording pipette. These findings support the existence of presynaptic NMDA-Rs and are
consistent with studies showing that presynaptic NMDA-Rs
tonically facilitate glutamate release as a result of increased
calcium influx into the terminals (Berretta and Jones 1996;
Sjostrom et al. 2003; Yang et al. 2006).
It was reported that mEPSCs events rely on presynaptic
Ca2⫹ transient either in hippocamcal area CA1 or in layer II
neurons of the rat barrel cortex (Rusakov 2006; Simkus and
Stricker 2002). Presynaptic Ca2⫹ transient could produce Ca2⫹
release from stores, named Ca2⫹-induced Ca2⫹ release
J Neurophysiol • VOL
(CICR), and then the postsynaptic EPSCs. The amplitude of
CICR from internal Ca2⫹ stores depends on the amount of
Ca2⫹ influx and probably on the cytosolic Ca2⫹ concentration
(Rusakov 2006). Activation of presynaptic NMDA-Rs could
induce the Ca2⫹ influx and then improve the CICR and finally
facilitate the neurotransmitter release.
The difference in saturation of the glycine site between preand postsynaptic NMDA-Rs seems to have different affinities
for glycine (Kew et al. 1998; Kutsuwada et al. 1992). There is
strong evidence that at P14 the postsynaptic NMDA receptors
are mostly NR1-NR2A, whereas the presynaptic ones are
mostly NR1-NR2B (Sjostrom et al. 2003; Woodhall et al.
2001; Yang et al. 2006). The EC50s of glycine and of D-serine
for NR1-NR2A receptors are known to be higher than those for
NR1-NR2B. Thus a concentration of glycine will be saturating
for the (presynaptic) NR2B-containing receptors and not saturating for the (postsynaptic) NR2A-containing receptors. Also
consistent with our findings, presynaptic NR1- and NR2Bcontaining NMDA-Rs are known to exist (Aoki et al. 1994)
and enhance neurotransmitter release in the visual cortex (Sjostrom et al. 2003).
The ability of glycine or D-serine to reverse the effects of
7-Cl KYNA when MK-801 was present in the recording
pipette argues against the possibility that glycine acts on
postsynaptic receptors to facilitate glutamate release via retrograde messengers. Additionally, the finding that D-serine is
able to reverse the effects of 7-Cl KYNA, combined with the
fact that strychnine was present in all experiments, makes it
unlikely that presynaptic, strychnine-sensitive glycine receptors facilitated glutamate release (Turecek and Trussell 2001).
In summary, our data show that glycine can act on glycine
binding sites at presynaptic and postsynaptic NMDA-Rs to
enhance the NMDA-R function, which could be indicative of a
general role for the glycine binding site of presynaptic
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FIG. 6. D-serine reverses the inhibitory effects of
7-Cl KYNA on mEPSCs frequency in the presence of
MK-801. A: NMDA-R open channel blocker, MK-801
(1 mM), was applied via the recording pipette, and
mEPSCs were recorded under control conditions (top)
and in the presence of only 20 ␮M 7-Cl KYNA
(middle) and along with 100 ␮M glycine (bottom).
The mEPSC tracings from the same neuron (holding
potential ⫽ ⫺60 mV) are shown. Blockade of
postsynaptic NMDA-Rs via MK-801 completely abolished NMDA-mediated mEPSCs. B: average mEPSCs
shown in A. C: pooled data from 9 neurons shows the
peak amplitudes of the mEPSCs were not significantly
changed under any of these conditions. D: in the
presence of MK-801, the frequency of mEPSCs was
reduced by 7-Cl KYNA, and this effect was reversed
by addition of D-serine (P ⬍ 0.001, n ⫽ 9).
GLYCINE TONICALLY REGULATES GLUTAMATE RELEASE
NMDA-Rs in regulating glutamate release in the CNS. Moreover, these findings may be clinically relevant to schizophrenia, where enhancing NMDA-R function is considered to be a
promising strategy for treatment of the disease (Coyle et al.
2002; Duncan et al. 1999).
GRANTS
This work was supported by the National Natural Science Fountation of
China Grant 30170310 and the key program of Xi’an Jiaotong University.
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