Báo cáo khoa học: acids in the brain: D-serine in neurotransmission and neurodegeneration ppt

MINIREVIEW
D-Amino acids in the brain: D-serine in neurotransmission
and neurodegeneration
Herman Wolosker, Elena Dumin, Livia Balan and Veronika N. Foltyn
Department of Biochemistry, B. Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel
N-methyl d-aspartate receptors (NMDARs) are key
excitatory neurotransmitter receptors in the brain and
are involved in many physiological processes, including
memory formation, synaptic plasticity and develop-
ment [1]. The NMDARs are composed of multiple
subunits and their activity is regulated by numerous
mechanisms, including different ligands and interacting
proteins [2]. The NMDARs display high permeability
to Ca
2+
, which is known to play a central role in syn-
aptic plasticity and many signal transduction mecha-
nisms [1]. NMDAR overstimulation promotes
neurotoxicity and is implicated in several pathological
conditions, such as stroke and neurodegenerative dis-
eases [3].
The NMDARs are unique in their requirement for
more than one agonist to operate. Glutamate, the main
NMDAR agonist, does not activate the receptors unless
a co-agonist binding site located at the NR1 subunit is
occupied [4,5]. d-Serine, an unusual d-amino acid pres-
ent in mammalian brain, is now recognized as a physio-
logical ligand of the NMDAR co-agonist site,
mediating several NMDAR-dependent processes [6–15].
At first, the NMDAR co-agonist site was thought
to be occupied by glycine. Hence, the co-agonist site

is also generally referred to as the ‘glycine site’. In
addition, to be essential for NMDAR activity, the
co-agonist site exerts neuromodulatory roles. Thus,
co-agonist binding increases the receptor’s affinity for
glutamate [16], decreases its desensitization [17] and
promotes NMDAR turnover by internalization [18].
Since its discovery, the role of the co-agonist site in
regulating the activity of the NMDAR has been
Keywords
D-serine; gliotransmitter; glutamate;
glycine;
L-serine; neurodegeneration;
neurotransmission; neurotoxicity;
NMDA receptor; schizophrenia;
serine racemase
Correspondence
H. Wolosker, Department of Biochemistry,
B. Rappaport Faculty of Medicine, Technion-
Israel Institute of Technology, Haifa 31096,
Israel
Fax: +972 4 8295384
Tel: +972 4 8295386
E-mail:
(Received 30 January 2008, revised 14 April
2008, accepted 22 May 2008)
doi:10.1111/j.1742-4658.2008.06515.x
The mammalian brain contains unusually high levels of d-serine, a d-amino
acid previously thought to be restricted to some bacteria and insects. In the
last few years, studies from several groups have demonstrated that d-serine
is a physiological co-agonist of the N-methyl d-aspartate (NMDA) type of

glutamate receptor – a key excitatory neurotransmitter receptor in the
brain. d-Serine binds with high affinity to a co-agonist site at the NMDA
receptors and, along with glutamate, mediates several important physio-
logical and pathological processes, including NMDA receptor transmission,
synaptic plasticity and neurotoxicity. In recent years, biosynthetic, degrada-
tive and release pathways for d-serine have been identified, indicating that
d-serine may function as a transmitter. At first, d-serine was described in
astrocytes, a class of glial cells that ensheathes neurons and release several
transmitters that modulate neurotransmission. This led to the notion that
d-serine is a glia-derived transmitter (or gliotransmitter). However, recent
data indicate that serine racemase, the d-serine biosynthetic enzyme, is
widely expressed in neurons of the brain, suggesting that d-serine also has
a neuronal origin. We now review these findings, focusing on recent ques-
tions regarding the roles of glia versus neurons in d-serine signaling.
Abbreviations
ALS, amyotrophic lateral sclerosis; AMPA, a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; ISH, in situ hybridization; LTP, long-term
potentiation; NMDA, N-methyl
D-aspartate; NMDAR, N-methyl D-aspartate receptor.
3514 FEBS Journal 275 (2008) 3514–3526 ª 2008 The Authors Journal compilation ª 2008 FEBS
controversial. The high extracellular concentration of
glycine was assumed to saturate the co-agonist site
in vivo, with some supporting studies demonstrating
that this site is indeed saturated in tissue slices [19,20].
However, most studies now agree that the co-agonist
site is not saturated under resting conditions, indicat-
ing that co-agonist binding exerts a dynamic regulation
of the NMDARs [1,21].
As the dispute about the degree of saturation of the
co-agonist site became abated, a new controversy
emerged regarding the identity of the physiological

