Glial D-serine gates NMDA receptors at excitatory synapses in prefrontal cortex.
ABSTRACT N-methyl-D-aspartate receptors (NMDARs) subserve numerous neurophysiological and neuropathological processes in the cerebral cortex. Their activation requires the binding of glutamate and also of a coagonist. Whereas glycine and D-serine (D-ser) are candidates for such a role at central synapses, the nature of the coagonist in cerebral cortex remains unknown. We first show that the glycine-binding site of NMDARs is not saturated in acute slices preparations of medial prefrontal cortex (mPFC). Using enzymes that selectively degrade either D-ser or glycine, we demonstrate that under the present conditions, D-ser is the principle endogenous coagonist of synaptic NMDARs at mature excitatory synapses in layers V/VI of mPFC where it is essential for long-term potentiation (LTP) induction. Furthermore, blocking the activity of glia with the metabolic inhibitor, fluoroacetate, impairs NMDAR-mediated synaptic transmission and prevents LTP induction by reducing the extracellular levels of D-serine. Such deficits can be restored by exogenous D-ser, indicating that the D-amino acid mainly originates from glia in the mPFC, as further confirmed by double-immunostaining studies for D-ser and anti-glial fibrillary acidic protein. Our findings suggest that D-ser modulates neuronal networks in the cerebral cortex by gating the activity of NMDARs and that altering its levels is relevant to the induction and potentially treatment of psychiatric and neurological disorders.
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ABSTRACT: Presynaptic NMDA receptors facilitate the release of glutamate at excitatory cortical synapses and are involved in regulation of synaptic dynamics and plasticity. At synapses in the entorhinal cortex these receptors are tonically activated and provide a positive feedback modulation of the level of background excitation. NMDA receptor activation requires obligatory occupation of a co-agonist binding site, and in the present investigation we have examined whether this site on the presynaptic receptor is activated by endogenous glycine or D-serine. We used whole-cell patch clamp recordings of spontaneous AMPA receptor-mediated synaptic currents from rat entorhinal cortex neurones in vitro as a monitor of presynaptic glutamate release. Addition of exogenous glycine or D-serine had minimal effects on spontaneous release, suggesting that the co-agonist site was endogenously activated and likely to be saturated in our slices. This was supported by the observation that a co-agonist site antagonist reduced the frequency of spontaneous currents. Depletion of endogenous glycine by enzymatic breakdown with a bacterial glycine oxidase had little effect on glutamate release, whereas D-serine depletion with a yeast D-amino acid oxidase significantly reduced glutamate release, suggesting that D-serine is the endogenous agonist. Finally, the effects of D-serine depletion were mimicked by compromising astroglial cell function, and this was rescued by exogenous D-serine, indicating that astroglial cells are the provider of the D-serine that tonically activates the presynaptic NMDA receptor. We discuss the significance of these observations for the aetiology of epilepsy and possible targeting of the presynaptic NMDA receptor in anticonvulsant therapy.Neuropharmacology 04/2014; · 4.11 Impact Factor
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ABSTRACT: The identification of the presence of active signaling between astrocytes and neurons in a process termed gliotransmission has caused a paradigm shift in our thinking about brain function. However, we are still in the early days of the conceptualization of how astrocytes influence synapses, neurons, networks, and ultimately behavior. In this Perspective, our goal is to identify emerging principles governing gliotransmission and consider the specific properties of this process that endow the astrocyte with unique functions in brain signal integration. We develop and present hypotheses aimed at reconciling confounding reports and define open questions to provide a conceptual framework for future studies. We propose that astrocytes mainly signal through high-affinity slowly desensitizing receptors to modulate neurons and perform integration in spatiotemporal domains complementary to those of neurons.Neuron 02/2014; 81(4):728-739. · 15.77 Impact Factor
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ABSTRACT: Schizophrenia is a complex and multifactorial disorder generally diagnosed in young adults at the time of the first psychotic episode of delusions and hallucinations. These positive symptoms can be controlled in most patients by currently-available antipsychotics. Conversely, they are poorly effective against concomitant neurocognitive dysfunction, deficits in social cognition and negative symptoms (NS), which strongly contribute to poor functional outcome. The precise notion of NS has evolved over the past century, with recent studies - underpinned by novel rating methods - suggesting two major sub-domains: "decreased emotional expression", incorporating blunted affect and poverty of speech, and "avolition", which embraces amotivation, asociality and "anhedonia" (inability to anticipate pleasure). Recent studies implicate a dysfunction of frontocortico-temporal networks in the aetiology of NS, together with a disruption of cortico-striatal circuits, though other structures are also involved, like the insular and parietal cortices, amygdala and thalamus. At the cellular level, a disruption of GABAergic-glutamatergic balance, dopaminergic signalling and, possibly, oxytocinergic and cannibinoidergic transmission may be involved. Several agents are currently under clinical investigation for the potentially improved control of NS, including oxytocin itself, N-Methyl-d-Aspartate receptor modulators and minocycline. Further, magnetic-electrical "stimulation" strategies to recruit cortical circuits and "cognitive-behavioural-psychosocial" therapies likewise hold promise. To acquire novel insights into the causes and treatment of NS, experimental study is crucial, and opportunities are emerging for improved genetic, pharmacological and developmental modelling, together with more refined readouts related to deficits in reward, sociality and "expression". The present article comprises an integrative overview of the above issues as a platform for this Special Issue of European Neuropsychopharmacology in which five clinical and five preclinical articles treat individual themes in greater detail. This Volume provides, then, a framework for progress in the understanding - and ultimately control - of the debilitating NS of schizophrenia.European neuropsychopharmacology: the journal of the European College of Neuropsychopharmacology 05/2014; 24(5):645-92. · 3.68 Impact Factor
Cerebral Cortex March 2012;22:595--606
Advance Access publication June 20, 2011
Glial D-Serine Gates NMDA Receptors at Excitatory Synapses in Prefrontal Cortex
Pascal Fossat1,2, Fabrice R. Turpin1,2, Silvia Sacchi3, Je ´ ro ˆ me Dulong1,2, Ting Shi4, Jean-Michel Rivet5, Jonathan V. Sweedler4,
Loredano Pollegioni3, Mark J. Millan5, Ste ´ phane H.R. Oliet1,2and Jean-Pierre Mothet1,2
1Institut National de la Sante ´ et de la Recherche Me ´ dicale U862, Neurocentre Magendie, 33077 Bordeaux, France,2Universite ´ de
Bordeaux, 33077 Bordeaux, France,3Dipartimento di Biotecnologie e Scienze Molecolari, Universita ` degli Studi dell’Insubria, and
The Protein Factory, Centro Interuniversitario di Biotecnologie Proteiche, Politecnico di Milano and Universita ` degli Studi
dell’Insubria, 21100 Varese, Italy,4Department of Chemistry and Beckman Institute, University of Illinois, Urbana 61801, Illinois,
USA and5Department of Psychopharmacology, Institut de Recherches Servier, 78290 Croissy-sur-Seine, France
Pascal Fossat and Fabrice R. Turpin have contributed equally to the work
Address correspondence to Jean-Pierre Mothet, Centre de Recherche en Neurobiologie et Neurophysiologie de Marseille, CNRS UMR 6231,
Universite ´ Aix-Marseille Faculte ´ de Me ´ decine Nord, Gliotransmission and Synaptopathies Team, 51 Boulevard Pierre Dramard, 13344 Marseille Cedex
15, France. Email: email@example.com.
