Genetic inactivation of D-amino acid oxidase enhances
extinction and reversal learning in mice
Viviane Labrie,1,2,6Steven Duffy,1Wei Wang,3Steven W. Barger,3,4
Glen B. Baker,5and John C. Roder1,2
1Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto M5G 1X5, Canada;2Institute of Medical Science,
University of Toronto, Toronto M5S 1A8, Canada;3Department of Neurobiology and Developmental Sciences,
University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA;4Department of Geriatrics, University of
Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA;5Neurochemical Research Unit and Bebensee
Schizophrenia Research Unit, Department of Psychiatry, University of Alberta, Edmonton T6G 2G3, Canada
Activation of the N-methyl-D-aspartate receptor (NMDAR) glycine site has been shown to accelerate adaptive forms of
learning that may benefit psychopathologies involving cognitive and perseverative disturbances. In this study, the effects
of increasing the brain levels of the endogenous NMDAR glycine site agonist D-serine, through the genetic inactivation
of its catabolic enzyme D-amino acid oxidase (DAO), were examined in behavioral tests of learning and memory. In the
Morris water maze task (MWM), mice carrying the hypofunctional Dao1G181Rmutation demonstrated normal acquisition
of a single platform location but had substantially improved memory for a new target location in the subsequent reversal
phase. Furthermore, Dao1G181Rmutant animals exhibited an increased rate of extinction in the MWM that was similarly
observed following pharmacological administration of D-serine (600 mg/kg) in wild-type C57BL/6J mice. In contextual
and cued fear conditioning, no alterations were found in initial associative memory recall; however, extinction of the
contextual fear memory was facilitated in mutant animals. Thus, an augmented level of D-serine resulting from reduced
DAO activity promotes adaptive learning in response to changing conditions. The NMDAR glycine site and DAO may
be promising therapeutic targets to improve cognitive flexibility and inhibitory learning in psychiatric disorders such as
schizophrenia and anxiety syndromes.
The N-methyl-D-aspartate receptor (NMDAR) has an important
role in excitatory neurotransmission and contributes to numerous
brain processes, including synaptic plasticity, learning, and mem-
ory formation (Nicoll 2003). Activation of NMDARs requires
membrane depolarization in addition to concurrent binding of
glutamate to NMDAR2 (NR2) and glycine to the NMDAR1 (NR1)
subunit (Johnson and Ascher 1987; Clements and Westbrook
1991). D-serine has also been shown to be an endogenous co-
agonist for the NR1 glycine site, acting with high selectivity and
a potency similar to or greater than that of glycine (Matsui et al.
1995). In the brain, the localization of D-serine closely resembles
that of NMDARs (Schell et al. 1997), and D-serine has been
reported to be the predominant physiologic co-agonist for the
maintenance of NMDAR-mediated currents in the hippocampus,
retina, and hypothalamus (Mothet et al. 2000; Yang et al. 2003).
Moreover, in vivo studies have demonstrated that the NMDAR
glycine site is not saturated at the synapses of several brain regions
(Fuchs et al. 2005). Consequently, increasing D-serine levels may
modulate neurotransmission and behavioral responses reliant on
The NMDAR glycine site has been implicated in the patho-
physiology and treatment of a number of psychiatric conditions
(Coyle and Tsai 2004; Millan 2005). Blockade of the NMDAR with
noncompetitive antagonists like phencyclidine results in the
production and exacerbation of schizophrenic-like symptoms in
humans and animals (Javitt and Zukin 1991; Krystal et al. 1994).
Genetic studies have associated genes that mediate D-serine syn-
thesis and degradation with a vulnerability to schizophrenia, and
levels of D-serine are decreased in the CSF and serum of schizo-
phrenic patients (Chumakov et al. 2002; Hashimoto et al. 2003,
2005; Schumacher et al. 2004; Morita et al. 2007). These observa-
tions prompted clinical trials with direct and indirect activators of
the NMDAR glycine site, including D-serine, and improvements
were revealed when these compounds were added to conventional
antipsychotic regimes, particularly with the negative and cogni-
tive symptoms of schizophrenia (Tsai et al. 1998; Coyle and Tsai
2004; Heresco-Levy et al. 2005). Furthermore, altered NMDAR
activation has also been shown to affect extinction, a learning
process that may be of benefit in anxiety illnesses, such as post-
traumatic stress syndrome and obsessive-compulsive disorder
(Davis et al. 2006). In rodents, extinction was shown to be
impaired following inhibition of NMDARs in contextual fear
conditioning, inhibitory avoidance, and eyeblink conditioning
tasks (Kehoe et al. 1996; Lee and Kim 1998; Szapiro et al. 2003). In
contrast, the partial NMDAR agonist D-cycloserine facilitated the
extinction of fear memories in rodents and individuals with
phobias and other anxiety disorders (Ressler et al. 2004; Ledger-
wood et al. 2005; Norberg et al. 2008). Thus, the NMDAR glycine
site and its related modulatory proteins may be important targets
for the amelioration of psychopathologies involving cognitive
dysfunction and maladaptive behaviors.
Endogenous levels of D-serine in the brain are regulated by its
catabolic enzyme, D-amino acid oxidase (DAO); by the D-serine
synthesis enzyme, serine racemase (Srr); and by neuronal and glial
transporters (Foltyn et al. 2005; Martineau et al. 2006). Agents
targeting such proteins may prove to be an effective method of
increasingcerebral D-serine and occupancy of the NMDAR glycine
site, which could overcome the difficulties D-serine and similar
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Article is online at http://www.learnmem.org/cgi/doi/10.1101/lm.1112209.
