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Behavioural Brain Research 271 (2014) 374–379

Contents lists available at ScienceDirect

Behavioural Brain Research

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esearch report

-cycloserine injected into the dorsolateral periaqueductal graynduces anxiolytic-like effects in rats

elipe V. Gomesa,b,∗, Alessandra M. Kakihataa, Ana Carolina G. Semedoa, Sara C. Hotta,aniela L. Ulianaa, Francisco S. Guimarãesa,b, Leonardo B.M. Resstela,b

Department of Pharmacology, Medical School of Ribeirão Preto, University of São Paulo, BrazilCenter for Interdisciplinary Research on Applied Neurosciences (NAPNA), University of São Paulo, São Paulo, Brazil

i g h l i g h t s

D-cycloserine (DCS) is a partial agonistpe of the NMDA receptor-associated glycine site.DCS, in lower doses, acts as agonist, but in higher acts as antagonist.Anxiolytic-like effect has been observed only after systemic administration of high doses of DCS.Glycine site associated with the NMDAR in the dlPAG modulates defensive behavior.Intra-dlPAG DCS administration induced anxiolytic-like effects in three different animal models.

r t i c l e i n f o

rticle history:eceived 17 February 2014eceived in revised form 3 June 2014ccepted 6 June 2014vailable online 13 June 2014

eywords:-cycloserineMDA receptorlycineeriaqueductal graynxiety

a b s t r a c t

d-cycloserine (DCS) is a partial agonist of the glycine site coupled to the NMDA receptor (NMDAR). Asa consequence, depending on the doses used it can function as an agonist or antagonist at this site. Inrodents, anxiolytic-like effects have been observed after the systemic administration of high doses of DCS.The brain sites of these effects have not been investigated. Direct brain injection of glycine site antagonistsor agonists into the dorsolateral periaqueductal gray (dlPAG), a brain structure involved in the modulationof defensive-related behaviors, produces anxiolytic- or anxiogenic-like effects, respectively. The presentstudy investigated if the dlPAG could be a brain site of the anxiolytic effects observed after DCS systemicadministration. Male Wistar rats received intra-dlPAG injections of DCS (25, 50, 100 or 200 nmol) andwere exposed to the elevated plus-maze (EPM) or to the light–dark box. DCS, at the dose of 200 nmol,increased open arm exploration and the time spent in the light compartment, respectively. Based onthis result we tested the effects of intra-dlPAG DCS (200 nmol) administration in animals submitted to

nimal model the Vogel conflict tests. Anxiolytic-like effect was also observed in this test indicated by the increaseof punished responses. The drug did not change locomotor activity, discarding potential confoundingfactors. These results indicated that administration of DCS, a partial agonist of the NMDAR-associatedglycine site, into the dlPAG induces anxiolytic-like effects in different models, pointing to a possible siteof action of this compound.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

The N-methyl-d-aspartate receptor (NMDAR) is one of theeveral receptors responsible for synaptic transmission mediatedy excitatory amino acids. In addition to the glutamate-binding

∗ Corresponding author at: Department of Pharmacology, Medical School ofibeirão Preto, University of São Paulo, 3900 Bandeirantes Ave, 14049-900, Ribeirãoreto, São Paulo, Brazil. Tel.: +55 16 36023325; fax: +55 16 36332301.

E-mail addresses: gomesfv@usp.br, gomesfv@yahoo.com.br (F.V. Gomes).

ttp://dx.doi.org/10.1016/j.bbr.2014.06.009166-4328/© 2014 Elsevier B.V. All rights reserved.

site, NMDAR contains modulatory sites for glycine, polyamines,protons and channel blockers, including the binding site for non-competitive antagonists such as phencyclidine and ketamine [1].These sites can modulate NMDAR function [2] and glutamate isunable to activate this receptor in the absence of glycine [3]. Thus,changes in the glycine concentration or competition for its bindingsite can alter NMDAR-mediated responses.

d-cycloserine (DCS), a glycine cyclic analog, acts as a partial

agonist of the glycine site coupled to the NMDAR, being able tofunction as an agonist or antagonist at this site, depending on thedoses used [4]. Anxiolytic-like effects have been observed after