NMDAR co-agonist. Like glycine, d-serine is a high-
affinity ligand of the co-agonist site, displaying up to
threefold higher affinity than glycine [22,23]. At first,
d-amino acids like d-serine were not thought to exist
at significant quantities in eukaryotes. Therefore, in
contrast to glycine, d-serine was not viewed as a physi-
ological ligand of NMDARs.
The serendipitous discovery of large amounts of
endogenous d-serine in the brain, by Hashimoto et al.,
quickly changed this view [24]. Following this discov-
ery, studies from several laboratories have shown that
endogenous d -serine is a physiological regulator of
NMDARs through binding to the co-agonist site.
Endogenous d-serine has been implicated in several
physiological and pathological NMDAR-dependent
processes, including normal NMDAR transmission
and synaptic plasticity [6,7,10–12,14,15], cell migration
[9] and neurotoxicity [8,25–29].
A structural explanation for the selective effects of
d-serine on NMDARs comes from inspection of the
crystal structure of the binding core of the NR1 sub-
unit of NMDARs. d-Serine binds more tightly to the
receptor in comparison with glycine because it makes
three additional hydrogen bonds and displaces a water
molecule from the binding pocket [22]. There is also
unique selectivity for the d-isomer of serine, as the
hydroxyl group of l-serine interacts unfavorably in the
binding pocket [22].
D-Serine – a physiological co-agonist
of NMDARs

d-Serine is present at very high levels in the mamma-
lian brain and at a much lower concentration in the
peripheral tissues (Fig. 1). Brain d-serine accounts for
one-third of the l-serine and its levels are higher than
most essential amino acids [24,30]. In contrast to
l-amino acids, d-serine is not incorporated into pro-
teins or peptides, thus constituting a free amino acid
pool. Experiments of brain microdialysis show that
the extracellular concentration of endogenous d-serine
is twice that of glycine in the striatum and compara-
ble to the concentration of glycine in the cerebral
cortex [31].
Hashimoto et al. initially observed that d-serine was
enriched in rat forebrain areas, where NMDARs are
abundant [32]. Subsequently, immunohistochemical
studies carried out by the Snyder group (Fig. 1) dem-
onstrated that the regional distribution of d-serine in
rat brain co-localized almost perfectly with that
of NMDARs [33,34]. The density of d-serine is much
lower in the caudal part of the brain, including the
adult cerebellum and brainstem (Fig. 1). This is
because of the emergence of d-amino acid oxidase
in adult animals, which degrades endogenous d-serine
almost completely in these regions [34,35].
In contrast with d-serine, glycine immunoreactivity
is higher in the caudal areas of the brain, where the
density of NMDARs is lower [36]. The inverse local-
izations of d-serine and glycine led Schell et al. to
propose that endogenous d-serine was physically closer
to NMDARs than glycine.

d-Serine is enriched in protoplasmic astrocytes, rais-
ing the possibility that it is released from astrocytes
ensheathing the synapse to activate neuronal NMDARs
[33,34]. d-Serine was subsequently shown to be present
also in neurons, where the d-serine biosynthetic
enzyme, serine racemase, is robustly expressed, indicat-
ing that d-serine also has a neuronal origin [25,37–41].
A more direct demonstration that d-serine is a phys-
iological NMDAR co-agonist arose from experiments
that employed d-serine metabolic enzymes to remove,
in a selective manner, endogenous d-serine from brain
slices and cultures, leaving the levels of glycine
unchanged. Using this strategy, endogenous d-serine
was shown to mediate a variety of physiological
NMDAR-dependent events.
In a pioneer study, Snyder, Mothet et al. depleted
endogenous d-serine from neural cultures by applying
d-amino acid oxidase, which specifically degrades
H
T
S
C
AON
OT
MOL
D-Serine
Fig. 1. Localization of D-serine in the rat brain. The highest D-serine
densities (white areas) are observed in the forebrain. AON, anterior
olfactory nuclei; C, cerebral cortex; H, hippocampus; MOL, mole-
cular layer of the cerebellum; OT, olfactory tubercule; S, striatum;

T, thalamus. Reproduced from Schell et al. [34].
H. Wolosker et al.
D-Serine in neurotransmission and neurodegeneration
FEBS Journal 275 (2008) 3514–3526 ª 2008 The Authors Journal compilation ª 2008 FEBS 3515
d-amino acids, but not l-amino acids. Depletion of
d-serine led to a 60% decrease in the spontaneous
activity attributed to the postsynaptic NMDAR,
whereas a-amino-3-hydroxy-5-methylisoxazole-4-propi-
onic acid (AMPA) responses were unaffected [10]. Sub-
sequent studies demonstrated that endogenous d-serine
is required for the NMDAR-mediated light-evoked
responses in the retina [6,12]. Likewise, along with glu-
tamate, endogenous d-serine mediates the long-term
potentiation of synaptic activity in the hippocampus, a
region associated with learning and memory
[7,14,15,42].
One concern with the experiments employing
d-amino acid oxidase to deplete d-serine is that the
enzyme displays a very low affinity for d-serine –
about 50 mm at physiological conditions [43]. As the
affinity of NMDARs to d-serine is at least five orders
of magnitude higher than that of d-amino acid oxi-
dase, it is conceivable that a significant fraction of
endogenous d-serine remains bound to the receptors.
Furthermore, some commercial preparations of
d-amino acid oxidase contain many impurities that
may negatively affect the tissue preparation, including
large quantities of d-aspartate oxidase, which quickly
degrades N-methyl d-aspartate (NMDA) itself [28].
To overcome some of the limitations of the d-amino