16:28–37 ? 2009 Cold Spring Harbor Laboratory Press
ISSN 1072-0502/09; www.learnmem.org
Learning & Memory
compounds have with penetrating the blood-brain barrier (Coyle
and Tsai 2004; Bauer et al. 2005). Inhibiting DAO function in the
brain is of particular interest as it would circumvent any nephro-
toxicity associated with high levels of systemic D-serine (Maekawa
et al. 2005a). DAO is a peroxisomal flavoprotein that at physio-
logical pH is highly selective for D-serine, and in the brain, DAO
is located predominantly in astrocytes (Mothet et al. 2000). An
inverse correlation between the brain distribution of DAO and D-
serine evincesthe efficacy of thisenzyme, withthe most abundant
DAO expression located in the D-serine-sparse hindbrain and
cerebellum (Schell et al. 1995; Moreno et al. 1999). To study the
effects of limiting DAO function, we tested a line of mice carrying
a single point mutation (G181R) that results in a complete lack of
DAO activity and consequently augmented D-serine in serum and
brain (Sasaki et al. 1992; Hashimoto et al. 1993). These mice have
previously been shown to exhibit an in vitro increase in NMDAR-
mediated excitatory postsynaptic currents in dorsal horn neurons
of the spinal cord and an in vivo elevation of cGMP that
is indicative of augmented NMDAR activity (Wake et al. 2001;
Almond et al. 2006). This demonstrates that reduced DAO
function is capable of augmenting NMDAR activation, and it
may follow that cognitive and extinction processes influenced by
NMDARs are enhanced in Dao1G181Rmutant mice. To investigate
this possibility, we assessed the effects of the Dao1G181Rmutation
on learning, memory, and extinction in Morris water maze
(MWM) and in contextual and cued fear conditioning paradigms.
D-serine measurements in the brains of Dao1G181Rmice
To determine the effect of the Dao1G181Rmutation on D-serine
levels in C57BL/6J mice, HPLC analysis was employed. D-serine
concentrations were found to be significantly elevated in
Dao1G181Rmutant animals (main effect of genotype; F(1,83) =
43.4, P < 0.001) compared with wild-type animals (Fig. 1). Higher
levels werefound in whole brain,whole cortex, hippocampus, and
especiallycerebellum (P< 0.05).In contrast,D-serinelevelsdidnot
differ in the prefrontal cortex and amygdala (P > 0.05). No changes
were detected when hippocampus was assayed for the concentra-
tion of other amino acids,includingaspartate, glutamate,L-serine,
glutamine, glycine, arginine, alanine, and GABA (P > 0.05) (data
not shown). A chemiluminescent assay measuring D-serine catab-
olism following exposure to the Rhodotorula gracilis DAO enzyme
was also used to quantify D-serine in the hippocampus (wild type:
3.7 6 0.1 nmol/mg protein; mutant: 4.8 6 0.2 nmol/mg protein)
and whole cortex (wild type: 2.5 6 0.2 nmol/mg protein; mutant:
3.8 6 0.2 nmol/mg protein) of Dao1G181Rmice. D-serine concen-
trations were again found to be elevated in mutant animals (main
effect of genotype: F(1,27)= 65.7, P < 0.001) in both of these brain
regions (P < 0.01).
The effects of a loss of DAO function in spatial reversal
learning and memory
Administration of exogenous D-serine has previously been shown
to improve spatial reversal learning in the MWM (Duffy et al.
2008). Consequently, we examined the effect of genetic inactiva-
tion of DAO and enhanced levels of endogenous D-serine in this
hippocampus-dependent behavioral task. Initially, a control ex-
periment was done where animals were placed into the MWM for
a 300-sec acclimatization period without the platform. This
verified that the Dao1G181Rmice did not have an inherent bias
for a quadrantlocation or alterations in swimming behavior(Table
1 , left column). Additionally, similar performance was observed
in a control experiment involving a visible platform session, indi-
cating comparable sensorimotor abilities and motivation (Table 1,
left column). Afterward, spatial acquisition and reversal learning
were tested in the MWM on a separate cohort of mice. The
Dao1G181Rmutation did not affect performance in a visible plat-
form sessionor duringthe7-dacquisitiontrainingphasewhenthe
platform was hidden, as demonstrated by a similar path length
and latency to reach the platform (Fig. 2A). Also, swim speed,
floating time, and thigmotaxis duration were not altered during
the acquisition trials or in the subsequent reversal training phase
(main effect of genotype in acquisition trials: swim speed: F(1,24)=
1.2, P = 0.3; floating time: F(1,24)= 0.5, P = 0.5; thigmotaxis time:
F(1,24)= 4.1, P = 0.1; in reversal trials: swim speed: F(1,24)= 0.2, P =
0.6; floating time: F(1,24)= 0.1, P = 0.8; thigmotaxis time: F(1,24)=
1.7, P = 0.2). Spatial memory in the acquisition probe, as measured
by the amount of time spent and number of crosses in an area 23
the platform diameter centered over its former location did not
differ between wild-type and mutant animals (P > 0.05; Fig. 2B).
Additionally, both genotypes spent more time (main effect of
platform location: F(1,24)= 44.1, P < 0.001) and made more crosses
over the target platform area (main effect of platform location:
F(1,24)= 42.7, P < 0.001) than the averaged analogous nontarget
areas (P < 0.05; wild-type nontarget: % time: 3.0 6 0.4, crosses:
1.9 6 0.3; mutant nontarget: % time: 2.7 6 0.4, crosses: 1.5 6 0.2).
A comparable performance between wild-type and mutant ani-
mals was also observed during reversal training, where the hidden
platform was switched to a new location (Fig. 2A). However,
memory for the new platform location was substantially improved
in Dao1G181Rmutant mice compared with wild-type animals in
the reversal probe. Mutant mice spent more time (main effect of
genotype: F(1,25)= 5.3, P < 0.05; genotype 3 platform location
interaction: F(2,50)= 7.9, P < 0.01) and had a greater number of
crosses (genotype 3 platform location interaction: F(2,50)= 4.4, P <
0.05; Fig. 2C). Wild-type and mutant mice both demonstrated
a preference for the reversal target area (main effect of platform
location: % time: F(2,50)= 32.3, P < 0.001; crosses: F(2,50)= 31.0, P <
0.001) compared with the acquisition target and the averaged
unbiased nontarget locations (P < 0.05), indicating that memory
for the reversal target location was present in both genotypes but
was particularly increased in mutant animals.