N-methyl-D-aspartate receptors (NMDARs) subserve numerous
neurophysiological and neuropathological processes in the cerebral
cortex. Their activation requires the binding of glutamate and also
of a coagonist. Whereas glycine and D-serine (D-ser) are candidates
for such a role at central synapses, the nature of the coagonist in
cerebral cortex remains unknown. We first show that the glycine-
binding site of NMDARs is not saturated in acute slices
preparations of medial prefrontal cortex (mPFC). Using enzymes
that selectively degrade either D-ser or glycine, we demonstrate
that under the present conditions, D-ser is the principle endogenous
coagonist of synaptic NMDARs at mature excitatory synapses in
layers V/VI of mPFC where it is essential for long-term potentiation
(LTP) induction. Furthermore, blocking the activity of glia with the
metabolic inhibitor, fluoroacetate, impairs NMDAR-mediated syn-
aptic transmission and prevents LTP induction by reducing the
extracellular levels of D-serine. Such deficits can be restored by
exogenous D-ser, indicating that the D-amino acid mainly originates
from glia in the mPFC, as further confirmed by double-immunostain-
ing studies for D-ser and anti-glial fibrillary acidic protein. Our
findings suggest that D-ser modulates neuronal networks in the
cerebral cortex by gating the activity of NMDARs and that altering
its levels is relevant to the induction and potentially treatment of
psychiatric and neurological disorders.
Keywords: astrocytes, coagonist, excitatory synapses, NMDA receptors,
The N-methyl-D-aspartate receptor (NMDAR) subfamily of
glutamate receptors is widely expressed in the central nervous
system (CNS) where it is indispensable to many of the activity-
dependent changes in synaptic strength and connectivity
underlying higher brain functions such as memory formation
and cognition (Malenka and Bear 2004; Rebola et al. 2010).
Dysfunction of NMDA receptors is considered central to the
pathophysiology of several neurologic and psychiatric disor-
ders. Indeed, NMDAR hyperactivity can cause cell death in
stroke and chronic neurodegenerative disorders such as
Parkinson, Alzheimer’s diseases, and HIV-associated dementia
(Kemp and McKernan 2002; Hardingham and Bading 2003). By
contrast, hypoactivity of NMDAR induces apoptosis during
brain development and may contribute to psychotic and
cognitive symptoms associated to schizophrenia (Millan 2005;
Ross et al. 2006).
In addition to the agonist glutamate, activation of the
NMDAR requires the binding of a coagonist which was
originally thought to be glycine (Johnson and Ascher 1987;
Paoletti and Neyton 2007). However, studies over the last
decade have shown that the CNS produces significant amount
of an atypical amino acid, D-serine (D-ser) (Hashimoto and Oka
1997; Martineau et al. 2006; Wolosker 2007). D-Ser is converted
from L-ser by serine racemase (SR), an enzyme enriched in the
CNS and is thought to be degraded by D-amino acid oxidase
(DAAO) (Martineau et al. 2006; Wolosker 2007; Pollegioni and
Sacchi 2010). Functional studies have demonstrated that the D-
amino acid appears to be the physiological ligand for the
coagonist site of NMDARs in different brain areas like the
hippocampus (Mothet et al. 2000; Yang et al. 2003; Mothet et al.
2006; Zhang et al. 2008; Basu et al. 2009), the retina (Stevens
et al. 2003; Kalbaugh et al. 2009), and the hypothalamus
(Panatier et al. 2006).
Compelling evidence suggests that besides its physiological
functions, D-ser may participate in excitotoxic events when
released in excess (Martineau et al. 2006). Indeed, SR deletion
confers neuronal protection to cerebral ischemia and excito-
toxicity (Mustafa et al. 2010) and to b-amyloid 1--42 peptide
injury in the forebrain (Inoue et al. 2008). By contrast, aging
and schizophrenia are associated with decreased D-ser levels
(Hashimoto et al. 2005; Mothet et al. 2006; Bendikov et al. 2007;
Sacchi et al. 2008; Turpin et al. 2009).
As such, D-ser modulation appears to be central to many
brain functions but also to play a role in the etiology of
neurodegenerative disorders and psychiatric diseases affecting
particularly the cerebral cortex. Nevertheless, whether D-ser
serves as the endogenous coagonist of synaptic NMDARs in the
cerebral cortex is unknown. Amongst the different cerebral
areas, the prefrontal cortex (PFC) is critical for social cognition,
conceptualization, and working memory whose disturbances
are evident in Alzheimer’s disease, stroke, or schizophrenia
(Goto et al. 2010). In this study, we examined the respective
roles of glycine versus
D-ser in governing the activity of
NMDARs localized in layers V/VI of PFC where they are
strongly expressed (Wang et al. 2008). We have developed an
acute brain slice preparation of medial prefrontal cortex
(mPFC) for recordings from prelimbic area, an analog structure
of primate PFC, from adult rat (Gabbott et al. 2005). We here
demonstrate that mPFC of adult rats contains high levels of D-
ser and that the coagonist site of NMDARs in layers V/VI is not
saturated. Using enzymes that selectively degrade either D-ser
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or glycine, we further report that under the present conditions
D-ser is the major endogenous coagonist of synaptic NMDAR.
Furthermore, we show that
astrocytes in layers V/VI of mPFC and that D-ser and glia are
both necessary for the induction of NMDAR-dependent long-
term synaptic plasticity. Our findings thus underpin therapeu-
tic strategies targeting D-ser availability in the treatment of
brain disorders such as schizophrenia or stroke where the
operation of NMDARs is disrupted.
D-ser is produced partly by
Materials and Methods
All experiments were conducted with respect to European and French
directives on animal experimentation.
Slice Preparation and Electrophysiological Recordings
Experiments were carried out on acute slices obtained from 45- to 60-
old-day Wistar rats. The rats were anaesthetized with isoflurane and
decapitated. The brain was then quickly removed and placed in ice-
cold artificial cerebrospinal fluid (ACSF) saturated with 95% O2and 5%
CO2. Coronal slices (300 lm) of the mPFC including the infralimbic
(area, 25) and the prelimbic (area 32) cortices were obtained and
allowed to recover for at least 45 min at 31 ?C in a submerged chamber
containing ACSF before recording. After 30--60 min recovery at room
temperature, one slice was transferred and submerged in a recording
chamber where it was continuously perfused (1--2 mL/min) with ACSF
composed of (in mM): NaCl, 123; KCl, 2.5; Na2HPO4, 1; NaHCO3, 26.2;
MgCl2, 1.3; CaCl2, 2.5; and glucose, 10 (pH 7.4; 295--300 mOsm kg–1).
Pyramidal PFC neurons in layers V/VI of prelimbic cortices (area 32)
were identified visually using infrared differential interference contrast
microscopy (Olympus BX51). Synaptically evoked excitatory currents
(EPSCs) were recorded under whole-cell voltage-clamp mode. The
patch-clamp recording pipettes (borosilicate glass, Harvard Apparatus,
2.5--5 MX) were filled with cesium chloride (CsCl) solution containing
(in mM): CsCl, 130; NaCl, 10; 4-(2-hydroxyethyl)-1-piperazineethane-
sulfonic acid (HEPES), 10; QX-314, 5; ethyleneglycol-bis(2-amino-
ethylether)-N,N,N#,N#-tetra acetic acid (EGTA), 1; and CaCl2, 0.1
(adjusted to pH 7.1--7.3 with CsOH; 292--296 mOsm kg–1). Neurons
were clamped first at –70 mV for 5 min after breaking the seal and then
depolarized at +40 mV. Membrane currents were recorded at 30 ?C
using a Multiclamp 700B amplifier (Axon Instruments, Inc.); signals
were filtered at 2 kHz and digitized at 5 kHz via a DigiData 1440A
interface (Molecular Devices). Series resistance (6--15 MX) and holding
current were monitored throughout the experiment. Cells with Ra >
25 MX or holding current > –200 pA at resting potential were excluded
from data analysis, as well as any cell for which a >20% change in those
parameters occurred during the course of the experiment. The paired-
pulse ratio (PPR) was calculated as the peak amplitude of the second
evoked EPSC (2)/peak amplitude of the first EPSC (1). The pulse
duration was 100 ls and the interval between the 2 pulses was 100 ms.