acid oxidase treatment, new enzyme preparations were
developed to allow direct comparison between the
effects of d-serine and glycine in stimulating
NMDARs. One of these new enzyme preparations is
the recombinant bacterial d-serine deaminase enzyme.
This enzyme displays both high affinity and high speci-
ficity to d-serine, and efficiently degrades it in organo-
typic hippocampal slices [28], neuronal cultures [25]
and retina preparations [6]. We found that depletion
of endogenous d-serine by d-serine deaminase virtually
abolished NMDA-elicited neurotoxicity in organotypic
hippocampal slices (Fig. 2). This indicates that d-ser-
ine, and not glycine, is the dominant co-agonist
required for the neuronal cell death elicited by
NMDAR stimulation in the hippocampus (Fig. 2).
Likewise, the essential role of d-serine for NMDAR
neurotoxicity was also observed in cortical slices
subjected to ischemic cell death induced by oxygen ⁄
glucose deprivation [8,26].
The dominant role of d-serine for NMDAR activity
observed in neurotoxicity experiments is also sup-
ported by electrophysiological experiments. d-Serine is
essential for the NMDAR-mediated light-evoked
responses in the rat retina, shown by Miller et al. to
contain endogenous d-serine [6,12,44]. A similar domi-
nant role for endogenous d-serine in NMDAR trans-
mission was observed in the supra-optic nucleus of the
hypothalamus [11]. In this study, Panatier et al. dem-
onstrated that a more efficient recombinant d-amino
acid oxidase preparation destroyed d

-serine from
hypothalamic slices and blocked the NMDAR
responses. By contrast, the degradation of endogenous
glycine by a glycine oxidase enzyme had no effect,
suggesting that d-serine, rather than glycine, is the
dominant NMDAR co-agonist in the supra-optic
nucleus [11].
The role of endogenous d-serine as the foremost
co-agonist of NMDARs, as suggested by some studies,
is at odds with the very high levels of extracellular
glycine [31]. In hippocampal organotypic slice cultures,
A B
C D
E F
Ctl
NMDA
NMDA + DsdA
(No D-serine)
(No D-serine)
NMDA + MK-801
NMDA +DNQ
DsdA
Fig. 2. The role of endogenous D-serine in
NMDAR-elicited neurotoxicity. The removal
of
D-serine by D-serine deaminase enzyme
(DsdA) completely prevented NMDA-elicited
cell death. (A) Control. (B) NMDA (500 l
M)
elicited robust cell death in all hippocampal

areas, as measured by propidium iodide (PI)
uptake. (C) Control treated with DsdA
(10 lgÆmL
)1
for 90 min). (D) Destruction of
D-serine by DsdA protected against NMDA-
elicited cell death. (E) The NMDA effect
was prevented by addition of the antagonist
MK-801. (F) The NMDA effect was not pre-
vented by addition of the AMPA receptor
antagonist, DNQX. Densitometric analysis of
PI uptake revealed almost complete neuro-
protection by the removal of endogenous
D-serine. Reproduced with slight
modifications from Shleper et al. [28].
D-Serine in neurotransmission and neurodegeneration H. Wolosker et al.
3516 FEBS Journal 275 (2008) 3514–3526 ª 2008 The Authors Journal compilation ª 2008 FEBS
the removal of endogenous d-serine completely
blocked the NMDAR-elicited neurotoxicity, even
though extracellular glycine was present at a level was
10-fold higher than d-serine [28]. Glycine alone was
very inefficient in promoting NMDAR neurotoxicity.
When d-serine was completely removed from the slice
cultures, the amount of glycine needed to cause maxi-
mal NMDAR neurotoxicity was two orders of magni-
tude higher than its dissociation constant from purified
NMDARs [28]. Likewise, in hypoglossal neurons,
exogenously applied d-serine was almost two orders of
magnitude more effective than glycine in stimulating
NMDAR responses [45].