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ystemic administration of DCS in rodents submitted to differ-nt animal models. Interestingly, those effects were only observedfter administration of high doses of DCS [5,6], suggesting thathe drug was acting as a functional antagonist of the NMDAR-ssociated glycine site. Several other pieces of evidence indicate thenvolvement of the glycine site on defensive behavior modulation.or example, administration of glycine site antagonists or agonistsnto the dorsolateral portion of the periaqueductal gray (dlPAG)nduced anxiolytic- [7,8] or anxiogenic-like effects [9], respectiv-lly. Together, these results suggest that the glycine site associatedith the NMDAR in the dlPAG modulates defensive behavior [10].

The dlPAG is a brain region proposed to be a key componentf a neural substrate responsible for the elaboration of anxiety-elated behaviors. Several lines of evidence indicate that underormal circumstances these responses are mediated by local glu-amate release [11]. Indeed, intra-dlPAG administration of NMDARnd non-NMDAR agonists induces escape reactions [12], whereaslocking these receptors cause anxiolytic-like effects in rodents13].

Thus, considering that high doses of DCS could act as an NMDARlycine-site antagonist and that intra-dlPAG administration ofntagonists of this site attenuate defensive responses, the presenttudy tested the hypothesis that this brain structure could be aossible action site of the anxiolytic-like effects observed after sys-emic administration of DCS.

. Material and methods

.1. Animals

The study was performed with male Wistar rats weigh-ng 220–240 g at the beginning of the experiments. Animals

ere housed in groups of four per cage (41 × 33 × 17 cm) in aemperature-controlled room (24 ± 1 ◦C) under standard labora-ory conditions with free access to food and water and a 12 hight/dark cycle (lights on at 06:30 a.m.). Procedures were con-ucted in conformity with the Brazilian Society of Neurosciencend Behavior guidelines for the care and use of laboratory animals,hich are in compliance with international laws and politics. The

nstitution’s Animal Ethics Committee approved the housing con-itions and experimental procedures (process number: 144/2012).

.2. Stereotaxic surgery

Seven days before the experiments the rats were anesthetizedith 2,2,2-tribromoethanol (250 mg/kg i.p., Sigma–Aldrich, USA)

nd fixed in a stereotaxic frame (Stoelting, USA). After scalpnesthesia with 2% lidocaine, the skull was surgically exposednd stainless steel guide cannula (26G) was implanted uni-aterally on the right side aimed at the dlPAG (coordinates:ntero-posterior = 0 mm from lambda; lateral = −1.9 mm at a lateralnclination of 16◦, dorsal = −4.2 mm from the skull, [14]). The can-ula was fixed to the skull with dental cement and a metal screw.n obturator was placed inside the cannula to prevent obstruction.fter surgery, the animals received a polyantibiotic (0.27 g/kg i.m.;entabiotico®, Fort Dodge, Brazil) to prevent infection and a non-teroidal anti-inflammatory (0.025 g/kg s.c.; Banamine®, Scheringlough, Brazil) for post-operative analgesia.

.3. Apparatus

.3.1. Elevated plus-maze (EPM)The EPM consisted of two opposite wooden open arms

50 × 10 cm), crossed at right angle by two arms of the same dimen-ions enclosed by 40-cm high walls with no roof. The maze was

Research 271 (2014) 374–379 375

located 50 cm above the floor and a 1-cm high edge made of Plex-iglas surrounded the open arms to prevent falls. It was located ina sound-attenuated, temperature-controlled (24 ± 1 ◦C) room andthe environment was illuminated by one 40-W fluorescent lightplaced 3 m away from the EPM. The Any-Maze software (Stoelting,USA) was employed for behavioral analysis in the EPM. It detectsthe position of the animal in the maze and calculates the numberof entries and time spent in open and enclosed arms. Each sessionlasted for 5 min and after each trial the maze was cleaned with analcohol solution.