Glass electrodes filled with ACSF were used to stimulate the layers I/II
of the prelimbic area in the mPFC (Fig. 1B,C). Assuming that mean
center-to-center distance between minicolumn in the prelimbic area is
45 lm in rats (Gabbott et al. 2005), the electrode of stimulation was
shifted in order to avoid direct stimulation of the dendritic bundles
of the recorded pyramidal neurons. For a-Amino-3-hydroxy-5-methyl-
4-isoxazolepropionic acid (AMPA) and NMDA-EPSCs, the pulse duration
was 100 ls and the interval between 2 pulses was, respectively, 15 s for
AMPA-EPSCs and 30 s for NMDA-EPSCs. In all figures, current curves are
mean of at least 10 successive responses.
For the induction of long-term potentiation (LTP), a 4 3 1 s pulse
protocol was used at resting membrane potential (holding current = 0),
with a 100-Hz stimulation period for each pulse. The interval between
each pulse was 15 s. In some experiment, current-clamp mode was
used to study the pattern of discharge of the pyramidal neurons from
which EPSCs were recorded. In those cases, the recording electrodes
were filled with (in mM): K-gluconate, 120; KCl, 20; HEPES, 10, EGTA, 1;
MgCl2, 1; CaCl2, 0.1 (adjusted to pH = 7.2--7.4; 290 ± 5 mOsm kg–1).
Spontaneous unitary excitatory postsynaptic currents (mEPSCs) were
obtained in the presence of tetrodotoxin (TTX, 0.5 lM) and picrotoxin
(50 lM), and in those case, the recordings electrodes were filled with
K-gluconate-based solution. mEPSCs were recorded by using Axoscope,
detected, and analyzed off-line using Axograph (Molecular Devices). At
least 200 events were analyzed for each cell in each condition.
Data Analysis and Statistics
Data were collected and analyzed using pClamp10 software (Axon
Instruments, Inc.). They are expressed as percentage values and are
reported as mean ± the standard error of the mean of n cells. Statistical
significance was evaluated via paired or unpaired Student’s t-test.
Significance was assessed at P < 0.05.
Determination of Tissue and Extracellular Amino Acid Levels
Levels of endogenous amino acids were determined on acute PFC slices
from the same rats. Because of the limited amount of tissue per slice, 2
slices (approximately 1.5 mg) were pooled together. Analyses of the
samples were performed using capillary electrophoresis with laser-
induced fluorescence (CE-LIF) (CE: Beckman Coulter, P/ACE MDQ; LIF:
Picometrics, LIF-UV-02, 410 nm, 15 mW) (Lapainis and Sweedler 2008).
Briefly, tissue samples were first deproteinized by addition of cold
trichloroacetic acid (TCA) to a 5% final concentration. The suspension
was centrifuged at 16,800 3 g for 10 min, and the TCA was extracted
from the supernatant with water-saturated diethyl ether and stored at
–80 ?C until analysis. Extracellular levels of amino acids (glycine, L-ser, D-
ser) were determined in control conditions and after exposure of acute
PFC slices for 40 min to fluoroacetate (FAC), Rhodotorula gracilis
DAAO (RgDAAO), or Bacillus subtilis glycine oxidase (BsGO) to
evaluate the effects of such treatments. The extracellular media were
retrieved and stored at –80 ?C until analysis. Liquid phase of amino acids
were processed for micellar CE-LIF (Zhao, Song, et al. 2005). Briefly, the
samples were fluorescently derivatized at room temperature for 2 h
with napthalene-2,3-dicarboxaldehyde (NDA) before being analyzed by
CE using a hydroxypropyl-b-cyclodextrin (HP-b-CD) based chiral
separation buffer. All data were collected and analyzed using Karat 32
software v8.0 (Beckman Coulter, Fullerton, CA).
The amount of amino acids in tissue was scaled to the protein
content determined by the Lowry method using the Pierce BCA protein
Assay kit (Thermo Scientific) assay with bovine serum albumin (BSA) as
standards. The quantity of amino acids in the samples was determined
from a standardized curve and peak identification was made by spiking
the fraction with the appropriate amino acid and by evaluating the
effect of selective peak removing for D-ser or glycine by enzymatic
treatment with RgDAAO or BsGO on standards.
Wistar rats 45--60 old days were deeply anesthetized with sodium
pentobarbital (50 mg/kg, intraperitonetally), transcardially perfused
with an ice-cold solution containing 4% paraformaldehyde in 0.1 M
phosphate-buffered saline (PBS), pH 7.4 supplemented with 0.25%
glutaraldehyde, and brain were explanted and subjected to an
overnight postfixation in the same solution and finally cryoprotected
with 30% sucrose in 0.1 M PBS (pH 7.4). Brain coronal 30 lm sections
were cut on a freezing microtome (Micron HM450). After several
washes in PBS, free-floating brain sections were immunostained as
described previously (Puyal et al. 2006). After a blocking/permeabiliza-
tion step, sections were probed for 36 h at 4 ?C with pairs of primary
antibodies diluted in PBS plus 4% normal goat serum and 0.1% Triton X-
100. After several washes, the slices were incubated for 1 h at room
temperature with pairs of Alexa secondary antibodies (Alexa 488 anti-
rabbit/Alexa 546 anti-mouse, Molecular Probes) at 1:2000 dilution in
PBS plus 1.5% normal goat serum and 0.1% Triton X-100. Finally, slices
were washed and mounted in Vectashield mounting medium (Vector
Laboratories). Controls were performed by avoiding the primary
antibodies. Immunofluorescence was analyzed using a laser scanning
confocal microscope (Leica TCS SP2; Leica Microsystems). The
confocal images were acquired using the Leica TCS software with
a sequential mode to avoid interference between each channel and
Functions of D-Serine at NMDARs in the PFC
Fossat et al.
at INIST-CNRS on January 7, 2013
without saturation of any pixel. Moreover, emission windows were
fixed for each fluorophore in conditions where no signal is detected
from the other fluorophore. Stack images were taken in the PFC area,
and a Z-projection was made with ImageJ 1.43 software (http://
rsb.info.nih.gov/ij) in standard deviation projection mode.
Drugs, Enzymes, and Antibodies
All drugs were bath applied. Appropriate stock solutions were made,
stored at –20 ?C and diluted to the final concentration with ACSF
containing vehicle (<1/1000) just before application. Picrotoxin, 2,3-
amino-5-phosphonopentanoic acid (D-AP5), ifenprodil, sarcosine,
D-ser, glycine, and TTX were obtained from TOCRIS bioscience.
Sodium fluoroacetate (FAC) was obtained from Fluka. D-Cycloserine
was from Sigma-Aldrich. NDA was obtained from Molecular Probes
(Eugene, OR). D-Cycloserine and HP-b-CD were from Sigma-Aldrich.
