ORIGINAL INVESTIGATION

Effects of low-dose D-serine on recognition and workingmemory in mice

Patricia Bado & Caroline Madeira & Charles Vargas-Lopes & Thiago C. Moulin &

Ana Paula Wasilewska-Sampaio & Luise Maretti & Ricardo V. de Oliveira &

Olavo B. Amaral & Rogério Panizzutti

Received: 28 July 2010 /Accepted: 24 April 2011# Springer-Verlag 2011

AbstractRationale D-Serine is an endogenous co-agonist of theN-methyl-D-aspartate (NMDA) receptor and has beensuggested to improve cognitive deficits in schizophrenia.Objectives The present study investigates the effects oftreatment with D-serine in mice on tasks that requirerecognition learning and working memory, two cognitivedomains that are impaired in schizophrenia.Methods We studied the effects of various regimens ofsystemic administration of D-serine (50 mg/kg/day) onBALB/c mice performing object recognition, T-mazealternation, and open-field exploration tasks. For the objectrecognition task, we also contrasted the effects of D-serineand D-cycloserine and investigated whether D-serine couldreverse alterations induced by subchronic injections of theNMDA antagonist MK-801. D-Serine levels after injectionswere measured by high-performance liquid chromatography.Results In the object recognition task, pre-training treatmentwith D-serine or D-cycloserine significantly enhanced recog-nition memory 24 h after training. A single administration ofD-serine 30 min (but not 6 h) after training produced similar

enhancement, suggesting an effect on memory consolidation.Daily treatment with D-serine enhanced both object recognitionand T-maze performance over multiple days and improvedshort-term memory in MK-801-treated mice. D-Serine treat-ment did not alter open-field exploration. Behavioral effectswere accompanied by increased levels of D-serine in thehippocampus of treated animals.Conclusions Our results show that treatment with D-serinecan improve performance in tasks related to recognitionlearning and working memory, suggesting that this agentcan be useful for the treatment of disorders involvingdeclines in these cognitive domains.

Keywords D-Serine .Memory . NMDA receptor . T-maze .

Object recognition

Introduction

Cognitive deficits are core symptoms of schizophrenia thatare closely correlated with functional outcome in affectedpatients (Green 1996) and are not adequately treated byantipsychotics (Keefe et al. 2007). Cognitive domainswhich are affected by schizophrenia include visual learning,working memory and spatial learning, among others (Greenet al. 2004; Folley et al. 2010). Ideally, effective treatmentsfor schizophrenia should include the management of thesecognitive symptoms. In this sense, both pre-clinical andclinical studies of cognitive enhancers targeting the affecteddomains are warranted (Young et al. 2009).

D-Serine is a co-agonist of the N-methyl-D-aspartate(NMDA) subtype of glutamate receptor and mediatesneurotransmission and synaptic plasticity through thisreceptor. Several lines of evidence suggest that decreasedD-serine availability contributes to the hypofunction of

P. Bado : C. Madeira :C. Vargas-Lopes : L. Maretti :R. Panizzutti (*)Instituto de Ciências Biomédicas, CCS, sala F1-03,Universidade Federal do Rio de Janeiro,Rio de Janeiro, Rio de Janeiro 21941-590, Brazile-mail: rogerio@anato.ufrj.br

P. BadoInstituto D’Or de Pesquisa e Ensino,Rio de Janeiro, Brazil

T. C. Moulin :A. P. Wasilewska-Sampaio :R. V. de Oliveira :O. B. AmaralInstituto de Bioquímica Médica,Universidade Federal do Rio de Janeiro,Rio de Janeiro, Brazil

PsychopharmacologyDOI 10.1007/s00213-011-2330-4

NMDA receptors thought to occur in schizophrenia (Coyle1996; Kristiansen et al. 2007). Decreased levels of D-serinehave been described in the serum (Hashimoto et al. 2003)and cerebrospinal fluid (Bendikov et al. 2007; Hashimotoet al. 2005) of patients with schizophrenia; moreover,changes in the expression and/or activity of enzymesmediating the synthesis and degradation of D-serine havebeen shown in postmortem analyses of patients’ brains(Steffek et al. 2006; Bendikov et al. 2007; Madeira et al.2008; Habl et al. 2009). Animal studies have also shownthat D-serine administration can reverse acute behavioralalterations induced by NMDA blockers such as phencyclidine(PCP) and MK-801, which are frequently used as models forantipsychotic testing (Hashimoto et al. 2008; Kanahara et al.2008; Nilsson et al. 1997).