2.3.2. Light–dark boxThe light–dark box (23 × 21 × 60 cm) had two compartments

separated by a wall with an open door in its inferior portion. Onecompartment was completely dark, with black walls, and anotherone had white wall and a superior transparent cover. The floor wasmade of metal grids. Each animal was initially placed in the darkcompartment and could freely explore the box for 5 min. The exper-iment was recorded and analyzed by the Any-Maze software. Aftereach trial the box was cleaned with an alcohol solution.

2.3.3. Vogel conflict testThe Vogel conflict test was performed in a Plexiglas box

(25 × 20 × 42 cm) with a stainless grid floor. A metallic spout of adrinking bottle containing water was projected into the box. Thecontact of the animal with the spout and the grid floor closed anelectrical circuit controlled by a sensor (Anxio-Meter model 102,Columbus, USA), which produced seven pulses per second when-ever the animal was in contact with both components. Each pulsewas considered as a lick, and at every 20 licks the animal received a0.5-mA shock for 2 s. The sensor recorded the total number of licksand shocks delivered during the test period. The whole apparatuswas located inside a sound-attenuated cage.

2.3.4. Open field testThe open field consisted of an acrylic circular arena (76.5 cm

diameter surrounded by 50 cm high wall). The animals were placedindividually in the center of the arena and the total distance trav-eled was measured during 5 min using the Any-Maze software.

2.4. Procedure

2.4.1. Intra-dlPAG injectionSeven days after surgery the animals were randomly assigned

to one of the treatment groups. Intracerebral injections were per-formed with a thin dental needle (30G 0.3 mm OD; Terumo DentalNeedle®, Brazil) introduced through the guide cannula until its tipwas 1 mm below the cannula end, connected to a 10 �L syringe(7001 KH, Hamilton Co., USA) through a polyethylene catheter(PE10). The needles were carefully inserted into the cannula, anda volume of 0.2 �L was injected over a 30 s period with a rate of0.4 �L/min with the help of an infusion pump (Kd Scientific Inc.,USA). The movement of an air bubble in the PE10 confirmed theeffectiveness of the injection. To prevent reflux, the needles wereleft in place for a 45–60 s period after the end of each injection. DCS(Sigma–Aldrich) was dissolved in saline. The solutions were pre-pared immediately before the tests and protected from light duringthe experimental sessions.

2.4.2. Experiment 1: intra-dlPAG DCS effects in rats submitted tothe EPM

The rats received intra-dlPAG injections of saline or DCS (25,

50, 100 or 200 nmol; doses based on previous studies employingintracerebral infusion of DCS at the dose of 10 �g (roughly equiva-lent to 100 nmol) [15,16]) and 10 min later were placed in the centerof the EPM facing an enclosed arm. The percentages of entries and

376 F.V. Gomes et al. / Behavioural Brain Research 271 (2014) 374–379

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As seen in Fig. 2, DCS (200 nmol) significantly increasedthe percentage of entries (n = 6–10/group; F5,44 = 2.6, P < 0.05;

Fig. 2. Anxiolytic-like effect of DCS (25, 50, 100 or 200 nmol) injected into the dlPAGof rats submitted to the EPM. The upper panel shows the number of entries into theenclosed arms whereas the lower panel shows the percentage of entries (white

ig. 1. Photomicrograph of a coronal brain section from a representative rat showintlas of Paxinos and Watson [14], indicating the injection sites into (black circles) a

ime spent in the open arms (100 × open/(open + enclosed)) duringhe 5 min sessions in the EPM were calculated for each animal.

.4.3. Experiment 2: intra-dlPAG DCS effects on the light–darkox

Animals received intra-dlPAG injections of saline or DCS (25, 50,00 or 200 nmol) and 10 min later were placed in the dark side ofhe light–dark box. The number of entries and time spent in eachompartment was recorded for 5 min.

.4.4. Experiment 3: intra-dlPAG DCS effects in rats submitted tohe Vogel conflict test

An independent group of animals was water-deprived for 48 hefore the test. After the first 24 h of deprivation, they were allowedo freely drink water for 3 min in the test cage in order to find therinking bottle spout. Some animals did not find the spout and wereot included in the experiment. Based on the results of experiments

and 2, 24 h later they received intra-dlPAG injections of saline orCS (200 nmol) and after 10 min were placed inside the appara-

us. The test lasted 3 min, and the animals received a 0.5 mA shockhrough the bottle spout every 20 licks. The number of punishedicks was registered.