Why is glycine much less efficient than d-serine in
slice preparations? Glycine and d-serine display com-
parable affinities to NMDARs, and therefore the dif-
ference in their functional efficiency may be related to
the availability of the two co-agonists at synaptic or
extra-synaptic sites. The synaptic glycine concentration
is efficiently regulated by a high-affinity glycine trans-
port that limits glycine access to NMDAR sites
[45,46]. Accordingly, the addition of a selective inhibi-
tor of the GlyT1 transporter potentiates NMDAR
responses [47] and elicits NMDAR neurotoxicity after
the endogenous d-serine has been enzymatically
destroyed [28].
In contrast to glycine, d-serine behaves as a poorly
transported analogue. Specific d-serine transporters
have not yet been identified, and neutral amino acid
uptake systems capable of transporting d-serine dis-
play only low-to-moderate affinity [48–50]. It is there-
fore conceivable that d-serine can more easily reach
synaptic or extrasynaptic NMDARs by evading the
neutral amino acid re-uptake systems. Nevertheless,
the relative contributions of d-serine versus glycine in
mediating NMDAR transmission are still largely
unknown. Further studies will be required to map the
relative contributions of d-serine and glycine for
NMDAR transmission in different brain regions.
The levels of d-serine are high in the cerebellum of
neonatal rats, decreasing to very low levels in the third
week of life as a result of the emergence of the
d-amino acid oxidase enzyme, which destroys endoge-

nous d-serine [34,35]. The transient presence of d-ser-
ine in the cerebellum coincides with the postnatal
cerebellar development, in which granule cells migrate
from the external to the internal granule cell layer in
an NMDAR-dependent manner [51]. Blockage of the
NMDAR at granule cells decreases the rate of migra-
tion [51]. Bergman glia cells, which contain high levels
of endogenous d-serine, serve as a scaffold for granule
cell migration. Endogenous d-serine, presumably
released by the Bergman glia, mediates the NMDAR-
dependent neuronal migration in the cerebellum [9].
As migrating granule cells do not make conventional
synaptic connections, the modulatory action of
glial-released d-serine reflects a novel mechanism for
neuromodulation [9].
Origin of brain D-serine
The role of d-serine as a possible regulator of
NMDARs was initially viewed with scepticism, or even
ignored, for d-amino acids were not thought to be syn-
thesized in mammals. The discovery of the biosynthetic
enzyme for d-serine paved the way for additional
advances in the field. We found that endogenous d-ser-
ine is synthesized from l-serine by serine racemase, a
brain-enriched enzyme [52–54]. Serine racemase
requires pyridoxal 5¢-phosphate as a cofactor and, in
addition to racemization, it de-aminates l-serine into
pyruvate and ammonia [52,55]. The enzyme is unique
among the pyridoxal 5¢-phosphate enzymes as a result
of its requirement for divalent cations and the
Mg.ATP complex for its activity [52,56–58].

The regional localization of serine racemase matches
those of endogenous d-serine, indicating a physiologi-
cal role in d -serine synthesis [53]. Preliminary reports
indicate that serine racemase knockout mice display an
80–90% decrease in brain d-serine levels, confirming
the role of serine racemase as the biosynthetic enzyme
for d-serine [59–62]. Serine racemase knockout animals
exhibit decreased NMDAR transmission, impaired
long-term potentiation of synaptic activity in the
hippocampus, and are more resistant to stroke damage
upon middle-cerebral artery occlusion [60,61]. These
preliminary reports support previous biochemical and
electrophysiological data, indicating that d-serine is
indeed a physiologically relevant endogenous co-
agonist of NMDARs.
Is D-serine a transmitter?
By adopting a more liberal conceptualization of a neu-
rotransmitter, Snyder and Ferris proposed that d-ser-
ine belongs to a new class of transmitters that only
partially fulfill the criteria previously used to define
classic neurotransmitters [63]. The existence of a bio-
synthetic pathway, a target receptor, an uptake system
and a degradative enzyme for d-serine favors the
notion that d-serine is indeed a neurotransmitter
(Table 1). Unlike classical chemical transmitters, how-
ever, d-serine was originally thought to be specifically
produced and released from astrocytes, suggesting that
d-serine is a glial-transmitter (also known as a glio-
transmitter) [33,34,64]. A boost to the notion that
H. Wolosker et al. D-Serine in neurotransmission and neurodegeneration

FEBS Journal 275 (2008) 3514–3526 ª 2008 The Authors Journal compilation ª 2008 FEBS 3517
d-serine is a gliotransmitter was recently provided by
Mothet et al. who demonstrated that cultured astro-
cytes are capable of the vesicular release of d-serine
[65]. In this study, AMPA receptor stimulation was
shown to promote the release of d-serine by exocyto-
sis, an effect blocked by cocanamycin A, an inhibitor
of the vesicular filling of neurotransmitters that blocks
the generation of the electrochemical proton gradient
across the vesicles [65]. In support for a possible vesic-
ular localization of d-serine, Pow et al. observed vesic-
ular-like structures containing d-serine in astrocytes in
situ, which may correspond to synaptic-like vesicles
[66]. The question remains, however, whether the vesic-
ular pathway in cultured astrocytes surpasses nonvesic-
ular forms of d-serine release and whether the
vesicular release of d-serine occurs in more physiologi-
cal preparations or in vivo.
In order to function as a gliotransmitter, d-serine
actions should depend on an intimate relationship
between astrocytes and neurons. In an elegant study,
Oliet et al. found that NMDAR transmission in the
supra-optic nucleus depends on the degree of astrocytic
coverage of neurons [11]. The neuronal centers in the
supra-optic nucleus undergo an extensive reduction of
astrocytic ensheathing of its neurons and synapses
under conditions such as lactation. Using this model,
the authors showed that lactating rats display reduced
NMDAR activity compared with virgin rats as a result
of reduced levels of d-serine release. The data indicate