Dao1G181Rmutant (G181R/G181R) mice. HPLC was used to measure D-
serine levels in whole brain and isolated brain regions including whole
cortex, prefrontal cortex, hippocampus, amygdala, and cerebellum. n =
7–9 per group; *P < 0.05, **P < 0.01, ***P < 0.001 compared with wild-
type mice, within the same brain structure.
D-serine concentrations in the brains of wild-type (+/+) and
Extinction in Dao1G181Rmice
Learning & Memory
Modulation of extinction learning by a
lack of DAO activity
Considering that the Dao1G181Rmutant mice expressed a specific
improvement in reversal memory and the recent literature de-
scribing an enhancing effect of exogenous D-cycloserine on the
extinction of learned responses (Davis et al. 2006), the Dao1G181R
mice were examined in an extinction task in the MWM using
repeated probe trials. The extinction procedure employed an
experimentally naı ¨ve cohort of mice and was a separate experi-
ment from the reversal learning study. In the visible and acquisi-
tion phases, wild-type and mutant mice demonstrated a similar
performance, as measured by the path length and latency to find
the visible or hidden platform (Fig. 3A). In the first probe trial
conducted 24 h after the completion of acquisition training, wild-
type and mutant mice did not differ in the time spent or number
of crosses over a target area 23 the platform diameter, signifying
that the memory for the target platform was initially equivalent in
both genotypes (P > 0.05; Fig. 3B,C). In this probe trial, wild-type
and mutant mice also displayed a greater amount of time (main
effect of platform location: F(1,14)= 24.7, P < 0.001) and crosses in
the target location (main effect of platform location: F(1,14)= 23.4,
P < 0.001) than in the averaged analogous nontarget areas (P <
0.05; wild-type nontarget: % time: 3.0 6 0.6, crosses: 1.8 6 0.3;
mutant nontarget: % time: 2.4 6 0.4, crosses: 1.6 6 0.2). During
the extinction probe trials, Dao1G181Rmutant mice demonstrated
enhanced extinction rates compared with the wild-type animals.
The mutant animals spent less time (main effect of genotype:
F(1,14)= 3.7, P < 0.05) and made fewer crosses over the target
location (main effect of genotype: F(1,14)= 5.3, P < 0.05), partic-
ularly on the fourth and fifth probe repeat (days 7 and 9, P < 0.05;
Fig. 3B,C). Though extinction of the learned response occurred
earlier in the mutant mice, it was demonstrated across trials in
both genotypes (wild type: main effect of trial: % time: F(7,49)= 2.7,
P < 0.05, crosses: F(7,49)= 5.2, P < 0.001; mutant: main effect of
trial: % time: F(7,49)= 4.6, P < 0.001, crosses: F(7,49)= 6.4, P < 0.001).
Swim speed, floating, and thigmotaxis time did not differ between
genotypes during the extinction phase (main effect of genotype:
swim speed: F(1,14)= 0.8, P = 0.4, floating time: F(1,14)= 0.2, P = 0.7,
thigmotaxis time: F(1,14)= 0.2, P = 0.7). Additionally, comparisons
were made between mutant animals that received several extinc-
tion sessions and control mutant animals given a single probe trial
on day 9. A greater time (P < 0.05) and number of crosses in the
target area (P < 0.01) were observed in the control mutant mice,
indicating that Dao1G181Rmutant animals exhibit a facilitation in
extinction rather than an impairment in memory duration.
The effects of D-serine treatments on extinction learning
To confirm that a loss of DAO activity produces an increase in the
extinction of a learned response, D-serine treatments were given
during the probe sessions to wild-type C57BL/6J mice. In the first
probetrialfollowingacquisitiontraining,a similaramountof time
and number of crosses over an area 23 the platform diameter was
observed in saline-injected and D-serine-injected mice, suggesting
that the initial memory for the platform location was comparable
in each group (P > 0.05; Fig. 4A,B). Accordingly, both treatment
groups spent more time (main effect of platform location: F(1,15)=
22.6, P < 0.001) and made more crosses over the target area (main
effect of platform location: F(1,15)= 33.3, P < 0.001) than in the
averaged analogous nontarget locations during the first probe trial
(P < 0.05; saline nontarget: % time: 3.7 6 0.7, crosses: 1.7 6 0.3;
D-serine nontarget: % time: 3.2 6 0.5, crosses: 2.0 6 0.3). In the
extinction probe sessions, D-serine-treated animals displayed an
enhancement in extinction, as measured by the reduced time
spent (main effect of genotype: F(1,15)= 6.8, P < 0.05) and lower
frequency of crosses over the target platform area (main effect of
genotype: F(1,15)= 7.9, P < 0.05; Fig. 4A,B). Compared with saline-
treated mice, animals given D-serine had a reduction in the
amount of time spent in the target location on days 3, 5, 9, and
15 (P < 0.05), and crossed the platform area less often on days 9
and 15 (P < 0.05). Though greater in the D-serine-injected mice,
extinction was progressively demonstrated across probe sessions
4.3, P < 0.001, crosses: F(7,56)= 5.7, P < 0.001; D-serine: main effect
of trial: % time: F(7,49)= 6.7, P < 0.001, crosses: F(7,49)= 7.1, P <
0.001). Additionally, D-serine-treated animals that received a sin-
gle extinction session on day 9 had a greater percentage of time
(P < 0.01) and number of crosses over the target location (P <
0.001) compared with mice that received multiple extinction
sessions.This indicates thattheeffectsof D-serinewerespecifically
related to extinction rather than forgetting over the passage of
Control experiments examining performance in acclimatization and visible platform sessions
Acclimatization% time in area NEc
% time in area SE (AT/ ExT)c,d
% time in area SWc
% time in area NW (RT)c,d
Path length (m)
Swim speed (cm/s)
Floating (% time)
Thigmotaxis (% time)
Path length to target (m)
Latency to target (sec)
Swim speed (cm/sec)
Floating (% time)
Thigmotaxis (% time)
0.9 6 0.3
1.0 6 0.3
1.5 6 0.6
1.0 6 0.4
47.3 6 3.6
16.0 6 1.2
6.6 6 5.7
77.3 6 6.6
7.8 6 0.7
43.8 6 5.5
17.6 6 1.0
9.2 6 1.8
43.1 6 2.6
0.7 6 0.2
1.1 6 0.4
1.7 6 0.3
1.9 6 0.4
47.1 6 2.9
15.8 6 1.0
7.4 6 2.7
71.0 6 3.1
8.3 6 1.0
40.6 6 4.1
19.7 6 0.9
8.4 6 2.7
40.6 6 4.0
1.7 6 0.4
1.1 6 0.3
2.3 6 0.4
1.7 6 0.5
54.1 6 2.2
18.1 6 0.8
2.3 6 0.6
63.3 6 5.0
6.5 6 1.1
33.3 6 5.2
19.1 6 0.6
8.3 6 1.4
31.2 6 3.6
1.4 6 0.5
1.4 6 0.4
1.8 6 0.4
1.8 6 0.6
53.1 6 2.7
17.7 6 0.9
2.9 6 0.6
66.9 6 6.7
5.8 6 0.8
31.9 6 4.5
18.2 6 0.9
7.1 6 1.1
34.2 6 2.9
Data are expressed as mean 6 SEM, n = 7–10 per group. In the control experiments, no differences were found between genotype or drug treatment
groups (P > 0.05 for all behavioral variables). In the acclimatization experiment, animals did not preferentially spend more time in any platform area (main
effect of platform location: for Dao1G181Rmice: F(3,45)= 2.7, P = 0.1, for C57BL/6J mice: F(3,36)= 1.8, P = 0.2).