In line with this evidence, clinical trials have shown thatoral administration of D-serine added to antipsychotics canimprove symptoms in patients with schizophrenia (Tsaiet al. 1998; Heresco-Levy et al. 2005; Kantrowitz et al.2010). Strikingly, these changes were observed not only inpositive and negative symptomatology but also in cognitivedeficits, which are not normally improved by treatmentwith antipsychotic medication (Keefe et al. 2007). This factcould likely be related to the action of D-serine on theNMDA receptor, which has widely been shown to benecessary for learning and memory in animals (Nakazawaet al. 2004; Riedel et al. 2003).

Behavioral studies in rodents have evaluated some ofthe specific cognitive effects of D-serine. Exogenousadministration of D-serine leads to improvements inspecific features of spatial memory in the Morris watermaze, such as reversal learning (Duffy et al. 2008), probetest retrieval (Zhang et al. 2008), and day-to-day savingsin the delayed-match-to-place version of the task (Stoufferet al. 2004). In addition, D-serine administration improvessocial memory in rats (Shimazaki et al. 2010). However,most of the above cited studies used doses of 600–1,300 mg/kg (with the exception of that by Stouffer et al.2004, which used 100 mg/kg), far above the range shownto be effective in clinical trials with schizophrenia patients(30–120 mg/kg). Nevertheless, studies of functionalmagnetic resonance imaging in rodents have shown thatparenteral administration of 50 mg/kg of D-serine isenough to induce an increase in hippocampal activation(Panizzutti et al. 2005).

Pre-clinical data on the effects of D-serine on cognition inother tasks relevant for schizophrenia are scarcer, however.Previous studies have investigated the effects of D-cycloserine,a partial agonist acting at the same site on the NMDAreceptor, on short-term object recognition (Kart-Teke et al.2006; Zlomuzica et al. 2007) and object location memory(Assini et al. 2009). Additionally, D-cycloserine was described

to reverse working memory deficits induced by muscarinic(Fishkin et al. 1993) and NMDA blockers (Meyer et al. 1998)in a T-maze alternation task. However, these behavioralstudies of D-cycloserine neither examined its effect on long-term object recognition memory nor evaluated the effects ofthe drug by itself on T-maze performance. Additionally,clinical trials using D-cycloserine for schizophrenia treatmenthave been less promising than those using D-serine (Goff et al.2008; Tsai and Lin 2010; Tuominen et al. 2005), possiblybecause of its partial agonist properties; thus, more studies onthe effects of D-serine on cognitive tasks are warranted.

In this study, we investigated whether a low dose of D-serine, in a range comparable to that used in human clinicaltrials, could have a beneficial effect on rodent modelsrelated to cognitive domains affected by schizophrenia. Weevaluated the effects of systemic treatment with D-serine(50 mg/kg) on recognition memory and working memorycorrelates in mice, both in normal animals and aftersubchronic injections of an NMDA antagonist, using thenovel object recognition and T-maze tasks. Additionally, wealso compared the effects of D-serine and D-cycloserine onlong-term memory of the object recognition task.