.4.5. Experiment 4: intra-dlPAG DCS effects on the open-field testIn order to control for a possible non-specific drug effect on

ocomotor activity, animals were submitted to the open field test.he animals received intra-dlPAG administration of saline or DCS200 nmol) and 10 min later were individually placed on the cen-er of a circular arena. The distance traveled by the animals was

easured for 5 min.

.5. Histological procedure

At the end of the experiments the rats were anesthetized withrethane (1.25 g/kg i.p., Sigma–Aldrich) and 0.2 �L of 1% Evan’slue dye was unilaterally infused into the dlPAG as an injectionite marker. The chest was surgically opened, the descending aortaccluded, the right atrium severed and the brain perfused with 10%ormalin through the left ventricle. Brains were post-fixed for 24 ht 4 ◦C, and 40 �m sections were cut using a cryostat (CM-1900,

eica, Germany). The injection site were located using the rat braintlas of Paxinos and Watson [14] as reference. Rats receiving injec-ions of the active dose of DCS outside the dlPAG were included inn additional group (OUT).

ction sites into the dlPAG and diagrammatic representation, based on the rat braintside (white circles) the dlPAG of all the animals used in the experiments.

2.6. Statistical analysis

Data were analyzed by one-way analysis of variance (ANOVA).Post-hoc analysis was performed using the Newman–Keuls test.Differences were considered significant at P < 0.05 level.

3. Results

Diagrammatic representations showing the injection sites inthe dlPAG and a representative photomicrograph are presented inFig. 1.

3.1. Experiment 1: intra-dlPAG DCS effects in rats submitted to

columns) and the time spent (black columns) in the open arms. Columns representthe means±SEM. Animals receiving DCS (200 nmol) injections outside the dlPAGwere joined in an additional group (OUT). Asterisk indicates significant differencefrom controls (SAL – saline, P < 0.05, ANOVA followed by the Newman–Keuls posthoc test; n = 9, 8, 10, 10, 7, 6, respectively).

F.V. Gomes et al. / Behavioural Brain Research 271 (2014) 374–379 377

Fig. 3. Effects of DCS (25, 50, 100 or 200 nmol) or saline injected into the dlPAG of ratssubmitted to the light–dark box. The upper panel indicates the number of transitionsinto the different compartments whereas the lower panel shows the time spentin the light compartment. Columns represent the means ± SEM. Animals receivingD(f

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Fig. 4. Effects of DCS (200 nmol) or saline injected into the dlPAG of rats submittedto the Vogel conflict test. Columns represent the means ± SEM of the total numberof punished licks in the 3-min session. Animals receiving DCS injections outside

CS (200 nmol) injections outside the dlPAG were joined in an additional groupOUT). Asterisk indicates significant difference from control group (P < 0.05, ANOVAollowed by the Newman–Keuls post hoc test; n = 18, 8, 10, 7, 10, 10, respectively).

ewman–Keuls, P < 0.05), but not the time spent (F5,44 = 1.5, > 0.05) in the open arms of the EPM as compared to saline-treatedats. Animals receiving the active dose of DCS (200 nmol) outsidehe dlPAG (OUT) were not different from controls (Newman–Keuls,

> 0.05). No effect on the number of enclosed arms entries wasound, suggesting that DCS is not affecting basal motor activityF5,44 = 0.8, P > 0.05).

.2. Experiment 2: intra-dlPAG DCS effects on the light–dark box

The administration of DCS (200 nmol) into the dlPAG increasedhe time spent in the light compartment (F5,57 = 3.8, P < 0.05;ewman–Keuls, P < 0.05) and the number of transitions into the

wo compartments (F5,57 = 2.8, P < 0.05; Newman–Keuls, P < 0.05;ig. 3) compared to the control group. No change was observedhen DCS at the dose of 200 nmol was administered into areasear to the dlPAG (group OUT; Newman–Keuls, P > 0.05).