that variations in the astrocytic environment of neu-
rons and synapses play a prominent role in the post-
synaptic control of excitatory neurotransmission by
releasing d-serine [11].
Key to the hypothesis that d-serine is a gliotransmit-
ter is the notion that the electrophysiological effects of
d-serine should be attributable to astrocytic rather
than neuronal release of d-serine. Most studies demon-
strating a role for d-serine in mediating NMDAR
activity attributed its effects solely to glial d-serine and
overlooked a possible neuronal origin. Although glial
d-serine is prominent, a number of recent studies have
reported the presence of d-serine also in neurons.
Thus, purified neuronal cultures were recently shown
to synthesize large amounts of d-serine [25]. d-Serine
was also identified in situ by immunohistochemistry in
neurons of the nervous system (Fig. 3), including the
cerebral cortex [25,39], some nuclei of the hindbrain
[38,39,66] and in ganglion cells of the retina [67].
Table 1. Some transmitter-like properties of brain D-serine. ASCT, alanine, serine, cysteine and thrionine transporter.
D-Serine Selected references
Occurrence Enriched in the mammalian brain and in some insects [24,30]
Target receptor NMDAR [10,22,93]
Actions Modulates NMDAR transmission, long-term potentiation of synaptic activity,
NMDAR-elicited neurotoxicity and NMDAR-dependent cell migration
[6–12,14,15,25–29]
Biosynthesis Serine racemase [52–54,56–58,94,95]
Metabolism
D-amino acid oxidase enzyme and b-elimination catalyzed by serine racemase [34,55,96]
Transport Neutral amino acid transporters; Asc-1 in neurons and ASCT-like in astrocytes [49,50,97,98]

Release Vesicular and nonvesicular release modes described in neurons and astrocytes [25,50,65,99,100]
D-ser
Fig. 3. D-Serine localizes to neurons and
astrocytes in the brain. Staining for
D-serine
was performed in pyramidal neurons of
layer V of the cerebral cortex and in astro-
cytes in the corpus callosum of a P9 rat.
The lower panels depict double-labeling
immunofluorescence for
D-serine (labeled
for SR in the original publication) and a
neuronal nucleus marker (NeuN) in layer VI
of the cerebral cortex of a P9 rat. Repro-
duced with slight modifications from
Kartvelishvily et al. [25].
D-Serine in neurotransmission and neurodegeneration H. Wolosker et al.
3518 FEBS Journal 275 (2008) 3514–3526 ª 2008 The Authors Journal compilation ª 2008 FEBS
Recently, Puyal et al. showed that d-serine displays a
developmental glia-to-neuron switch. In the vestibular
nuclei of young rats, d-serine is predominantly glial,
whereas in adult rats, d-serine is exclusively present in
neurons in these regions [38].
Neuronal D-serine in NMDAR regulation
Is there a role for neurons in synthesizing and releas-
ing d-serine? Although present at a lower level than in
astrocytes, d-serine is detectable in pyramidal neurons
of the cerebral cortex in situ (Fig. 3) [25,39]. Originally
regarded as an elusive or nonimportant source of
d-serine, the extent of the neuronal pool of d-serine

became apparent when we re-investigated the expres-
sion of serine racemase using new antibodies [25]. We
observed widespread and prominent neuronal serine
racemase in situ, especially in the cerebral cortex and
hippocampal formation, in which neuronal serine race-
mase predominates (Fig. 4A–C). Furthermore, recent
studies indicate that cultured neurons contain both ser-
ine racemase mRNA and protein, and catalyze the
synthesis of d-serine to levels comparable to that
observed with astrocytes [25,40,41]. Neuronal staining
for serine racemase was also recently observed in
ganglion cells of the retina [67].
The neuronal expression of serine racemase was con-
firmed by in situ hybridization (ISH), which revealed
prominent serine racemase mRNA in neurons of the
brain [41] and in neuronal ganglion cells of the retina
[67]. Like the immunohistochemistry for serine race-
mase, the ISH of rat brain shows striking neuronal
predominance [41]. The neuronal-like distribution of
serine racemase mRNA in the hippocampus is evident,
AB C
D
E
F
G
Fig. 4. Localizations of serine racemase protein and mRNA in the brain. (A) Staining for serine racemase in the cerebral cortex (Ctx) of a P7
rat (layers IV–VI). (B) Staining of neurons in the stratum pyramidale (Pyr) of the CA1 region of the hippocampus. (C) Staining for serine race-
mase in the pyramidal cell layers and dentate gyrus of the hippocampus. (D) In situ hybridization (ISH) for serine racemase in the hippocam-
pus of adult mice, showing the highest serine racemase mRNA levels in pyramidal cell and dentate gyrus layers (saggital image series
392945, Srr_110, Allen Brain Atlas). (E) ISH of adult mouse brain (saggital image series 392945, Srr_110, Allen Brain Atlas). (F) Dark-field ISH