Recombinant RgDAAO (EC 184.108.40.206) was overexpressed in Escherichia
coli cells and purified as reported earlier (Fantinato et al. 2001); the final
enzyme preparation had a specific activity of 100 ± 15 U/mg protein on
D-ser as substrate. Recombinant BsGO (EC 220.127.116.11) was overexpressed
in E. coli cells as well (Job et al. 2002); the final enzyme preparation had
a specific activity of 0.9± 0.2 U/mg protein on glycine as substrate. These
flavoenzymes specifically degrade D-ser (RgDAAO) and glycine (BsGO),
as demonstrated by the corresponding apparent kinetic efficiency (kcat/
Km ratio) values: kcat/Km ratios of 3.0 and 0.058 mM–1s–1were
Figure 1. Properties of NMDA currents of layers V/VI pyramidal neurons in the mPFC. (A) Typical electropherogram (right) of NDA-derivatized amino acids showing the presence
of D-ser in the mPFC of mature rats. L-S: L-serine, D-S: D-serine, E: glutamate, G: glycine, Eth: ethanolamine (internal standard). (B and C) Schematic representation (B) of the
mPFC and micrograph (C) from the prelimbic area showing the patched pyramidal neurons in layers V/VI. Stimulating electrode is placed in layers I/II. (D) Characteristic I/V
pyramidal cell response in current-clamp configuration. (E) In voltage clamp, pyramidal cells (lower trace) present at ?70 mV a typical evoked AMPA current in presence of
GABAAinhibitor picrotoxin; this current is blocked by application of 10 lM NBQX. (F) A typical outward NMDA current (upper trace) is revealed at þ40 mV under both picrotoxin
and NBQX. This current is blocked by application of AP5. An AMPA/NMDA ratio was measured by dividing the NMDA peak current and the AMPA peak current. This ratio is close
to 1 in control conditions. (G) Time course of the effect of ifenprodil on NMDA-EPSCs. Layers V/VI pyramidal cell NMDA receptors contain a mixed NR2A/NR2B subunits.
Application of 3 lM ifenprodil reduced NMDA currents by about 30%.
Cerebral Cortex March 2012, V 22 N 3 597
at INIST-CNRS on January 7, 2013
determined for RgDAAO on D-ser and glycine, respectively (Fantinato
et al. 2001; Pollegioni et al. 2007), while the kcat/Kmratios determined
for BsGO were 0.00025 and 0.867 mM–1s–1on D-ser and glycine,
respectively (Job et al. 2002). To degrade D-ser or glycine, slices were
incubated for at least 45 min and then continuously perfused with aCSF
Affinity-purified rabbit polyclonal antibody against conjugated D-ser
was from GemacBio (France) and affinity-purified mouse monoclonal
anti-glial fibrillary acidic protein (GFAP) antibody (clone G-A-5) and
mouse monoclonal S100b antibody (clone 1B2) were from Sigma.
Evoked NMDA Receptor--Mediated EPSCs in Layers V/VI
We first showed that both D-ser and glycine are present in
mPFC in significant amounts (D-ser: 0.144 nmol/mg prot;
glycine: 0.805 nmol/mg prot) as revealed by capillary electro-
phoresis (Fig. 1A). The retrieved amounts and their ratio are
comparable to the ones found in others brain areas such as the
hypothalamus (Panatier et al. 2006), the hippocampus (Mothet
et al. 2006), or the retina (Stevens et al. 2003). This result
indicates that both amino acids, glycine, and D-ser could serve
as NMDAR endogenous coagonist in the prelimbic area of the
Synaptic currents in layers V/VI pyramidal cortical neurons
were recorded in the whole-cell patch-clamp configuration
(Fig. 1B,C). These neurons displayed a mean resting membrane
potential of –64 ± 0.4 mV and were able to sustain firing when
depolarized above spike threshold (Fig. 1D) We studied
glutamatergic transmission in layers V/VI mPFC neurons by
stimulating excitatory afferents through a glass electrode
placed in layers I/II (Fig. 1B,C). When recorded at –70 mV
and under picrotoxin (50 lM), EPSCs were entirely mediated
by AMPA/kainate receptors since they were insensitive to AP5
(50 lM) and blocked by NBQX (10 lM) (Fig. 1E). To isolate the
NMDA component of the EPSCs, we clamped the cells at +40
mV in the presence of NBQX. Under these conditions, the
evoked current had much slower rise time and decay time as
expected for NMDARs (Wang et al. 2008) and was inhibited by
AP5 (Fig. 1F), confirming that it was mediated by NMDARs.
These responses were also partially blocked with ifenprodil (3
lM; 73.5 ± 7.4% of control, n = 7, P = 0.012) (Fig. 1G), a NR2B
subunit--containing NMDAR antagonist (Panatier et al. 2006;
Paoletti and Neyton 2007; Wang et al. 2008) indicating that
synaptic NMDARs are mainly composed of NR2A subunits
(Massey et al. 2004). No changes in the holding current and in
the series resistance (Ra) were observed during the time
course of ifenprodil application arguing against a possible
effect of ifenprodil on the integrity of the membrane
(Supplementary Fig. 1).
We next checked for the level of occupancy of the NMDAR
glycine-binding site. Exogenous application of D-ser (100 lM)
to the bathing solution induced a significant and reversible
increase in the amplitude of the NMDAR-mediated EPSCs
(125.0 ± 6.2% of control, n = 17, P = 0.001, Fig. 2A,F). D-ser had
no effect on AMPAR-mediated EPSCs (Fig. 2B). Surprisingly,
glycine by itself (10--500 lM) failed to potentiate NMDA-EPSCs
(104.6 ± 8.7% of control, n = 10, P =0,43, Fig. 2C,F) and only
a modest but significant potentiating effect (115.6 ± 1.9% of
control, n = 7, P < 0.001) was also observed when glycine
transporters (GlyTs) were blocked with 0.5 mM sarcosine
(Fig. 2D,F). Preincubation of mPFC slices with D-serine (100
lM) occluded the potentiating effect of sarcosine (Fig. 2E,F)
arguing that D-serine and not glycine is the most effective
coagonist in the PFC. Finally, we used another different specific
partial agonist of the glycine site,
application (500 lM) significantly increases the NMDA
component to 121.3 ± 5.1% (Supplementary Fig. 2). A dose--
response curve shows that D-ser is the most effective coagonist
at lower doses (EC50for D-ser: 26.5 lM, D-cycloserine: 202.3
lM). Taken together, these results indicate that the glycine site
of NMDARs in layers V/VI mPFC pyramidal neurons is not fully
saturated by the endogenous coagonist at least in acute brain
D-Serine is the Endogenous Coagonist of NMDARs in
Layers V/VI mPFC Neurons
To identify whether D-ser, glycine, or both amino acids acted as
pyramidal neurons, we investigated the action of 2 enzyme
(BsGO), that selectively degrade D-ser and glycine, respectively
(Fantinato et al. 2001; Job et al. 2002; Panatier et al. 2006). To
assess the action of these enzymes on synaptic NMDARs, slices
were incubated for at least 45 min with either RgDAAO or BsGO
(0.2 U/mL), and the AMPA/NMDA ratios of peak currents were
compared between these different conditions (Fig. 3A,B). In
slices treated with RgDAAO, this ratio was largely increased
(control vs. RgDAAO: 1.09± 0.18 vs. 2.20± 0.26, n = 6, P = 0.006)
compared with control conditions, whereas inactive catalytic
mutant RgDAAO had no effect (data not shown). While NMDA-
EPSCs were decreased by RgDAAO, AMPAR-mediated EPSCs
were not impacted (control vs. RgDAAO: –118± 11 vs. –132± 24
pA,n =27forcontrolandn =10forRgDAAO,P =0.57,Fig.3A,C).