.3. Experiment 3: intra-dlPAG DCS effects in rats submitted tohe Vogel conflict test

Consistent with the previous experiments, an anxiolytic-likeffect of DCS was also observed. In this experiment DCS (200 nmol)njection into the dlPAG increased the number of punished licksn = 6–13/group; F2,29 = 3.90, P < 0.05; Newman–Keuls, P < 0.05) asompared to saline-treated rats (Fig. 4). Moreover, animals receiv-ng the active dose of DCS (200 nmol) outside the dlPAG (OUT) wereot different from controls (Newman–Keuls, P > 0.05).

.4. Experiment 4: intra-dlPAG DCS effects on the open-field test

Intra-dlPAG injection of DCS (200 nmol) did not modify the loco-otor activity of rats in the open field test (vehicle: 10.6 ± 0.97;CS: 11.93 ± 0.61; n = 5/group, t8 = 1.16, P > 0.05).

the dlPAG were joined in an additional group (OUT). Asterisk indicates significantdifference from controls (P < 0.05, ANOVA followed by the Newman–Keuls post hoctest; n = 13, 13, 6, respectively).

4. Discussion

The present study investigated the effects induced by the admin-istration of DCS into the dlPAG of rats submitted to three animalmodels of anxiety, the EPM, light–dark and Vogel conflict tests. Thedrugs caused anxiolytic-like effects in all these models. Althoughthose tests have been extensively used to study anxiety, they arebased on different aversive contingencies that could involve differ-ent neurobiological substrates [17]. While the light–dark test andthe EPM are related to the innate aversion of rodents for lighterand open and high spaces, respectively [18,19], the Vogel conflicttest involves the suppression of punished responses [20]. Typically,anxiolytic drugs increase the exploration of the open arms in theEPM and the light compartment in the light–dark test [18,19], andthe number of licks in the Vogel test, even if they resulted in pun-ishment [21].

Intra-dlPAG administration of the higher dose (200 nmol) ofDCS increased the frequency of open arms exploration in the EPM,indicating an anxiolytic-like effect. A similar anxiolytic effect wasobserved in the light–dark test, where DCS increased the timespent in the light compartment. The light–dark test is based on theobservation that, even if rodents usually tend to explore novel envi-ronments, lighted areas have aversive properties that inhibit thisexploratory behavior. When given the choice between exploring alighted or a dark and enclosed area, the rodent will favor the latter[19]. Additionally, DCS administration into the dlPAG increased thenumber of transitions between the compartments induced. Besidesbeing an index associated with locomotion, the number of transi-tions may also reflect changes in anxiety [17]. However, DCS failedto change two usual measures of locomotor activity, the distancemoved in the open field test and the number of enclosed arm entriesin the EPM, discarding motor effect as a possible confounding factor.

In the Vogel conflict test, water deprived rats were allowedto freely drink water for 3 min. However, during this period, thisbehavior was punished by an electrical shock (0.5 �A) delivered onthe animal tongue by the drinking bottle spout. This paradigm isthought to induce a conflict situation that inhibits drinking behav-ior [20]. Similar to anxiolytic drugs, the administration of DCS intothe dlPAG increased the number of punished licks. Together, theseresults indicate that administration of DCS into the dlPAG inducesanxiolytic-like effect in animal models widely used for the studyof anxiety. Although the extent of DCS diffusion from the injectionsite cannot be established precisely, the absence of any effect whenthe effective dose of DCS was administered in neighboring struc-tures (group OUT) suggests that the effects were mediated by a drugaction in the dlPAG.

Our results agree with previous works showing that systemicadministration of DCS produces anxiolytic-like effects in rats sub-mitted to the EPM and Vogel conflict test [6,22]. Moreover, thepresent findings indicate that the dlPAG could be one of the brain

3 Brain

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ites responsible for these effects. Few studies have investigatedhe brain structures involved in DCS effects. Although DCS injec-ions into the amygdala, hippocampus and medial prefrontal cortexndices facilitation of fear conditioning extinction [16,23], cocaine-nduced place preference extinction [24] and radial maze learning25], this is the first study, to the best of our knowledge, that investi-ated the effects of intra-cerebral administration of DCS in animalsubmitted to classical animal models of anxiety.