Materials and methods

Animals

Three- to 4-month-old male BALB/c mice were used in theexperiments. This strain of mice was chosen because it wasshown to have good short-term memory of the novel objectrecognition task at 4 h, but no long-term retention at 24 h(Brooks et al. 2005; Sik et al. 2003). Mice were housed fiveor six per cage under controlled light/dark cycles andtemperature. Food and water were supplied ad libitum to allmice, except for those used in the appetitively motivatedtask (T-maze), which were kept at 90% of their free-feedingweight. All experimental protocols were approved by theCommittee for the Use of Experimental Animals of ourInstitution and were carried out in accordance with the NIHGuide for Care and Use of Laboratory Animals.

Drug administration

D-Serine, D-cycloserine, and MK-801 (Sigma ChemicalCo., St. Louis, MO, USA) were dissolved in 0.9% saline inorder to inject 10 ml/kg of body weight in each mouse.Injections of D-serine (50 mg/kg), D-cycloserine (20 mg/kg),or saline were normally performed 30 min before behavioralsessions, unless otherwise specified. Injections of MK-801(0.5 mg/kg) were performed twice daily for seven consecutivedays as described below.

Psychopharmacology

Behavioral tasks

Novel object recognition

The novel object recognition task was performed in order toevaluate short- and long-term recognition memory. Duringa 4-day habituation period, animals were allowed to freelyexplore the task arena (50×50×39 cm chamber of whiteplywood, with a frontal glass wall) in the absence of objectsfor a single 20-min session on each day. On the trainingday, two identical objects made of aluminum, plastic orglass were placed in the arena, equidistantly of its walls,and mice were allowed to explore the environment for5 min. The exploration time for both objects (defined assniffing or touching the stimulus object with the nose and/or forepaws) was recorded by the experimenter. Simplysitting on top of objects in the absence of active explorationwas not counted as exploration time.

In the test session, mice were left to explore the openfield for 5 min in the presence of one of the familiarobjects, as well as a novel one of similar size. The testsession was performed either 4 h after training (to evaluateshort-term memory) or 24 h after training (to evaluate long-term memory). Exploration times for the familiar and novelobject were recorded during this session, and the ratio ofexploration of the novel object to total exploration time wasused as a measure of recognition memory.

In the initial experiments, D-serine, D-cycloserine, or salinewas injected 30 min before the training session. Additionally,to investigate whether the effects observed in these experi-ments were related to modulation of memory consolidation,independent experimental groups were trained in the task asdescribed above but received only a single D-serine or salineinjection either 30 min or 6 h after the training session.Finally, to investigate whether chronic daily injections wouldalso be effective in improving memory, D-serine or salinewas injected 30 min before all behavioral sessions (i.e.,habituation, training, and testing) in both normal animals andin those treated subchronically with MK-801 (see below).

T-maze

Working memory was assessed in a plywood T-maze witharms measuring 35 cm in length, 10 cm in width, and25 cm in height. The start box (10×12 cm) was separatedfrom the stem by a horizontal sliding door. Horizontalsliding doors were also placed at the entrance of each arm.Low-intensity diffuse illumination was provided above theapparatus. Four days before the first habituation session, micewere lightly food deprived until they reached 90% of theirinitial weight. In the habituation sessions, which lasted 4 days,mice were placed in the starting arm of the maze and allowed

to explore it freely for 10 min, with a piece of cereal providedas a reward at the end of both side (goal) arms.

After the habituation phase, mice were trained for 15 trialsper day over 8 days. In the first trial, the reward was placed inboth goal arms. Over the subsequent 15 trials, only one of thegoal arms was baited; if the mouse had successfully found thebait on the previous trial, the arm opposite to the one it had justentered was baited; if it had gone to the empty arm on the lasttrial, the reward was left in the same place. Thus, animals hadto learn to alternate between both arms to find the reward.After the animal had chosen one of the goal arms, the slidingdoor was used to keep the animal in it for 20 s; subsequently,the mouse was placed again in the starting arm for 10 s beforethe door was opened for another trial.

D-Serine or saline was injected 30 min before eachhabituation and training session on the T-maze. Thepercentage of correct arm choices during each training daywas recorded and used as a measure of memory in the task.