aControl experiments conducted for reversal and extinction studies with Dao1G181Rmice (Figs. 2, 3).
bControl experiments conducted for extinction study with D-serine treatments (Fig. 4).
cChance level for each platform location is 2.6% (the ratio of the target area to the total pool area).
dAT/ExT is the target platform in acquisition and extinction experiments, RT is the target platform in the reversal experiment.
Extinction in Dao1G181Rmice
Learning & Memory
time. In a control experiment conducted on a different cohort of
mice, saline-treated and D-serine-treated mice did not express
a quadrant bias or abnormalities in swimming behavior (Table 1,
right column). Furthermore, D-serine treatments did not affect
performance in a visible platform session (Table 1, right column),
indicating that D-serine does not impair motor coordination,
vision, or search motivation.
Absence of DAO activity affects extinction of
contextual fear memory
Dao1G181Rmice were examined in a fear-conditioning paradigm to
determine whether our results could extend to another hippo-
campus-dependent task with different sensory, motivational, and
performance requirements. In fear conditioning experiments, the
hippocampus is known to be required for the formation and
retrieval of context–fear associations, while the amygdala is
necessary for conditioning and recall of contextual and cued/tone
associations (Holland and Bouton 1999). An assessment of freez-
ing time in the training phase revealed that the Dao1G181R
mutation did not produce any nonspecific effects on fear condi-
tioning, as no differences in freezing time were observed during
the 2-min baseline activity period, the 30-sec auditory tone
exposure, or the 30-sec interval following shock presentation (P
> 0.05; Fig. 5A). In subsequent sessions, context– and tone–shock
associations were evaluated, in which contextual freezing re-
sponses were normalized to the baseline activity period, while
tone–freezing responses were relative to the pretone period and
assessed in an altered context. Wild-type and mutant mice re-
exposed 24 h later to the training context did not demonstrate
differing freezing responses, indicating a similar learning of the
context-shock association (P > 0.05; Fig. 5B). However, when the
animals were exposed to the context in repeated extinction trials,
the Dao1G181Rmutant mice demonstrated an enhanced extinction
rate (main effect of genotype: F(1,29)= 4.6, P < 0.05) that was
especially significant on the second exposure (day 3, P < 0.05; Fig.
5B). Contextual extinction across trials was present earlier in
mutant animals (main effect of trial: F(4,64)= 3.9, P < 0.01) but
also eventually occurred in wild-type animals (main effect of
trial: F(4,52)= 6.6, P < 0.001). Supporting the interpretation of
facilitated fear extinction, the control mutant group exposed to
the context only on day 7 showed a greater freezing time than
did mice given several extinction trials (P < 0.05). In contrast, the
tone–shock association and its extinction were not significantly
altered in Dao1G181Rmutant mice compared with wild-type
littermates (P > 0.05; Fig. 5C). Since altered pain sensitivity can
potentially influence performance in fear conditioning tests
(Clapcote et al. 2005), the tail-flick assay was used to examine
antinociceptive responses in the Dao1G181Rmice. Latencies fol-
lowing immersion in hot water were similar in wild-type and
mutant mice, suggesting an equivalent pain sensitivity in both
a target platform were examined in wild-type (+/+) and mutant (G181R/G181R) mice during a visible platform session (day 1), an acquisition training
phase (days 2–8), and a reversal training phase (days 10–12). In the acquisition (B) and reversal (C) probe trials, the percentage of time spent (left panels)
and the number of crosses (right panels) over the target area were measured in wild-type and mutant animals. The dashed line represents chance level,
corresponding to the ratio of the target area to the total pool area (2.6%). Platform locations include the reversal target (RT) in the NW quadrant, the
acquisition target (AT) in the SE quadrant, and the nontarget areas in the NE and SW quadrants. n = 11 wild types and 15 mutants; *P < 0.05, **P < 0.01
compared with wild-type mice within the same platform location; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with the reversal target within the same
Dao1G181Rmutant mice have improved reversal memory in the MWM task. The path length (A, left panel) and latency (A, right panel) to reach
Extinction in Dao1G181Rmice
Learning & Memory
genotypes (wild type: 14.6 6 1.6 sec, n = 17; mutant: 13.2 6 1.7
sec, n = 15; P > 0.05).
Diminished DAO function was associ-
ated with elevated levels of D-serine in
the brain and enhancements in reversal
memory and extinction learning in the
MWM and contextual fear conditioning
procedures. Moreover, pharmacological
administration of D-serine substantially
accelerated extinction of a learned spatial
response in the MWM. These findings
indicate that D-serine and decreased
DAO activity improves performance un-
der changing conditions, and predict
their therapeutic utility for the treatment
of psychiatric disorders characterized by
cognitive inflexibility and aberrant repet-
DAO inhibition may be a well-toler-
ated and effective means of modulating
D-serine levels and NMDAR glycine site
(Coyle and Tsai 2004; Maekawa et al.