The specific action of RgDAAO on NMDAR-EPSCs was further
investigated by theanalysis of spontaneous EPSCs (mEPSCs) that
are driven by AMPAR (Supplementary Fig. 3). RgDAAO did not
the specific action of DAAO on NMDA-EPSCs as reported earlier
(Mothet et al. 2000; Yang et al. 2003). Subsequent application of
exogenous D-ser restored the AMPA/NMDA peak current ratio
(1.40 ± 0.15, Fig. 3A,D) to control values indicating that these
changes induced by RgDAAO on NMDAR-mediated currents are
most likely due to the degradation of D-ser by the enzyme. To
ensure that this is the case, we next evaluated the effect of
RgDAAO on extracellular levels of D-ser. Using CE-LIF, we
measured the levels of D-ser before and after exposure of PFC
slices to RgDAAO (Supplementary Fig. 4B). As expected,
RgDAAO (0.2 U/mL, 40 min) decreased extracellular D-ser levels
by 77.03 ± 9.51% (P = 0.0098) without impacting significantly
glycine levels (–14.40 ± 12.29% of control, P = 0.139).
Collectively, these findings strongly support the idea that in the
mPFC, D-ser is an endogenous coagonist of synaptic NMDARs as
reported for other brain areas (Stevens et al. 2003; Yang et al.
2003; Panatier et al. 2006; Zhang et al. 2008).
We next investigated the contribution of endogenous
glycine to NMDARs activity at the same synapses using BsGO.
Unlike what we observed following RgDAAO incubation, we
did not detect any significant changes in the AMPA/NMDA ratio
(control vs. BsGO: 1.24 ± 0.25, n = 11, P = 0.62, Fig. 3B,C)
although BsGO (0.2 U/mL, 40 min) is effective in reducing
significantly and specifically the extracellular levels of glycine
by 64.1 ± 7.5% (P
< 0.001, Supplementary Fig. 4C).
Functions of D-Serine at NMDARs in the PFC
Fossat et al.
at INIST-CNRS on January 7, 2013
Furthermore, the application of 100 lM D-ser had an effect
similar to that observed in the absence of BsGO (120.4 ± 10.1%
of control, n = 10, P < 0.05) indicating that degradation of
glycine did not affect NMDA-EPSCs (Fig. 3C,D). Interestingly,
glycine (10--500 lM) failed to induce any change in NMDA-
EPSCs (Fig. 2C for 100 lM and data not shown), an effect that
may be related to the presence of highly efficient GlyTs for this
amino acid that quickly clear up glycine from the synaptic cleft
(Betz et al. 2006). Indeed, inhibition of GlyTs by sarcosine
increased modestly NMDA-EPSCs (Fig. 2D,F) in the absence of
added D-ser (Fig. 2E,F), an effect that was prevented by BsGO
(Supplementary Fig. 5A,D) further demonstrating that the
enzyme is active and not degraded by peptidase during
incubation. Still, because sarcosine has been reported to be
an excellent substrate for BsGO (Job et al. 2002), we
conducted another set of controls to ascertain that the
occluding effect of BsGO was due to degradation of glycine
rather than sarcosine. When glycine was first enzymatically
removed by BsGO (Supplementary Fig. 5B,D), sarcosine no
longer potentiated NMDA-EPSCs. These results support the
idea that GlyTs do remove efficiently glycine preventing it to
enter synaptic cleft and also show that the observed
potentiating effect of sarcosine was not due to a direct agonist
action of sarcosine on NMDARs as reported (Zhang et al. 2009).
These data support the idea that glycine, unlike D-ser, is not an
endogenous coagonist at synaptic NMDARs on layers V/VI
mPFC pyramidal neurons and that glycine and D-ser compete at
synaptic NMDARs. Accordingly, glycine induced a significant
potentiation of NMDA-EPSCs (+37.0 ± 9.6% vs. control, n = 4,
P < 0.03) only when D-ser has been removed enzymatically by
Figure 2. Saturation of NMDA coagonist site. (A and B) Glycine site of NMDA receptors is not saturated under control conditions. Indeed, application of 100 lM D-ser reversibly
increases NMDA currents of about 20% while no effect is observed on AMPAR-mediated EPSCs (panel B). (C) The application of 100 lM glycine has no effect on NMDA-EPSCs
while, (D) the application of 0.5 mM sarcosine (a specific GlyT1 inhibitor) induces a slow but significant increase in NMDA currents. (E) The potentiating effect of sarcosine was
abolished when sarcosine was applied in presence of 100 lM D-ser. Numbers in panels A--E represent the example of currents for the corresponding condition. (F) Bar graphs
indicate mean ± standard error of the mean for the different experimental conditions. All NMDA-EPSCs and AMPA-EPSCs were recorded at þ40 and ?70 mV holding potentials,
respectively. **P \ 0.01, ***P \ 0.001, comparison with Student’s t-test.
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RgDAAO (Supplementary Fig. 5C,D) as described previously in
the hippocampus (Shleper et al. 2005).
Because endogenous D-ser regulates the activity of NMDARs
in mPFC, we made the assumption that the D-amino acid may
contribute to the induction of long-term changes in synaptic
plasticity notably the LTP whose induction in the cerebral
cortex like in others brain areas requires NMDAR activation
(Massey et al. 2004; Banerjee et al. 2009). LTP was induced by
high-frequency stimulation of the excitatory inputs in layers I/
II. This protocol reliably induced a long-lasting increase in the
amplitude of the evoked EPSCs (162.4 ± 18.3% of control, n = 7,
P = 0.007, Fig. 4A). Such an LTP was abolished in the presence
of D-AP5 in the bath (96.1 ± 18.7% of control, n = 5, P = 0.44, Fig.
4A), as expected from an NMDAR-dependent process. In-
terestingly, the same induction protocol yielded no potentia-
tion in RgDAAO-incubated slices (81.0 ± 13.0% of control, n = 5,
P = 0.12). Such impairment of synaptic plasticity could be
rescued by providing exogenous glycine to the brain slices
(163.4 ± 19.8%, n = 5, P = 0.016, Fig. 4B) in line with our above
observations that glycine can modulate NMDA-EPSCs when D-
ser is not present at the synaptic cleft (Supplementary Fig. 5).
These results support the notion that D-ser is governing the
plasticity of excitatory synapses in the CNS as observed in the
hippocampus (Yang et al. 2003; Mothet et al. 2006) and the
hypothalamus (Panatier et al. 2006).
Glia as a Main Source for D-Ser in the mPFC
Recent data revealed that in the cerebral cortex and other
brain regions, neurons in addition to astrocytes are an
important source of D-ser (Kartvelishvily et al. 2006; Miya
et al. 2008; Rosenberg et al. 2010). To test whether PFC D-ser
originates from astrocytes or neurons in layers V/VI mPFC, we
performed immunostaining for D-ser (Martineau et al. 2006).
We showed that the amino acid was evenly present in all layers
of the mPFC of adult rat with a strong staining evident in
astrocyte-like cells (Fig. 5A). Double immunostaining for D-ser
with GFAP in layers V/VI of mPFC further identified astrocytes
as the cells where the amino acid is strongly present (Fig. 5B)
with labeling in the soma and processes of those cells. No
particular laminar staining for GFAP was observed in the mPFC
(data not shown). We also found moderate and infrequent
staining of D-ser in the cell bodies of neurons throughout the
mPFC layers (Fig. 5B).
The presence of D-ser in mPFC glia cells strongly suggested
that those cells contribute to the regulation of NMDAR
functions in the mPFC through the release of D-ser. Thus, to
test this hypothesis, we next assessed the effect of FAC (5 mM,
40 min), a glia-specific metabolic inhibitor (Hassel et al. 1997;
Hu ¨ lsmann et al. 2003; Andersson et al. 2007; Zhang et al. 2008;
Okada-Ogawa et al. 2009) on synaptic transmission and LTP. As
expected if glia was the source of
a significant reduction in D-ser (FAC vs. control: –55.46 ±
18.80%, n = 2, P = 0.02) but not in glycine (FAC vs. control:
–12.32 ± 12.39%, n = 2, P = 0.20) extracellular levels measured
when acute PFC slices were exposed to the toxin (Supple-
mentary Fig. 4B,C). Accordingly, FAC increases the AMPA/
NMDA ratio (FAC vs. control: 3.15 ± 0.70, n = 6, P = 0.03,
Fig. 6A) while it did not affect AMPA-EPSCs (data not shown).