Open-field exploration

During the habituation period of the object recognition task,the behavior of the animal in the open-field arena wasrecorded in order to evaluate the effect of D-serineadministration on locomotion, anxiety-related place prefer-ence, and habituation memory. The floor of the above-described arena was divided into 16 equal squares, and thenumber of square crossings and rearings of each animal wasmeasured over the initial 5 min of the first session to evaluateboth locomotion and day-to-day habituation to the arena.Additionally, the proportion of time spent in the central25×25 cm of the arena was recorded and used as a measure ofanxiety in the task. As described previously, D-serine or salinewas injected 30 min before the session.

Subchronic MK-801 model

To induce learning deficits mimicking those observed inschizophrenia, we used a model of subchronic NMDAantagonist treatment followed by withdrawal, which hasbeen proposed as a model for the disorder’s negative andcognitive symptoms (Mouri et al. 2007; Neill et al. 2010).First, mice received two daily injections of MK-801(0.5 mg/kg) for 7 days. After treatment cessation, theanimals were habituated to the open-field arena for 4 daysfollowed by training in the object recognition task toevaluate short-term memory after 4 h, as described above.

High-performance liquid chromatography

In order to evaluate whether systemic treatment with D-serine was able to increase its levels in the hippocampus,

Psychopharmacology

we sacrificed animals after the test session of the objectrecognition task performed to evaluate short-term memory(i.e., 35 min after the last injection). The hippocampi weredissected and homogenized in buffer containing 50 mMTris–HCl (pH 7.4), 150 mM NaCl, 1.5 mM MgCl2, 1.5 mMEDTA, 1% Triton X-100, 10% glycerol, a cocktail ofprotease inhibitors (10 μg/ml each of aprotinin, leupeptin,and pepstatin), and 1 mM phenylmethylsulfonyl fluoride.D-Serine levels in the homogenates were measured by high-performance liquid chromatography (HPLC) as previouslydescribed (Hashimoto et al. 1992).

Statistical analysis

Comparisons of performance between two groups in theobject recognition and open-field exploration tasks, as wellas comparisons of hippocampal D-serine levels measuredby HPLC, were performed using a two-tailed Student’st test. When more than two groups were compared, thesame analysis was done by a one-way ANOVA with aBonferroni post hoc test. Evaluation of learning inindividual groups in the object recognition task was donethrough a two-tailed one-sample t test comparing novelobject exploration to chance performance (i.e., 50% of theexploration time spent on the novel object).

Comparisons between the D-serine and saline groups inthe T-maze task were performed using a two-way repeatedmeasures ANOVA (drug treatment versus time) and aBonferroni post hoc test to evaluate differences betweengroups in individual days. A one-way repeated measuresANOVA was used to evaluate learning within individualgroups in this task, and Dunnett’s post hoc test was used toevaluate differences between individual days and the initialday of testing. Statistical significance in all tests wasdefined as a p value of less than 0.05.

Results

Figure 1 shows the effect of treatment with D-serine(50 mg/kg) and D-cycloserine (20 mg/kg) 30 min beforetraining on long-term memory in the novel object recognitiontask. Control animals showed no significant preference for thenovel object 24 h after training (t=0.56, p=0.59, n=8), whileboth D-serine- and D-cycloserine-treated groups showed astrong bias for the novel object (t=4.85, p=0.0009, n=10and t=6.71, p=0.0002, n=9, respectively), which wassignificantly different from that of the control group(F=7.74; p=0.002 for general comparison; p<0.01 drugvs. control post hoc comparisons). This result is consistentwith previous studies evaluating the same dose of D-cycloserine in shorter-term memory protocols (Zlomuzicaet al. 2007; Assini et al. 2009) and shows that the 50-mg/kg

dose of D-serine has an enhancing effect comparable to thatof an optimal dose of D-cycloserine on long-term objectrecognition memory.