2005a; Davis et al. 2006). Animals that
chronically lack DAO activity exhibit normal development, lon-
gevity, and reproductive potential (Konno and Yasumura 1983).
Furthermore, chronic administration of D-serine at therapeutic
doses has not been shown to produce adverse effects in humans
a target platform were evaluated in wild-type (+/+) and mutant (G181R/G181R) mice during the visible platform session (day 1) and acquisition training
phase (days 2–8). Multiple probe trials were performed on alternate days (1–15), allowing extinction to be examined in the Dao1G181Rwild-type and
mutant mice. In a control group of mutant mice, a probe trial was given only on day 9. During the probe trials, the time spent (B) and frequency of crosses
(C) over the target area were determined. Chance levels are depicted as a dashed line. n = 8 wild types, 8 mutants, and 6 mutant controls; *P < 0.05, **P <
0.01 compared with wild-type mice within the same day; #P < 0.05, ##P < 0.01 compared with mutant mice that received several extinction trials.
The Dao1G181Rmutation augments extinction responses in the MWM. The path length (A, left panel) and latency (A, right panel) to attain
treated with saline or D-serine (600 mg/kg) before each probe trial (alternate days 1–15), and
extinction was evaluated in these mice. Also, a control group of mice were administered D-serine
injections in the home cage (alternate days 1–7) and given a probe trial only on day 9. The amount of
time spent (A) and number of crosses (B) over the target area were determined for each treatment
group. The dashed line indicates chance level. n = 9 saline, 8 D-serine, and 7 D-serine controls; *P <
0.05, **P < 0.01 compared with saline-treated mice within the same day; ##P < 0.01, ###P < 0.001
compared with D-serine-treated mice that received several extinction trials.
D-serine treatment enhances extinction rates in the MWM. Male C57BL/6J mice were
Extinction in Dao1G181Rmice
Learning & Memory
(Tsai et al. 1998). However, under conditions that promote
excitotoxicity and neuroinflammation, D-serine can potentially
compromise neuronal survival through excessive NMDAR activa-
tion (Wu et al. 2004; Martineau et al. 2006), suggesting that
modest rises in D-serine, as observed following DAO reduction,
may be advantageous over dramatic increases. Though D-serine
elevations in Dao1G181Rmutant mice were shown to be greatest in
the cerebellum, as expected based on DAO expression patterns
(Moreno et al. 1999), increases were also demonstrated in the
hippocampus, an area crucial to the behavioral tasks studied
(Whishaw and Tomie 1997; Holland and Bouton 1999). Previous
reports have indicated more subtle D-serine increases in the
forebrain of Dao1G181Rmutant mice (Hashimoto et al. 1993), but
this difference may be related to background mouse strain. Impor-
tantly, reducedDAOactivityin mutantmice produced increases in
D-serine that enhanced NMDAR function (Hashimoto et al. 1993;
Wake et al. 2001; Almond et al. 2006) without producing func-
tional changes in [3H]-D-serine reuptake or alterations in the
expression of proteins relevant to NMDAR signaling, including
NR1, serine racemase, glycine transporter 1, and alanine-serine-
cysteine transporter 1 (Almond et al. 2006). This suggests a lack of
obvious compensatory changes in the Dao1G181Rmice. Hence,
these animals may be useful preclinical models for the study of
behavioral endophenotypes specifically related to diminished
This study is the first to demonstrate that inactivation of
DAO leads to improved behavioral flexibility in response to
changing environmental contingencies. In the reversal phase of
the MWM,memory forthe novelplatformlocationwas selectively
enhanced in Dao1G181Rmutant mice. Prior studies investigating
genetically modified and wild-type mice have attributed perfor-
mance in the reversal MWM task to the capacity for cognitive
flexibility, i.e., the ability to simultaneously inhibit a previously
acquired spatial navigation strategy and develop a new strategy
(Malleretet al.1999;Duffy etal. 2008).Deficitsin reversallearning
and perseveration have been reported in both rodents and
humans given NMDAR antagonists (Krystal et al. 2000; van der
Meulen et al. 2003; Andersen and Pouzet 2004), as well as in rats
with lesions to the fimbria-fornix or hippocampus (Whishaw and
Tomie 1997). Acute administration of D-serine, on the other hand,
has been shown to enhance reversal learning in the MWM in wild-
type mice (Duffy et al. 2008) and in rats treated with the NMDAR
antagonist phencyclidine (Andersen and Pouzet 2004). Addition-
ally, nitric oxide (NO) has been shown to promote DAO function
and suppress serine racemase activity, and antagonism of NO
synthase was found to rescue phencycline-induced impairments
in MWM reversal learning (Shoji et al. 2006; Wass et al. 2008). By
directly targeting DAO function, our study indicates that chron-
ically enhanced D-serine levels can also promote appropriate
reversal behaviors, and suggests that DAO inhibition may atten-
uate symptoms of cognitive inflexibility, such as those commonly
observed in patients with schizophrenia (Pantelis et al. 1999).
Indeed, a clinical study demonstrated that D-serine administra-
tion improved the performance of schizophrenic individuals in
the Wisconsin card sorting task, a widely used measure of
flexibility (Tsai et al. 1998).
Extinction is regarded as a distinct form of learning that acts
to suppress, but not erase, previously learned responses (Davis
et al. 2006). Like other forms of learning, extinction has been
shown to depend on glutamate NMDAR-associated signaling
pathways, gene expression, and protein synthesis (Davis et al.
2006). A growing body of evidence indicates that the NMDAR
glycine site may be particularly important in mediating the
extinction of fear memory (Davis et al. 2006; Norberg et al.
2008). The partial NMDAR agonist D-cycloserine has been shown
to facilitate fear extinction and exposure therapy, though it is not
yet clear whether the improvements are related to an increase
of NMDAR function during the extinction process (Ressler et al.