This result was similar to that observed when
degraded with RgDAAO (Fig. 3A,C,D). At the same time, release
probability was not impaired by FAC as revealed by its lack of
action on PPR of AMPAR-EPSCs (control: 100.1 ± 7.9% vs. FAC:
110.8 ± 10.9%, n = 5, P = 0.19, Fig. 6B). Furthermore, we did not
observe any effect of FAC on the membrane properties of the
D-ser, FAC produced
Figure 3. D-serine but not glycine is an endogenous coagonist of NMDA receptors in layers V/VI pyramidal cells of mPFC. (A--D) AMPA/NMDA ratio after 45 min incubation of
slices in RgDAAO or BsGO. (A) After RgDAAO, NMDA current, but not AMPA current, was strongly decreased. As a consequence, the AMPA/NMDA ratio was significantly
increased (RgDAAO, open bar and circle in panels C and D, respectively). Subsequent application of 100 lM D-ser increased NMDA current (panel A, right). RgDAAO was not
completely washed out since AMPA/NMDA ratio was brought back only to control value (panel D, compare control in black circle to RgDAAO in open circles). (B) By contrast,
NMDA current was not reduced in slices incubated in BsGO. Thus, AMPA/NMDA ratio of slices incubated in BsGO was not modified (panels C and D, compare control to BsGO;
gray circle). Subsequent application of D-ser slightly increased NMDA current (~20% see text) as in control conditions (panel D, gray circle). All NMDA-EPSCs and AMPA-EPSCs
were recorded at þ40 and ?70 mV holding potentials, respectively. *P \ 0.05, comparison with Student’s t-test.
Functions of D-Serine at NMDARs in the PFC
Fossat et al.
at INIST-CNRS on January 7, 2013
cortical neurons as indicated by the absence of changes
in membrane resting potential (–65.3 ± 1.3 mV at t = 0 min
and –65.36 ± 1.8 mV at t = +45 min, n = 11), amplitude of action
potential (77.2 ± 2.6 mV at t = 0 min and 73.5 ± 2.4 mV at
t = +45 min, n = 11), and spike threshold (–36.5 ± 1.7 mV at t = 0
min and –37.7 ± 2.2 mV at t = +45 min, n = 11). The increase in
AMPA/NMDA ratio induced by FAC application could be
completely restored to its control values by adding exogenous
D-ser (100 lM) to the slice (FAC + D-ser vs. control: 1.60 ± 0.25,
P = 0.14) (Fig. 6A). These data strongly suggest that glia,
through the release of D-ser, gates NMDARs in layers V/VI
mPFC pyramidal neurons of the prelimbic area.
Given the contribution of glia to synaptic transmission, we
next examined their role in the induction of LTP which relies on
D-ser and which is NMDA dependent (Fig. 4). No LTP could be
induced in FAC-treated slices (91.4 ± 14.3% of control, n = 6, P =
0.54, Fig. 6C), a result similar to what was observed in the
presence of AP5 or RgDAAO and in agreement with the
requirement of glia and D-ser to activate NMDARs. Exogenous
applications of D-ser reversed this LTP impairment allowing the
induction of a potentiation equivalent to that obtained under
control conditions (158.4 ± 31.2% of control, n = 7, P = 0.03, Fig.
6B). Therefore, these data indicate that glia, through the release
of D-ser, regulates activity of excitatory synapses in the mPFC.
The data documented in this study suggest that, under the
present experimental conditions at least, in layers V/VI PFC
pyramidal neurons of prelimbic area, D-ser, but not glycine, is
the endogenous ligand of glycine-binding site of synaptic
NMDARs. This D-amino acid appears to be mainly derived from
astrocytes rather than neurons and is required for long-term
synaptic plasticity induction in the mature PFC of rodents.
Relevance of the Rat Prelimbic Area as an Analog
Structure of Primate PFC
We focused our study on the prelimbic area (area 32) inside
the mPFC of rats (Gabbott et al. 2005). Although the existence
of a proper prefrontal cortex in rodents is controversial, there
Figure 5. Cellular distribution of D-serine in the mPFC layers V/VI. (A) Low-
magnification view of the distribution of D-ser (1/1000, Alexa 488) and S100b (1/
2000, Alexa 546) throughout the PFC layers. (B) Double immunostainings showing the
cellular distribution of GFAP (1/1 000, Alexa 546) versus D-serine (1/1 000; Alexa
488) in mPFC layers V/VI. Scales: 25 lm. Arrowheads highlight the presence of
labeled soma of neurons.
15 20 2530
EPSCs amplitude (%)EPSCs amplitude (%)
Figure 4. Pyramidal cell LTP depends on the saturation level of glycine site of NMDA receptors. (A) LTP is induced in layers V/VI pyramidal cells by high-frequency stimulation
(HFS). This LTP is NMDA dependent since it is blocked by application of AP5. (B) LTP is suppressed if slices are incubated in RgDAAO and is maintained in slices incubated in
RgDAAO and glycine.
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is strong evidence that prelimbic and infralimbic areas of mPFC
in rats are homologue of the dorsolateral prefrontal cortex of
primates (Uylings et al. 2003). First, anatomical studies have
shown that projections of mPFC in rats to subcortical target
nuclei are very similar to that of ventromedial PFC in primates
in particular for monoaminergic and cholinergic systems
(Uylings and van Eden 1990; Jodo and Aston-Jones 1997; Hajo ´ s
et al. 1998; Jodo et al. 1998; Carr and Sesack. 2000). Second, the
mPFC in rats and the dorsolateral PFC in primates share similar
structural and laminar organizations and functions (Gabbott
et al. 2005). Indeed, prelimbic area is implicated in working
memory, selection of information, response selection and
implementation, and dynamic goal-directed behavior (Uylings
et al. 2003; Gabbott et al. 2005). Then, we can conclude that
although rat PFC is not as differentiated as it is in primates,
prelimbic area is centrally involved in some dorsolateral-like
characteristics allowing using it as an analog structure of
A Role for D-Ser in NMDAR-Dependent Functions at
Activation of NMDARs requires binding of a coagonist which
was first proposed to be glycine (Johnson and Ascher 1987). It
has, however, become clear over the last decade that D-ser can
play an equivalent role (Wolosker 2007). In hippocampal
neurons cocultured with astrocytes, degradation of D-ser with
DAAO affected NMDAR-mediated responses (Mothet et al.
2000). This finding was subsequently extended to acute slice
preparations from hippocampus (Yang et al. 2003; Mothet et al.
2006; Zhang et al. 2008) and to other structures such as the
retina (Stevens et al. 2003; Kalbaugh et al. 2009) and the
hypothalamus (Panatier et al. 2006). Moreover, studies in SR-
knockout (KO) mice also suggest that D-ser is an endogenous
coagonist of synaptic NMDARs in the hippocampus (Basu et al.
2009). Correspondingly, NMDAR-mediated synaptic currents
are impaired and LTP is blunted in SR-KO mice. We here report
that selectively degrading D-ser with RgDAAO causes a pro-
nounced but incomplete inhibition of NMDAR-mediated EPSCs,
an effect that is neutralized by application of exogenous D-ser.