After confirming that low dose of D-serine was able toimprove long-term memory in the novel object recognitiontask, we asked whether this effect could be related tostrengthening of memory consolidation. To test thishypothesis, we examined the effects of D-serine injectioneither 30 min or 6 h after training on the preference for thenovel object 24 h after training. Animals that received D-serine administration 30 min after training (Fig. 2a) showeda significant preference for the novel object (t=7.78,p<0.0001, n=10) which was comparable to that obtainedwith pre-training injections. This preference was notobserved in control animals (t=1.06, p=0.32, n=10),leading to a significant difference between the groups (t=11,p<0.0001). In contrast, animals that received D-serine only6 h after training (Fig. 2b) did not show a significantpreference for the novel object 24 h after training (t=1.52,p=0.17, n=10), and their performance was similar tocontrols (t=1.19, p=0.25, n=10). Taken together, theseresults indicate that D-serine administration can improverecognition memory by acting within a time-limited, post-training consolidation period.

Since the use of cognitive enhancers such as D-serine ina clinical setting is likely to be chronic, we then askedwhether daily injections of the drug during the wholebehavioral protocol would lead to a similar effect. When

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Fig. 1 Effects of D-serine and D-cycloserine treatment on long-termobject recognition memory. Arrows indicate the time of drug/vehicleadministration (30 min before training); Hab habituation, Traintraining session, Test test session. Bars (mean±SEM) indicatepercentage of object exploration (A familiar object, B novel object)during test sessions. *p<0.001 indicates a significant preference forthe novel object. #p<0.01 indicates a significant increase in novelobject exploration in the D-serine and D-cycloserine groups whencompared to the saline group

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injections were performed 30 min before every behavioralsession from habituation to testing (Fig. 3a), animals treatedwith D-serine again showed a strong preference for thenovel object 24 h after training (t=4.24, p=0.006, n=17),while control animals did not (t=1.67, p=0.11, n=18),leading to a significant difference between the two groups(t=6.00, p<0.0001). Using the same administration protocol,we also evaluated short-term memory in the task, tested 4 hafter training. In this case, both the D-serine and saline groupsshowed a significant preference for the novel object (t=3.54,p=0.003, n=16 and t=3.4, p=0.005, n=14 for the D-serineand saline groups, respectively), with no difference inperformance between groups (Fig. 3b, t=0.7, p=0.49).

The effects of daily treatment with D-serine were alsoevaluated on the T-maze task (Fig. 3c). Animals thatreceived D-serine daily 30 min before behavioral sessionsimproved their performance in the task over multiple days oftraining (F=5.33, p<0.0001, n=13), with daily performancefrom the third day onward differing from that of the initialday (q=2.72–4.93, p<0.05). In contrast, the control groupdid not improve their performance over the period of training(F=0.4, p=0.9, n=13). Throughout all days, the D-serinegroup achieved 63% correct alternation, as opposed to 40%correct alternation in the control group, and this difference inperformance was strongly significant (F=22.72, p<0.0001for treatment effect). Post hoc analysis of individual daysshowed that the difference between the D-serine and controlgroups was significant on the last day of testing (t=2.81,p<0.05).

Next, to evaluate whether this memory-enhancing effectcould also be observed in a model of cognitive impairmentrelated to schizophrenia symptoms in humans, we treated

mice with the NMDA antagonist MK-801 for 7 days. Aftercessation of treatment, the animals went through the sameobject recognition protocol, again receiving daily D-serineor saline injections 30 min before behavioral sessions (as inFig. 3b). When mice previously treated with MK-801 weretested 4 h after training, no short-term memory in the taskwas observed in the control saline-treated group (t=0.86,p=0.41, n=15; Fig. 4). On the other hand, D-serine-treatedanimals showed preservation of the preference for the novelobject (t=5, p=0.003, n=13), leading to a significantdifference between groups (t=2.48, p=0.02).