2004; Ledgerwood et al. 2005; Norberg et al. 2008). Here, we show
that elevated D-serine can accelerate extinction in spatial and fear-
based tasks. The improvements were demonstrated to be specifi-
cally related to an increase in extinction learning rather than to
a deficit in memory duration. Furthermore, the ability of acute
D-serine treatments to replicate these findings in wild-type mice
indicates this behavioral phenotype is not likely the result of
potential abnormalities due to increased D-serine levels during
development. Also, facilitated extinction in Dao1G181Rmutant
mice could not be attributed to changes in motivation or anxiety,
as no differences were observed in the incentive to find a visible
platform, or in behaviors indicative of anxiety, such as thigmo-
taxis and baseline freezing. Although we did not detect differing
responses in an assessment of antinociception, mutant mice have
previously been reported to have elevations in pain sensitivity
fear memory. Freezing responses in wild-type (+/+) and Dao1G181R
mutant (G181R/G181R) animals were measured during a training session
(A) that contained a baseline activity period, followed by a 30-sec
exposure to an auditory tone that coterminated with a foot shock (US).
The percentage time spent freezing to the context (B) and to the tone in
an altered context (C) was evaluated in wild-type and mutant mice during
the extinction trials (days 1, 3, 5, 7, and 13). Freezing responses were also
measured in a control group of mutants exposed to the context and tone
on day 7 only. n = 14 wild types, 17 mutants, and 17 mutant controls; *P <
0.05 compared with wild-type mice within the same day; #P < 0.05
compared with mutant mice that received multiple extinction trials.
Loss of DAO activity facilitates the extinction of contextual
Extinction in Dao1G181Rmice
Learning & Memory
(Wake et al. 2001), which might bias toward the persistence of
a fear response.
Genetic perturbation of DAO activity specifically improved
reversal memory and extinction without altering performance
during the acquisition trials. Activation of the NMDAR glycine site
has been shown to be necessary for the acquisition and memory
retrieval of a single platform location (Watanabe et al. 1992).
However, the ability of glycine site activators to potentiate this
type of spatial learning and memory beyond normal capacity
remains controversial (Pitka ¨nen et al. 1995; Andersen and Pouzet
2004; Maekawa et al. 2005b; Duffy et al. 2008; Zhang et al. 2008),
and discrepancies may reflect different procedural demands,
dosing regimes, or species. In the acquisition phase of our study,
animals were trained to achieve asymptotic performance in order
to assess reversal learning and extinction from a common base-
line. Consequently, we cannot preclude a possible enhancement
of acquisition by D-serine under some conditions, and others
have shown that elevated D-serine can enhance acquisition of
spatial learning (Maekawa et al. 2005b). Regardless, we demon-
strate that elevations in D-serine are not required during acquisi-
tion sessions in order to improve extinction learning, further
supporting a vital role in adaptive behaviors necessary for chang-
Activation of NMDARs is critically involved in the synapto-
plastic processes underlying the acquisition of novel behaviors
(Nicoll 2003). Recent studies suggest that behavioral alterations in
response to changing environmental conditions are also depen-
dent on NMDAR activity (Sotres-Bayon et al. 2007; Duffy et al.
2008; Nicholls et al. 2008), though the neurocellular mechanisms
are unclear. Activation of pre-existing circuits encoding memory
traces renders them subject to modification, a process termed
reconsolidation (Tronson and Taylor 2007). NMDAR antagonists,
including compounds that target NR2B receptors prevent the
induction of memory lability (Ben Mamou et al. 2006). Conse-
quently, increased D-serine may augment the propensity for
NMDAR-dependent reconsolidation. NMDAR-mediated long-
term depression (LTD) is one form of synaptic plasticity that
may contribute to behavioral flexibility by facilitating the remod-
eling of labile circuits. Alternatively, LTD may facilitate learning
under changing environmental conditions by suppressing the
expression of previously encoded memory traces. In either case,
there is compelling evidence for the role of LTD in behavioral
flexibility and inhibition. Transgenic mice that exhibit deficits in
hippocampal LTD demonstrated a parallel disruption in the ability
to learn novel platform locations in the MWM (Zeng et al. 2001;
Morice et al. 2007; Nicholls et al. 2008) and a deficiency in
acquisition and recall of fear extinction (Dalton et al. 2007).
Blockade of NMDA-NR2B receptors has been reported to inhibit
hippocampal LTD in the adult mouse and has also been found
to disrupt behavioral inhibition in a serial reaction task, spatial
reversal learning, and fear extinction learning and retention
(Higgins et al. 2003; Sotres-Bayon et al. 2007; Duffy et al. 2008).
Conversely, mice overexpressing NR2B receptors exhibit better
learning and memory in tasks that include extinction of fear
associations (Tang et al. 1999). Likewise, exogenous application of
D-serine was shown to strongly enhance NR2B-dependent LTD in
rodent hippocampal slices in vitro (Duffy et al. 2008; Zhang et al.
2008). Finally, the propensity for LTD has been shown to increase
with exposure to novel environments and when salient environ-
mental cues are relocated (Manahan-Vaughan and Braunewell
1999; Kemp and Manahan-Vaughan 2004).
In conclusion, elevated D-serine and genetic inactivation of
DAO function ameliorated cognitive flexibility and inhibitory
learning in the MWM and in a contextual fear-conditioning task.
Growing evidence indicates that reduced occupancy of the
NMDAR glycine binding site may be involved in the pathophys-
iology of schizophrenia, and activators that target this site may
have therapeutic potential (Coyle and Tsai 2004; Millan 2005).