for serine racemase in the hippocampus of adult mice using a
33
P-labelled RNA probe and silver grain emulsion (saggital image 38687, Gen-
sat project). (G) Dark-field ISH for serine racemase in the hippocampus of adult mice (coronal image 36854, Gensat project). (A–C) Repro-
duced with slight modifications from Kartvelishvily et al. [25]. (D–E) ISH images from the Allen Institute of Brain Science [101,102], and the
Gensat project [103]. bs, brainstem; cb, cerebellum; cc, corpus callosum; ctx, cerebral cortex; DG, dentate gyrus; H or Hipp, hippocampus;
ob, olfactory bulb; st, striatum.
H. Wolosker et al.
D-Serine in neurotransmission and neurodegeneration
FEBS Journal 275 (2008) 3514–3526 ª 2008 The Authors Journal compilation ª 2008 FEBS 3519
with little or no serine racemase message in astrocytes
at the corpus callosum, as revealed by ISH from both
the Allen Institute for Brain Science and the Gensat
project (Fig. 4D–G).
Does neuronal d-serine activate NMDARs? We
found that endogenous d-serine released by neuronal
cultures lacking significant levels of astrocytes mediates
a considerable fraction of NMDAR-elicited neurotox-
icity [25]. Like astrocytes, cultured neurons release
d-serine in a regulated manner, involving ionotropic
glutamate receptor stimulation and depolarization by
KCl [25]. In contrast to that previously reported with
cultured astrocytes [65], however, the neuronal d-serine
was not released through exocytosis of synaptic vesi-
cles under our experimental conditions [25]. It remains
to be established whether neuron-derived d-serine
affects normal NMDAR transmission and if neurons
indeed release d-serine in more physiological prep-
arations.
In light of the widespread expression of serine race-

mase in forebrain neurons, which lack significant levels
of d-amino acid oxidase, one would predict that d-ser-
ine should be present in all neurons. A few studies
detected the presence of d-serine in some neuronal
populations in situ, including the pyramidal neurons of
the cerebral cortex [25,38,39]. Neuronal d-serine, how-
ever, is scarcely seen in most studies. We speculate that
this may be attributed to technical difficulties or to
low sensitivity of the immunohistochemical methods to
detect d-serine. Being a small amino acid, d-serine
may be poorly fixed by the commonly used fixatives,
or even released from cells during the perfusion of the
brain. In this framework, it is conceivable that the
antibodies against d-serine miss many neuronal popu-
lations that contain significant levels of d-serine.
Many questions remain to be solved regarding the
relative roles of glia versus neurons in the synthesis
and release of d-serine. In the original model of d-ser-
ine signaling, d-serine was thought to be exclusively
released from astrocytes. The predominance of serine
racemase expression in neurons led us to propose an
alternative model of d-serine signaling, in which d-ser-
ine may be released from both neurons and astrocytes
(Fig. 5). This model assumes that the neuronal serine
racemase enzyme is active towards d-serine synthesis
and, like astrocytes, neurons release d-serine in a regu-
lated manner (Fig. 5).
The notion that neurons play a role in d-serine sig-
naling does not exclude a role of glia in releasing
d-serine, as d-serine is clearly enriched in protoplasmic