In addition, to test whether D-serine treatment affectedlocomotion or anxiety in normal mice, we evaluated theirbehavior during open-field exploration. D-Serine did notaffect exploratory behavior, as measured by the number ofcrossings (87±20 in the control group; 89±21 in the D-serine group; t=0.08, p=0.94, n=9/group) and rearings(15.8±4.9 in the control group; 17.6±5.3 in the D-serinegroup; t=0.24, p=0.81, n=9/group) over 5 min ofexploration. D-Serine also did not affect anxiety-relatedplace preference, as measured by the percentage of timespent in the center of the arena (6.3±0.9% in the controlgroup; 5.4±1.3% in the D-serine group; t=0.56, p=0.58,n=14/group). These findings suggest that the observedeffects of D-serine in the memory tasks were not due toaltered locomotion or anxiety.

Finally, to investigate if the effects of peripheral D-serineadministration could be related to increased D-serineavailability in the hippocampus, we sacrificed a subset ofanimals just after the novel object recognition task andmeasured hippocampal D-serine content. D-Serine-treatedanimals showed significantly higher levels of D-serine in

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Fig. 2 Effects of D-serine on consolidation are limited to a specifictime window after training. Arrows indicate the time of D-serine orsaline administration; Hab habituation, Train training session, Test testsession. Bars (mean±SEM) indicate percentage of object exploration(A familiar object, B novel object) during test sessions. a *p<0.0001

indicates a significant preference for the novel object. #p<0.0001indicates a significant increase in novel object exploration in theD-serine group compared to the saline group. b Mice treated 6 h aftertraining with D-serine or saline show no preference for the novelobject; no difference is observed between groups

Psychopharmacology

the hippocampus (96.9±10.9 nmol/g WT in the controlgroup; 159.4±33.2 nmol/g WT in the D-serine group,t=3.52, p=0.005, n=7/group), while other amino acidssuch as glutamate and L-serine showed no significantchange (data not shown).

Discussion

Improving cognition is a major challenge in the treatmentof schizophrenia, since the results of currently availabletherapies have been disappointing (Keefe et al. 2007).Preliminary clinical studies have suggested that D-serinecan improve cognitive symptoms in schizophrenia subjects(Tsai et al. 1998; Heresco-Levy et al. 2005; Kantrowitzet al. 2010). In this work, we show that D-serine treatmentimproved the performance of BALB/c mice in the novel

object recognition and T-maze tasks, both of which requirecognitive domains that are impaired in schizophrenia, suchas recognition learning and working memory (Green et al.2004). Importantly, learning in both tasks was improved bymuch lower doses of D-serine than those used in previousstudies reporting its effects on spatial memory (Duffy et al.2008; Zhang et al. 2008), a domain also recently shown tobe affected in patients with schizophrenia (Folley et al.2010).

The improvement in long-term object recognition memoryinduced by a pre-training injection of D-serine was similar inmagnitude to that induced by D-cycloserine at 20 mg/kg(a dose which was shown to yield optimal enhancing effectson memories of shorter duration in previous studies;Zlomuzica et al. 2007; Assini et al. 2009). Thus, stimulationat the co-agonist site of the NMDA receptor on the objectrecognition task seems to allow this memory to become

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Fig. 3 Effects of daily D-serine administration on novel objectrecognition and T-maze alternation. Arrows indicate the time of D-serineor saline administration (30 min before behavioral sessions); Habhabituation, Train training session, Test test session. Bars (mean±SEM)indicate percentage of object exploration (A familiar object, B novelobject) during test sessions, and *p<0.01 indicates a significantpreference for the novel object in both A and B. a Effects of D-serineon long-term memory 24 h after training. #p<0.0001 indicates a

significant increase in novel object exploration in D-serine-treatedanimals when compared to controls. b Effects of D-serine on short-term memory 4 h after training. No difference is observed betweengroups. c Effects of D-serine administration on T-maze alternation.Points (mean±SEM) denote percentage of correct alternations in micetreated with D-serine (circles) and saline (squares). Performance acrossdays is significantly better in the D-serine group (p<0.0001). *p<0.05indicates a significant difference between groups in day 8

Psychopharmacology

longer lasting. This was shown to involve the modulation ofpost-training consolidation processes, as injections of D-serine 30 min (but not 6 h) after training led to a similareffect. Although we cannot exclude that treatment with D-serine could also have an effect on initial memory encoding,our results point out that at least part of its effect is due tostrengthening of memory consolidation during a time-limitedpost-training window.