This finding indicates that D-ser is an endogenous coagonist of
NMDARs expressed by layers V/VI PFC pyramidal neurons
(Wang et al. 2008). The incomplete blockade of synaptic
transmission by RgDAAO may reflect incomplete depletion of
synaptic D-ser by RgDAAO or a complementary contribution of
endogenous glycine to the gating of synaptic NMDARs in the
In fact, although several studies have examined the
possibility that D-ser is the endogenous coagonist of synaptic
NMDARs, few have investigated whether or not glycine plays
such a role. Using BsGO, an enzyme that specifically degrades
glycine, it was established that this amino acid was not an
endogenous ligand of the NMDAR glycine-binding site in
supraoptic neurons of the rat hypothalamus (Panatier et al.
2006). We report here similar findings at intrinsic excitatory
synapses in layers V/VI pyramidal PFC neurons since BsGO,
unlike RgDAAO, did not affect NMDAR-mediated EPSCs
although the levels of glycine were significantly reduced by
the enzyme as we confirmed it by CE-LIF analysis. These
findings are consistent with previous observations that D-ser is
the dominant endogenous ligand of synaptic NMDARs during
adulthood in the CNS (Mothet et al. 2000; Yang et al. 2003;
Shleper et al. 2005; Panatier et al. 2006). They also suggest that
glycine might not usually be present at sufficient concen-
trations within the synaptic cleft to serve as an endogenous
coagonist of synaptic NMDARs, in contradiction to what is
often described in the literature. CE-LIF measurements of the
PFC content of amino acids revealed a significantly higher
concentration of glycine than D-ser that may question this
assertion. However, it is plausible that high affinity glycine
transporters GlyT1 and GlyT2 expressed by PFC glia and
neurons (Jursky and Nelson 1996; Chen et al. 2003; Cubelos
et al. 2005) suppress glycine levels in the local microenviron-
ment of pyramidal synaptic NMDARs well below their affinity
for the coagonist (range 0.36--3.7 lM) (Laurie and Seeburg
1994). Coexpression of both GlyT1 and NMDARs in Xenopus
oocytes dramatically reduces the level of glycine capable of
reaching the coagonist site (Supplisson and Bergman 1997).
Similarly, inhibition of GlyTs potentiates NMDA-mediated
responses in the prefrontal cortex (Chen et al. 2003).
Accordingly, we observed a modulatory effect of endogenous
glycine on NMDA-EPSCs only when GlyTs were initially
inhibited with sarcosine. Although the potentiating effect of
sarcosine on synaptic NMDA-EPSCs was only modest, this is
Mean PPR (%)
EPSCS amplitude (%)
Figure 6. Glial cells control NMDA receptors in layers V/VI pyramidal cells. (A) In slices incubated for 40 min in FAC (a metabolic glial inhibitor), AMPA/NMDA peak current ratio
is strongly increased. This effect was abolished by further application of 100 lM D-ser (black bar). All NMDA-EPSCs and AMPA-EPSCs were recorded at þ40 and ?70 mV
holding potentials, respectively. (B) NMDA-dependent LTP is under glial control. Indeed, LTP is suppressed in slices incubated in FAC and maintained in slices incubated in FAC
plus D-ser. (C) PPR is not affected when glia is functionally eliminated by FAC.
Functions of D-Serine at NMDARs in the PFC
Fossat et al.
at INIST-CNRS on January 7, 2013
likely due to the fact that endogenous D-ser competes with
glycine for the coagonist site of synaptic NMDARs (Paoletti and
Neyton 2007). Indeed, we showed in the present study that
endogenous glycine could exert its modulatory action on
synaptic NMDARs only if D-ser has been first removed (Figs 4
and 5). In other cases, glycine has no modulatory effect when
D-ser is present in the synaptic cleft under basal conditions or
at saturating levels (Fig. 2). It is conceivable that GlyTs prevent
the amino acid from entering the synaptic cleft under
physiological conditions (Betz et al. 2006). Nevertheless, we
cannot discard the possibility that glycine may reach the
synapse and then gates the synaptic NMDARs in pathological
conditions wherein activity of GlyTs is altered/disrupted. The
likelihood of this scenario is supported by studies showing that
GlyTs inhibitor displays antipsychotic profiles in rodents (Tsai
et al. 2004). Furthermore, genetically induced reduction of
GlyTs expression in mice leads to impairment in glutamatergic
neurotransmission in the hippocampus (Tsai et al. 2004;
Martina et al. 2005) and retina (Reed et al. 2009). Thus, the
relative contribution of glycine versus
NMDARs implies that the release of D-ser is operating locally
at synaptic sites to underpin synaptic functions (Schell et al.
1997; Mothet et al. 2000; Henneberger et al. 2010).
Our demonstration that D-ser is the endogenous ligand for the
glycine site of synaptic NMDARs prompted an evaluation of its
participation in long-term synaptic plasticity since LTP at
excitatory synapses relies on NMDAR in particular in the
cerebral cortex (Feldman 2009). Not surprisingly perhaps, we
observed a complete blockade of LTP when D-ser was depleted
by RgDAAO. As LTP and its counterpart long-term depression
have been proposed as cellular substrate of information
processing and memory (Malenka and Bear 2004; Feldman
2009; Rebola et al. 2010), the observation that LTP depends on D-
ser further expands the notion that D-ser plays an important
function in the cognitive processes. In the present study, we
observed that the blockade of NR2B-containing synaptic
NMDARs with ifenprodil reduced the total synaptic NMDA
current by ~30%. These results indicate that NMDARs at
excitatory synapses at PFC layers V/VI pyramidal neurons are
preferentially composed of NR2A subunits as previously shown
(Zhao, Toyoda, et al. 2005) and that NR2B subunits represent
only a small fraction (Wang et al. 2008). Noteworthy, both NR2B
and NR2A NMDAR subunits contribute to the formation of LTP
in the PFC where NR2B-containing NMDARs are engaged in the
formation of contextual memory (Zhao, Toyoda, et al. 2005).
D-ser at synaptic
Astrocytes Are Involved in the Control of NMDARs
Many lines of evidence suggest that D-ser is synthesized and
released from glia. Immunostaining for D-ser and SR revealed
the presence of the D-amino acid and its synthesizing enzyme
in astrocytes from different brain regions in adulthood (Schell
et al. 1997; Stevens et al. 2003; Panatier et al. 2006; Williams
et al. 2006). Using neuron--glia cocultures and pure neuronal
cultures treated with glia medium, it was originally established
that glia was the source of D-ser in the hippocampus (Mothet
et al. 2000; Ribeiro et al. 2002; Yang et al. 2003; Zhang et al.
2008). Release of D-ser from (Mothet et al. 2005) and trafficking
within (Martineau et al. 2008) cortical glial cells in culture
indicated that astrocytes could release D-ser through a Ca2+-
dependent vesicular pathway. The idea that glia represents an
important source for D-ser was strengthened by observations
made in the hypothalamus where a physiological withdrawal of
astrocytic processes was found to be associated with a de-
ficiency in D-ser-mediated NMDAR EPSCs (Panatier et al. 2006).
Still, it has been also established that in brain areas such as the
hippocampus, the cerebral cortex, or the vestibular nuclei, D-
ser may originate from neurons (Puyal et al. 2006; Kartvelishvily
et al. 2006; Miya et al. 2008; Rosenberg et al. 2010).
In the present study, we show by immunostaining that D-ser
is mainly present in astrocytes (see Fig. 5). By contrast, PFC
neurons seem to represent only a minor source of D-ser in
a physiological context. Nevertheless, the localization of SR
remains controversial. In some areas like the hypothalamus
(Panatier et al. 2006), SR could be found localized mainly to glia
which is consistent with the fact that only glia and notably
astrocytes have the ability to synthesize L-serine, the substrate
of SR (Yamasaki et al. 2001). But SR is also found in neurons in
the cerebral cortex or in the hippocampus (Kartvelishvily et al.