The dlPAG is a structure located in the midbrain that plays anmportant role in the elaboration of active defensive responseso dangerous situations [26,27]. Under natural conditions, theseesponses are probably mediated by local glutamate release.ccordingly, intra-dlPAG administration of glutamate ionotropiceceptors (NMDA or non-NMDA) agonists in rats induces escapeeactions [12,28], whereas blockade of these receptors causesnxiolytic-like effects in models such as the EPM and the Vogelonflict test [13,29,30].

As mentioned in the Introduction section 1, in vitro studies indi-ate that DCS acts as a partial agonist at the glycine binding site ofhe NMDAR [22,31,32]. This site is activated by endogenous glycinend is required for receptor activation by glutamate [3]. In con-equence, NMDAR function can be altered by administration oflycine site ligands [33]. Indeed, antagonists and partial agonistsf the glycine site inhibit the function of the NMDAR complex androduce effects similar to those produced by competitive and non-ompetitive NMDAR antagonist [22,34,35]. In this context, in vivotudies have indicated that DCS produces agonistic and antagonis-ic effects predominating at low and high doses, respectively [4,34].onsistent with these findings, the anxiolytic-like activity of DCSfter systemic injection appears only at high doses, an effect con-istent with antagonist activity. Our results are also similar to thenxiolytic-like effects observed after administration of antagonistsf the glycine site [7,8] or NMDAR in the dlPAG [13,30]. On the otherand, it is rather unlikely that the anxiolytic-like effects of intra-lPAG DCS administration depend on stimulation of the glycineite coupled to the NMDAR, since the intra-dlPAG administrationf the glycine site full agonists, d-serine and glycine itself, inducednxiogenic-like effects [9]. Together, these pieces of evidence sug-est that DCS effects in the present study depend on its antagonistroperties at the glycine site.

Given that DCS shows agonistic effects predominating at lowose, we could be expected that at low doses (25, 50 or 100 nmol)CS would induce anxiogenic-like effects, but no such effect wasbserved. However, DCS has been reported to have an efficacy of0–60% of that of glycine [31,32,34]. This may help to explain the

ack of anxiogenic effects of this drug in the dlPAG compared toull agonists such as glycine or d-serine [9]. Additionally, it haseen suggest a differential participation of the rostral and cau-al areas of the dPAG in the anxiogenic-like effects induced bylycine ligands. Whereas an anxiolytic-like effect was observedfter administration of glycine antagonists in all levels of thePAG (rostral, IA = 3.12 mm; intermediate, IA = 2.28 mm; caudal,

A = 1.32 mm), glycine induced an anxiogenic-like effect only whendministrated to the caudal levels [10]. This could also help toxplain the lack of anxiogenic effects of low DCS doses, since ournjections sites were located largely at intermediate levels of theAG (as shown in the Fig. 1).

Recent successes as a pharmacological adjunct to exposure ther-py suggest that DCS has therapeutic potential in certain clinicalnxiety disorders. For example, DCS facilitates psychotherapy forcrophobia [36], social phobia [37,38], obsessive-compulsive dis-rder [39], panic disorder [40], and post-traumatic stress disorder

41]. Although believed to reflect a facilitation of extinction learn-ng, our results and previous research with DCS and other glycineartial agonists suggests the additional possibility of intrinsic anx-

olytic activity.

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Research 271 (2014) 374–379

In summary, the present study shows that administration ofDCS, a partial agonist of the NMDAR-associated glycine site, into thedlPAG induces anxiolytic-like effects in different animal models.These results are consistent with the proposal that the anxiolytic-like effects induced by this drug depend on the use of high dosessuggesting that DCS acts by antagonizing the glycine site of theNMDAR. Moreover, the results also support of the involvement ofthe NMDAR-associated glycine site within the dlPAG in the modu-lation of defensive behaviors.

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgments

The authors thank J.C. de Aguiar and L. Camargo for technicalsupport. This work was supported by grants from CNPq, CAPES,and FAPESP. FVG is a recipient of a FAPESP doctoral fellowship(2010/17343-0).

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