Treatment of patients with NMDA co-agonists inconditions such as schizophrenia is likely to be chronic.Since previous work with D-cycloserine in memory extinc-tion showed that chronic treatment can induce desensitiza-tion to the drug’s effect (Quartermain et al. 1994; Parnaset al. 2005; Werner-Seidler and Richardson 2007), it wasimportant to test whether this could also happen with D-serine treatment. In our protocol, nevertheless, the effect ofdaily injections of D-serine was similar to that of a singlepre-training injection. This effect was observed 24 h but not4 h after training, perhaps due to a ceiling effect, as controlmice showed good memory in the task at 4 h, but not at24 h. Using the daily injection protocol, we also testedmemory on a multiday learning task using the T-maze,which required animals to use both working memory (tocorrectly alternate between trials) and long-term memory(to remember the alternation rule from 1 day to the next).The fact that performance improved significantly over timein the D-serine group suggests that at least part of theimprovement was due to an increase in memory savingsfrom previous days, an effect on long-term memory similarto that which occurred in the object recognition task.

Finally, to show that memory enhancement occurred notonly in normal mice but also in conditions thought to berelated to schizophrenia in humans, we used a subchronicNMDA antagonist model to mimic cognitive deficitsoccurring in this disorder. Subchronic treatment withNMDA antagonists such as MK-801 or PCP has beenshown to cause deficits in cognitive domains related toschizophrenia even after withdrawal of the drug (Jentschand Roth 1999; Brigman et al. 2009; Mouri et al. 2007;Neill et al. 2010). In subchronically MK-801-treatedanimals, we could no longer observe memory in the objectrecognition task 4 h after training as we did in normal mice;importantly, D-serine administration reversed this deficitand restored short-term memory of the task. Reversal of thedeficits induced by acute MK-801 treatment on recognitionmemory (Karasawa et al. 2008) and prepulse inhibition(Lipina et al. 2005; Kanahara et al. 2008; Hashimoto et al.2009) by higher doses of D-serine had been previouslydescribed. Nevertheless, the long-term effects of chronicNMDA antagonism have a more specific profile ofbehavioral deficits than acute treatment and are thought tobe better models of behavioral and neurochemical alter-ations in schizophrenia, especially deficits in frontal lobefunction (Jentsch and Roth 1999). Thus, the reversal ofcognitive impairments in this model by a low dose of D-serine, as we report here, seems to be more specific andrelevant for the deficits that occur in schizophrenia.

It is naturally tempting to attribute the effects of D-serineon these behavioral tasks to the modulation of the NMDAreceptor; D-serine, after all, is increasingly thought to be themain ligand of the co-agonist site of the NMDA receptor inthe forebrain (Mothet et al. 2000; Henneberger et al. 2010).Moreover, there is evidence from behavioral (Nilsson et al.1997) and functional imaging (Panizzutti et al. 2005)studies pointing out that the site is not saturated in vivo inthe hippocampus. D-Serine is required for long-termpotentiation in in vitro models (Yang et al. 2003;Henneberger et al. 2010), and its endogenous productioncan be stimulated by behavioral tasks (Vargas-Lopes et al.2011). Taken together, these data suggest that D-serinecould act physiologically as a memory modulator, althoughthis hypothesis has yet to be experimentally demonstrated.