2006; Miya et al. 2008; Rosenberg et al. 2010). In an elegant
model developed recently, Herman Wolosker proposed that
glia synthesizes and exports L-ser which is used by neuronal SR
for the synthesis of D-ser. Once released by neurons, D-ser will
be uptaken, stored, and released in an activity-dependent
manner by glia (Wolosker 2011). According to this model,
neurons and not glia cells represent the main locus for the
synthesis of D-ser. Although neurons express ASCT1 and ASCT2
allowing for the potential shuttling of L-ser from glia to neurons
(Gliddon et al. 2009; Shao et al. 2009), PFC pyramidal neurons
are probably unable to synthesize large amount of D-ser. Thus,
the low amount of D-ser in PFC neurons could be attributed to
the presence of DAAO which degrades the amino acid once
taken up by neurons (data not shown). We cannot exclude that
PFC neurons may synthesize and release D-ser when depolar-
ized (Rosenberg et al. 2010) through a nonexocytotic mech-
anism. Nevertheless, the relative contribution of neuronal
versus glial-derived D-ser on NMDARs-mediated EPSCs and
LTP could be unmasked pharmacologically using FAC, an
astroglial aconitase inhibitor (Hassel et al. 1997; Hu ¨ lsmann et al.
2003; Andersson et al. 2007; Okada-Ogawa et al. 2009). In
addition, we have shown by CE-LIF that FAC specifically
reduced D-ser levels, supporting the idea of a control of NMDA-
EPSCs by glial D-ser. FAC poisoning of glia reduces NMDA-
EPSCs and completely blocks LTP induction, in line with the
idea that astrocytes are involved in the control of NMDARs
activity through the release of D-ser (Stevens et al. 2003; Yang
et al. 2003; Zhang et al. 2008). Still, we cannot rule out fully
a contribution of neuronal D-ser in regulating synaptic NMDARs
as FAC does not totally block NMDA-EPSCs. One possible
explanation is that the residual currents are modulated by the
neuronal reserve of D-ser released through a sodium dependent
antiport as proposed by the group of Wolosker (Ribeiro et al.
2002; Kartvelishvily et al. 2006; Rosenberg et al. 2010). It is also
conceivable that FAC indirectly affects the release of D-ser from
neurons rather than inhibiting the release of the coagonist from
glia. Indeed, inhibition of the glial aconitase by FAC leads to
accumulation of citrate and to a reduction in the formation of
glutamine, an important precursor for neurotransmitter gluta-
mate and c-aminobutyric acid (GABA) (Hamilton and Attwell
Under conditions of sustained GABAA receptor blockade,
FAC might theoretically inhibit glial glutamine levels thereby
affecting the neuronal synthesis of glutamate and in turn the
release of D-ser from glia or neurons (Kartvelishvily et al. 2006).
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However, this latter scenario can be ruled out since FAC did
not affect the release of neuronal glutamate as suggested by the
absence of any changes in the amplitude of AMPA-EPSCs or PPR
as expected if FAC was impacting the glutamine--glutamate
cycle (Fig. 6C). If there was a neural source of D-ser in the PFC,
functionally eliminating glia with FAC would not be expected
to impact synaptic transmission as neuronal
compensate (at least partially) the pharmacologically induced
deficit in extracellular D-ser and then result in unaffected
synaptic transmission, but the opposite was observed. These
observations indicate that the use of FAC selectively targeted
the glial contribution to synaptic transmission and unmasked
the role of glia-derived D-ser. It has been shown that in situ
administration of FAC decreases the level of D-ser by 25% in the
PFC (Kanematsu et al. 2006) supporting our observations that
FAC impacts synaptic transmission by selectively reducing the
levels of extracellular D-ser and not glycine (Supplementary Fig.
3). Interestingly, we observed that FAC also impacts the levels
of extracellular glutamate indicating that glutamate is also
released by glia in the PFC as demonstrated in the hippocam-
pus (Fellin et al. 2006). Still, the inhibitory effect of FAC on
synaptic transmission and plasticity is fully prevented when D-
ser is added showing that D-ser is the limiting extracellular
factor for NMDA receptors activity in the PFC. Nevertheless, we
cannot rule out the possibility that neuronal D-ser may also be
important for the function of NMDARs when neuronal DAAO
activity is impaired as observed in schizophrenia (Schumacher
et al. 2004; Millan 2005; Almond et al. 2006; Ross et al. 2006;
Verrall et al. 2007; Sacchi et al. 2008; Labrie et al. 2010). The
nature of the interplay between neuronal and glia-derived D-ser
will require further explorations.
Physiological and Pathological Relevance
In the prefrontal cortex, NMDARs dynamically control neuronal
circuitry and subserve cognitive functions such as working
memory, decision making, and experience-dependent plasticity
(Malenka and Bear 2004; Feldman 2009). Therefore, the control
D-ser and its metabolic enzymes may be crucial for
physiological processes depending on NMDARs in the mature
cerebral cortex (Malenka and Bear 2004; Feldman 2009).
Moreover, alterations of D-ser levels and function should have
important consequences for the activity of neuronal circuitry
in the PFC. Accumulating experimental evidence shows that
cognitive symptoms of schizophrenia are related to a hypo-
function of NMDARs in the PFC (Millan 2005; Ross et al. 2006).
Interestingly, convergent lines of evidence suggest an in-
volvement of perturbed D-ser transmission in schizophrenia
(Ross et al. 2006; Bendikov et al. 2007; Verrall et al. 2007; Sacchi
et al. 2008; Labrie et al. 2009). Indeed, single polymorphisms
for SR and DAAO have been linked to schizophrenia
(Schumacher et al. 2004; Detera-Wadleigh and McMahon
2006; Pollegioni and Sacchi 2010). In rodents, genetic loss
of DAAO activity reverses schizophrenia-like phenotypes
(Almond et al. 2006; Labrie et al. 2010). Coadministration in
D-ser with conventional neuroleptics partially
ameliorated the negative and cognitive symptoms of schizo-
phrenia in certain though not all studies (Tsai et al. 1998;
Heresco-Levy et al. 2005). Noteworthy, Kantrowitz et al. (2010)
have reported recently that administration of high doses of
D-ser alone (>60 mg/kg/day) in a 4-week trial displays
effectiveness in treatment of both persistent symptoms and
neurocognitive functions in antipsychotics-stabilized patients
with schizophrenia or schizoaffective disorder. Although yet to
be confirmed, the results already opened novel perspectives for
treatment of refractory symptoms in schizophrenia by D-serine-
based therapy. The demonstration that
activity of NMDARs and long-term synaptic plasticity supports
therapeutic strategies targeting
schizophrenia and other brain disorders involving either under
or over activity of cortical populations of NMDARs.
D-ser controls the
D-ser in the treatment of
INSERM, Conseil Re ´ gional d’Aquitaine, Agence Nationale pour
la Recherche (to J.P.M.); Human Frontier Science Program (to
S.H.R.O.); Institut de Recherches Servier (to J.P.M. and S.H.R.O.).
L.P. and S.S. were supported by grants from Fondo di Ateneo
per la Ricerca (Universita ` dell’Insubria) and from MIUR prot.
We thank Dr Philippe Ciofi and Vincent Ducourneau for their technical
assistance and advices in immunohistochemistry.
We thank also the Bordeaux Imaging Center for the training and
assistance in using the acquisitions systems. Conflict of Interest : None
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