Furthermore, modulation of the NMDAR glycine site has also
been proposed to benefit extinction and exposure therapy, which
are used to treat several psychopathologies, including obsessive-
compulsive disorder and post-traumatic stress syndrome (Ressler
et al. 2004; Norberg et al. 2008). The improvement of adaptive
responses through the inhibition of DAO activity may be of
substantial clinical utility for the treatment of these and other
psychiatric illnesses involving an inability to attenuate persistent
Materials and Methods
The Dao1G181Rmice were obtained from the Konno laboratory
that initially identified this spontaneous missense mutation
(glycine to arginine at amino acid 181) in the Dao1 gene of
the ddY strain, resulting in a completeloss of DAO activity(Konno
and Yasumura 1983; Sasaki et al. 1992). The Dao1G181Rmutation
was transferred onto a C57BL/6J genetic background using
a marker-assisted speed congenic strategy (Wong 2002). The
resultant Dao1G181Rmice contained >99% of the C57BL/6J
genome after six generations of backcrossing. Experimental sub-
jects were then bred from heterozygous intercrosses of Dao1G181R
mice in the animal colony at the Samuel Lunenfeld Research
Institute (Toronto, Canada). Animals carrying the Dao1G181R
mutation were genotyped using a PCR-amplicon restriction endo-
nuclease protocol that involved the amplification of a 263-bp PCR
product (59-TGATGTACGAAGCTGGAGGACA-39 and 59-TGTA
GTGGCACCAGCTTT-39), which lacked an HpaII (Fermentas)
restriction site in the homozygous state. Male C57BL/6J mice
were purchased from the Jackson Laboratory (Bar Harbor, ME,
USA) and were acclimatized to the animal colony for $1 wk prior
Groups of three to five littermates were housed by sex in
polycarbonate cages and given ad libitum sterile food (Purina
mouse chow) and water. The vivarium was maintained under
a controlled temperature (21°C 6 1°C) and humidity (50%–60%),
with a 12-h diurnal cycle (lights on: 0700–1900 h). All animal
procedures strictly followed the requirements of the Province of
Ontario Animals for Research Act 1971 and the Canadian Council
on Animal Care.
High-performance liquid chromatography
The high-performance liquid chromatography (HPLC) procedure
was adapted from a previously described protocol (Grant et al.
2006). Brain samples were homogenized in 5 vol of ice-cold
double-distilled water. An aliquot was mixed with 100% methanol
to give a final dilution of 603 and then centrifuged at 12,000g for
4 min at 4°C. A 5 mL aliquot of the supernatant was mixed with
5 mL of the derivatizing reagent (2 mg N-isobutyryl-L-cysteine and
1 mg o-phthaldialdehyde dissolved in 0.1 mL methanol, followed
by addition of 0.9 mL 0.1 M sodium borate buffer), and then was
placed into a sample management system (Waters Alliance
2690XE, Waters Corp.). HPLC separation was achieved on a Sym-
metry C18 column (4.6 mm 3 150 mm; 3.5-mm particle diameter)
coupled with a guard column of the same stationary phase (Waters
Corp.). The column heater was set at 30°C, and the sample cooler
was held at 4°C. To separate the derivatized amino acids of
interest, a gradient was established from equal parts of solvent A
(850 mL of 0.04 M sodium phosphate buffer and 150 mL
methanol at pH 6.2) and B (670 mL of 0.04 M sodium phosphate
buffer, 555 mL methanol and 30 mL tetrahydrofuran, pH 6.2) to
only solvent B by ;45 min, with a flow rate of 0.5 mL/min. The
runtime was 60 min for column washout and equilibrium, and
30 min to elute all compounds. AWaters 2475 fluorescence detector
(Waters Corp.) was used to quantify the eluted compounds
(excitation 344 nm; emission 433 nm).
Extinction in Dao1G181Rmice
Learning & Memory
The procedure for the chemiluminescent assay was modified from
a described protocol (Wolosker et al. 1999). Brain samples were
homogenized in ice-cold buffer (50 mM Tris-HCl, pH 8.8, 10 mM
KCl) and then centrifuged at 14,000g for 10 min at 4°C. From the
supernatant, protein concentrations were measured and standard-
ized. Samples were heated at 100°C for 20 min to eliminate
endogenous DAO activity and then cooled to 4°C. Assay buffer
(50 mL of 100 mM Tris, 50 mM NaCl, pH 8.8) with 0.1 U
horseradish peroxidase (Biochemika, Sigma), 0.8 nmol luminol
(Sigma), and 0.048 nmol flavin adenine dinucleotide (EMD
Chemicals Inc.) was added to each sample (10 mL). A Veritas
Microplate Luminometer (Turner Biosystems Inc.) was used to
detect the production of H2O2before and after the addition of
0.002 U R. gracilis DAO (recombinant courtesy of L. Pollegioni,
University of Insubria, Varese, Italy). Triplicates of each sample
were measured, and the content of D-serine was quantified using
a calibration curve of D-serine standards.
Behavioral testing was performed during the light phase between
1100 and 1700 h on experimentally naı ¨ve mice that were 11–16
wk old. Experiments were sex-balanced, except where stated. Since
no sex differences were found in the measured behaviors, male
and female data were pooled for greater subject numbers. Experi-
menters were blind to genotype. Subjects were handled for 2 min/
d on each of the five consecutive days prior to testing. During the
experimental days, mice were initially left undisturbed in the
room for 30 min before the start of the procedure to allow for
Morris water maze
The Morris water maze (MWM) consisted of a white Plexiglas,
cylindrical pool (1.85-m diameter) that was filled with opaque
water (26°C 6 1°C), as described by Duffy et al. (2008). The pool
was arbitrarily divided into four equal quadrants: northeast,
northwest, southeast, and southwest. The circular escape platform
(10-cm diameter) was made of clear Plexiglas. Distal visual cues
were fixed on each wall ;1 m from the pool edge. Activity in the
water maze was recorded using a CCD camera on the ceiling above
the center of the pool attached to an automated tracking system
(HVS Image Ltd.) that extracted and stored the x–y coordinates of
the subject every 0.01 sec. The HVS Water 2020 software (HVS
Image Ltd.) was used to establish experimental parameters and
analyze performance. Behavioral measures in the MWM included
latency to target (sec), path length (m), thigmotaxis (percent time
within 12.5 cm of the pool wall), swim speed (cm/sec), floating
(percent time), number of platform crosses, and percent time
within the target area.