The fact that D-serine was able to exert its effects eventhough it was administered 30 min after the learning sessioncan provide some insights on its mechanisms and site ofaction. Although NMDA antagonists have usually beenshown to have effects when administered before orimmediately after training, as shown in studies using theobject recognition task (Akirav and Maroun 2006; de Limaet al. 2005; Winters and Bussey 2005), some studies oftheir action in areas such as the entorhinal cortex haveshown more prolonged involvement of NMDA receptors inconsolidation which lasted up to 3 h after training

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Fig. 4 Effects of D-serine administration on short-term recognitionmemory after MK-801 subchronic treatment. Scheme shows theprotocol for MK-801 treatment and object recognition; arrowsindicate D-serine or saline administration 30 min before behavioralsessions; Hab habituation, Train training session, Test test session. Bars(mean±SEM) indicate percentage of object exploration (A familiarobject, B novel object) during test sessions. *p<0.01 indicates asignificant preference for the novel object. #p<0.05 indicates asignificant increase in novel object exploration in the D-serine groupwhen compared to the control group

Psychopharmacology

(Izquierdo et al. 2006; Rossato et al. 2004). Moreover,conditional knockout studies have shown that activity ofNMDA receptors in the hippocampus can play a role up toseveral days following learning (Shimizu et al. 2000).Accordingly, we observed that our injection protocolincreased the levels of D-serine in the hippocampus, raisingthe possibility that this structure is involved in theimprovement of long-term memory caused by the treatment.

Our memory effects also seemed to be unrelated toalterations in anxiety, as neither of the memory tasks usedhad a strong aversive component. Moreover, the analysis ofopen-field exploration did not show an effect of this dose ofD-serine in anxiety-related behavior, such as locomotion orplace preference within the arena. This is in contrast toprevious studies describing an anxiogenic effect of D-serineinjected systemically (Labrie et al. 2009) or in specificbrain areas such as the dorsal periaqueductal gray matter(Schmitt et al. 1995). This difference could be related to themuch lower dose used in our study (50 mg/kg as opposed to600mg/kg in Labrie et al. 2009). It is also in contrast with theincrease in locomotion reported to occur with D-cycloserineinjection in mice (Sunyer et al. 2008). The fact that positiveeffects on memory can be observed in the absence ofanxiogenic or motor effects is significant for the treatment ofcognitive symptoms in patients with schizophrenia, whohave a high prevalence of anxiety disorders.

Naturally, we do not know whether the effects observedin rodents can be transposed to humans in general, or tosubjects with schizophrenia in particular. However, it issignificant that the administration of D-serine in humans hasbeen shown to improve cognitive symptoms in subjects withschizophrenia (Tsai et al. 1998; Heresco-Levy et al. 2005). Inaddition, a recent report showed that D-serine administrationimproves the composite score in the MATRICS consensuscognitive battery, a battery of neuropsychological testsdesigned specifically for the assessment of subjects withschizophrenia (Kantrowitz et al. 2010). This suggests that theco-agonist site in the NMDA receptor is not fully saturated inhumans and opens up the possibility that D-serine might alsoshow significant cognitive effects in specific domains relevantfor schizophrenia treatment. Future studies are needed, thus, totest whether the improvement in memory and learning tasksobserved with D-serine administration in animals can beeffectively translated into useful treatments for cognitivedysfunction in schizophrenia and other disorders.

Acknowledgments This work was supported by grants fromConselho Nacional de Desenvolvimento Científico e Tecnológico(CNPq), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro(FAPERJ), Fundação Universitária José Bonifácio, and InternationalSociety for Neurochemistry to R.P. and by grants from FAPERJ andCNPq to O.B.A. R.P. is recipient of a Long-Term Fellowship from theHuman Frontier Science Program. P.B. and L.M. were supported byundergraduate scholarships from FAPERJ. C.V.L. and C.M. weresupported by pre-doctoral fellowships from CNPq. We are grateful to

Ana Cláudia Rangel for technical assistance and to Jean ChristopheHouzel for encouragement.

Conflicts of interest The authors declare that all experimentscomplied with current Brazilian laws and report no conflicts ofinterest concerning the work.

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