astrocytes in the forebrain [33,34]. One possibility is
that the higher level of d-serine in astrocytes reflects
the glial uptake of d-serine synthesized and released by
neurons. In this case, one would expect that d-serine
Fig. 5. Proposed roles of glia and neurons in D-serine signaling. The scheme depicts two modes of D-serine release. A glia to neuron D-ser-
ine flux would be achieved through activation of glial AMPA receptors by glutamate (reaction 1) [34]. This leads to the release of astrocytic
D-serine, possibly from a vesicular pool [65], to activate neuronal NMDARs (reaction 2). Because serine racemase (SR) occurs predominantly
in neurons [25,41], astrocytes may obtain
D-serine by re-uptake from the extracellular medium. Alternatively, the higher ability of astrocytes
to synthesize
L-serine from glucose [69] might also allow the synthesis of D-serine by some astrocytes containing serine racemase; the rela-
tive importance of each pathway leading to astrocytic accumulation of
D-serine is unknown. A neuron to glia D-serine flux would be achieved
by the release of
D-serine from neurons, presumably by membrane depolarization (reaction 3) [25]. Released D-serine will activate NMDARs
or be taken up by astrocytes (reaction 4). It is not clear whether neuronal
D-serine synthesis and release occur at presynaptic or postsynaptic
sites. Because neurons are mostly devoid of the ability to synthesize
L-serine from glucose [69], they should rely on the export of L-serine
from astrocytes (reaction 5) [70].
D-Serine in neurotransmission and neurodegeneration H. Wolosker et al.
3520 FEBS Journal 275 (2008) 3514–3526 ª 2008 The Authors Journal compilation ª 2008 FEBS
uptake by astrocytes in vivo will be more efficient than
in neurons or, alternatively, that the d-serine half-life
would be longer in astrocytes. Nevertheless, experi-
mental data demonstrating specific vesicular or non-
vesicular release of d-serine from astrocytes in brain
slices or in vivo are still lacking.
The levels of l-serine relative to serine racemase
expression will also influence the distribution of d-ser-

ine. l-Serine can be synthesized from either glycine or
glucose or be obtained by uptake from the extracellular
medium [68,69]. It is known that, along with many
growth factors, astrocytes release l-serine to neurons
[70,71]. Astrocytes have a higher l-serine content and
can synthesize it from the glycolytic intermediate
3-phosphoglycerate, an ability that neurons apparently
lack [72]. The importance of the 3-phosphoglycerate
pathway for the synthesis of d-serine arose from the
observation that children exhibiting 3-phosphoglycerate
dehydrogenase deficiency display severe neurodevelop-
mental problems associated with lower levels of d-serine
in the cerebrospinal fluid [73]. Thus, the ability of astro-
cytes to synthesize l-serine by the 3-phosphoglycerate
pathway might allow higher synthesis of d-serine, even
if the expression of serine racemase in astrocytes is lower
than in neurons. On the other hand, neuronal synthesis
of d-serine will require conversion of glycine into l-ser-
ine by the serine hydroxymethyltransferase enzyme [74]
or uptake of l-serine from the extracellular medium.
Serine racemase knockout mice will be a valuable tool
to ascertain definitively the cellular origin of d-serine.
In light of the new data indicating a neuronal source of
d-serine, serine racemase knockout mice will be useful
in defining the specificity of serine racemase and d-ser-
ine antibodies previously employed, hopefully providing
a more definitive answer as to whether d-serine origi-
nates from neurons or astrocytes, or from both. Indeed,
a preliminary study by Mori et al. demonstrated, using
serine racemase knockout mice as controls, that serine

racemase is present mainly in neurons [62].
D-Serine in disease
As well as being important for normal NMDAR trans-
mission, NMDAR-dependent plasticity and develop-
mental processes (Fig. 6), d-serine signaling
dysregulation might also be involved in the NMDAR
dysfunction that occurs in several pathologies, includ-
ing neuro-psychiatric and neurodegenerative diseases
(Fig. 6).
An important pathological aspect of d-serine signal-
ing relates to NMDAR hypofunction thought to occur
in schizophrenia [75]. NMDA antagonists, such as
phencyclidine, induce schizophrenic-like symptoms in
healthy volunteers, and precipitate thought disorder
and delusions in schizophrenia patients [75,76]. In
mice, d-serine antagonizes the stereotypical behavior
and ataxia caused by NMDAR antagonists [77]. Mice
expressing lower levels of the NMDAR1 (NR1) sub-
unit display behavioral abnormalities, including
increased motor activity and stereotypy, and deficits in
social and sexual interactions, which are ameliorated
by conventional antipsychotic treatment [78].
Based on the NMDA hypofunction hypothesis, sev-
eral clinical trials were carried out to evaluate the effi-
cacy of stimulation of NMDAR in schizophrenia. The
administration of d-serine greatly ameliorated the posi-
tive, negative and cognitive symptoms of schizophrenia
when associated with conventional neuroleptics
[79–81]. Currently, five additional clinical trials are
evaluating the effects of d-serine administration in