Each MWM procedure began with a 1-d stationary visible
platform task. Mice were given 4 trials with an ;1-h intertrial
interval (ITI). In each trial, mice were released facing the pool wall
from one of four pseudorandomized locations (N, S, W, E) at the
pool periphery. The platform was at the center of the target
quadrant (SE) and 25 cm from the pool wall. In the visible trials,
the platform was raised 0.5 cm above the water surface and
demarcated with a 10-cm vertical pole. The maximum duration
for a platform search was 90 sec. Animals that found the platform
remained on it for an additional 15 sec, whereas unsuccessful
animals were assigned a 90-sec latency and gently placed onto the
platform for 15 sec. The acquisition phase began 1 d later, and
lasted for seven consecutive days (4 trials/d, 1 h ITI). Each day was
performed similarly to the visible platform task, except the
platform was now submerged ;1 cm below the surface of the
water (hidden) in the SE quadrant. Retention of spatial memory
was assessed in a 60-sec probe trial that occurred 24 h after the last
acquisition trial. In the probe trial, the platform was removed and
mice were released from the point furthest (NW) from the former
platform location. Performance in the probe trial was quantified
by examining the percentage of time spent and number of crosses
overan areathatwas twicethe platform diameter, centeredoverits
Reversal learning and memory
The reversal experiments entailed three additional days of training
(four trials/d, 1 h ITI) that began 1 d after the acquisition probe.
Reversal trials were conducted as described in the above acquisi-
tion phase, with the exception that the platform was located in
the center of the NW (opposite) quadrant. Memory for the new
platformlocation wasexaminedin a 60-secreversalprobe that was
assessed 24 h after the last reversal trial (SW release point).
The extinction trials were performed as described in the acquisi-
tion probe, and occurred on the following days after the last
acquisition trial: 1, 3, 5, 7, 9, 11, 13, and 15 (48 h ITI). To verify
that the Dao1G181Rmutationor D-serinetreatments did not impair
memory duration, control groups of Dao1G181Rmutant and D-
serine-injected mice were tested for the first time 9 d after the
completion of acquisition training. The D-serine-treated control
group was injected in the home cage on the same days as those
mice exposed to multiple probe trials.
Contextual and cued fear conditioning were conducted as pre-
viouslydescribed (Clapcoteet al. 2005; Young et al. 2008). Thefear
conditioning apparatus (MED Associates Inc.) consisted of four
test chambers (25 cm height 3 30 cm width 3 25 cm length) that
each had a grid floor and were connected to a shock generator,
amplifier, and speaker. Experimental parameters were controlled
by automated fear conditioning software (FreezeFrame v. 1.6e,
Actimetrics) that also recorded freezing activity (presented as
a percentage of total time). In the training phase, each chamber
had a white curtain covering the front exterior and was cleaned
with 70%ethanolthat left an odor. Allsurfacesin contact withtest
subjects were carefully dried, and odor cues were placed on
a surface that was unattainable by the animals. The training phase
began with a 2-min period in the chamber to monitor baseline
activity. Afterward, a 30-sec auditory tone was delivered (3600 Hz,
95 dB),andin the last2 secof tonepresentation, a continuous foot
shock (0.75 mA scrambled) was administered. The animals were
given an additional 30-sec period (post-US) before being returned
to their home cages. Freezing to the context was assessed for 5 min
without tone or foot shock on the following days after training:
1 (24 h later), 3, 5, 7, and 13. The fear conditioning extinction
sessions were conducted over days in order to resemble the
extinction procedure completed in the MWM. The percentage of
time freezing specifically to the context was calculated for each
subject [% time freezing to context = % time freezing during
context exposure ? % time freezing during training baseline
period], and these values were averaged for each genotype (Val-
entinuzzi et al. 1998). Freezing response to the tone cue was
measured in an altered chamber 2 h after each contextual freezing
session. Ventilation of the testing room, in addition to careful
cleaning and drying of the chambers during this delay eliminated
the odor used in the contextual freezing session. For the tone test,
the sensory environment of the chamber was altered using a 1%
acetic acid odor, a smooth white Plexiglas floor, a black curtain on
the front exterior, and a clear Plexiglas insert that gave the interior
of the chamber a prism shape. These chamber alterations did not
affect the tone frequency but did reduce the tone amplitude by
2 dB. Animals were placed in the chamber and given 3 min to
explore (pretone) followed by a 3-min exposure to the tone. The
percentage of time freezing specifically to tone was determined
for each subject [% time freezing to tone = % time freezing during
tone exposure ? % time freezing during pretone period], and
these values were then averaged for each genotype (Graves et al.
The warm water tail-flick test was performed as previously de-
scribed (Bohn et al. 2002). Mice were carefully held by the
examiner, and ;3 cm of the distal end of their tails were immersed
Extinction in Dao1G181Rmice
Learning & Memory
in 50°C water. The latency to respond to the thermal stimulus by
removing the tail from the warm water (tail flick) was measured
with a stopwatch and used to evaluate antinociception.
In the drug treatment experiment (Fig. 4), D-serine (Sigma) was
administered 30 min before each probe trial to male C57BL/6J
mice. No treatments were given during visible or acquisition
training. D-serine (600 mg/kg) was dissolved in 0.9% NaCl saline
solution and injected subcutaneously at a volume of 10 mL/kg.
The dose of D-serine was based on previous work with this
compound in the MWM (Duffy et al. 2008).
Statistical analyses were completed using Statistica (Statsoft Inc.).
Biochemical and behavioral data were analyzed using one-
way, two-way, or repeated-measures ANOVA with the appro-
priate between- and within-subjects factors. Significant main
effects or interactions were followed by Fisher’s least significant
difference (LSD) post-hoc comparisons. Significance was set at
P < 0.05.
V.L. was supported by Natural Sciences and Engineering Research
Council (NSERC, Canada) studentship. J.C.R. is a Canadian Re-
search Council (CRC) chair. The research was supported by the
Canadian Institutes of Health Research (CIHR) and the United
States National Institutes of Health (P01AG12411). We thank Gail
Rauw and Edward Weiss for expert technical assistance.
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Received June 18, 2008; accepted in revised form October 27, 2008.
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Learning & Memory