schizophrenia in larger patient groups, which include
both phase II and phase III studies.
In addition to being a promising pharmacological
treatment for schizophrenia, a number of recent stud-
ies indicate that the level of endogenous d-serine may
also be altered in the disease. Schizophrenic patients
display a higher ratio of l-serine to d-serine in the
blood and cerebrospinal fluid [82–84]. The possible
involvement of d-serine in schizophrenia was also
highlighted by genetic studies showing polymorphisms
in the genes of serine racemase [85] and of the d-serine
metabolic enzyme, d-amino acid oxidase [86]. Confir-
mation of the above studies in larger populations will
be important to ascertain the role of endogenous d-ser-
ine in the pathophysiology of schizophrenia.
Fig. 6. Multitude of D-serine functions. D-Serine has been impli-
cated in several physiological NMDAR-dependent processes, includ-
ing normal transmission, synaptic plasticity and cell migration in the
developing cerebellum.
D-Serine dysregulation may also play patho-
logical roles in schizophrenia, ageing and acute and chronic
neurodegeneration (see the text for references).
H. Wolosker et al.
D-Serine in neurotransmission and neurodegeneration
FEBS Journal 275 (2008) 3514–3526 ª 2008 The Authors Journal compilation ª 2008 FEBS 3521
Is d-serine dysregulation linked to cognitive deficits?
The long-term potentiation (LTP) of the synaptic
activity in the hippocampus has been thought to play
a role in memory formation [87]. The role of endo-
genous d-serine in LTP [7,14,15,42] raises the possibility

that d-serine dysfunction might cause cognitive deficits.
Although this possibility has not been directly investi-
gated, aged rats display a sharp decrease in hippocam-
pal d-serine and serine racemase expression [42]. This
is associated with impaired LTP, which is reversed by
the addition of exogenous d-serine in aged rats [7].
By contrast, in young rats, LTP is not enhanced by
exogenous d-serine. Thus, it is possible that the LTP
impairment observed in aged rats is caused by specific
deficits in local d-serine synthesis.
The overproduction or excessive release of glutamate
has been widely implicated in a large number of acute
and chronic degenerative diseases. The harmful effects
of excessive glutamate occur mainly through activation
of the NMDARs and consequently by massive calcium
influx into the cell [1]. NMDAR over-activation is the
main culprit in the cell death that occurs following
stroke and in neurodegenerative diseases [3].
Blockers of NMDARs are neuroprotective in animal
models of stroke, but they were not well tolerated in
clinical trials because of the side effects caused by
NMDAR blockage, such as hallucinations [88,89].
Recently, low-affinity NMDAR inhibitors, like
memantine, have been proposed as an alternative to
high-affinity NMDAR blockers, and are indeed well
tolerated by patients [88,89]. Similarly to low-affinity
NMDAR antagonists, serine racemase inhibitors offer
a more gentle approach to decrease NMDAR activa-
tion, and are likely to be better tolerated than high-
affinity antagonists. In this framework, selective serine

racemase inhibitors provide a new strategy to prevent
stroke damage and cell death in neurodegenerative dis-
eases.
Excessive production or release of d-serine may also
be involved in chronic neurodegeneration. The levels
of d-serine and its biosynthetic enzyme, serine race-
mase, are greatly increased in the spinal cord of
patients with familiar and sporadic forms of amyo-
trophic lateral sclerosis (ALS) [27]. Although the
motoneuronal cell death in ALS is widely attributed to
excessive AMPA receptor stimulation [90], a recent
study indicates that endogenous d-serine mediates
motoneuron cell death by excessive stimulation of
NMDAR in the spinal cord of ALS mice [27]. In ALS
transgenic mice harboring the G93A mutation in
superoxide dismutase 1, activated microglia seem to be
the main source of spinal d-serine, constituting a
potential therapeutic target for ALS [27]. Activation of
microglia by inflammatory stimuli induces overexpres-
sion of serine racemase, an effect mediated by the
c-Jun terminal kinase [27,91]. Additionally, overexpres-
sion of the G39A mutant, superoxide dismutase 1, pro-
motes the upregulation of serine racemase in a c-Jun
terminal kinase-independent manner [27]. Removal of
endogenous d-serine from spinal cord cultures of ALS
transgenic mice protects the motoneurons against
NMDAR-mediated cell death, linking d-serine to
motoneuron degeneration [27]. The overproduction of
d-serine by glia in ALS fits the notion that glial activa-
tion ⁄ dysfunction plays a role in the disease [92]. In this

context, inhibitors of serine racemase may provide a
new neuroprotective strategy against ALS.
Conclusion
d-Serine is now widely recognized as an important
player in NMDAR transmission and in pathologies
linked to NMDAR dysfunction. Whether or not d-ser-
ine satisfies all the criteria for a transmitter, its role in
regulating NMDARs indicates an important physio-
logical role. There is still much to be learned regarding
the regulation of d-serine signaling, including its bio-
synthesis regulation and mechanisms of release. While
the experimental data so far favor a role of d-serine as
a transmitter, many of the effects previously attributed
to astrocytic d-serine release may also be caused by
neuronal d-serine. Furthermore, it is unclear whether
d-serine is physiologically released in a tonic manner
or in a fast and activity-dependent manner. Further
studies will be required to define the release pathways
for d-serine from both neurons and astrocytes, and to
clarify their relative contributions in d-serine-mediated
NMDAR signaling.
Acknowledgements
HW is supported by a grant from Israel Science Foun-
dation.
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