Behavioural Brain Research 164 (2005) 52–60

Research report

Metabotropic glutamate receptor 1 blockade impairs acquisitionand retention in a spatial Water maze task

Thomas Steckler∗, Ana F.M. Oliveira, Chistophe Van Dyck, Hansfried Van Craenendonck,Ana M.A. Mateus, Xavier Langlois, Anne S.J. Lesage, Jos Prickaerts1

Division of Psychiatry, Research and Early Development Unit Europe, Johnson & Johnson Pharmaceutical Research & Development,Turnhoutseweg 30, B-2340 Beerse, Belgium

Received 24 March 2005; received in revised form 25 May 2005; accepted 27 May 2005Available online 25 July 2005

Abstract

Metabotropic glutamate receptors, including the mGlu1 receptor, have received considerable attention as potential targets for anxiolytic,antidepressant, antipsychotic and antinociceptive drugs. mGlu1 receptors have also been suggested to play a role in the modulation of cognitivep ihydro-2H-p mandingl 1 receptorb lready at thel o locate thep 16259685.T nformationr ose tested(o torsa entral mGlur on.©

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rocesses, but knowledge is still very limited. In the present study the effects of the selective mGlu1 receptor antagonist 3,4-dyrano[2,3]�-quinolin-7-yl)(cis-4-methoxycyclohexyl)methanone (JNJ16259685, 0.63–10 mg/kg s.c.) on more or less spatially de

earning and spatial memory (retention and re-acquisition) were investigated in mice performing in a water maze. Selective mGlulockade with JNJ16259685 impaired spatial acquisition processes, irrespective of spatial load, as well as spatial re-acquisition, a

owest dose tested (0.63 mg/kg). In contrast, effects on spatial retention performance were relatively mild in mice that had learned tosition of the escape platform prior to treatment. Thigmotaxic behaviour and locomotor activity appeared to be unaffected by JNJhese data suggest that blockade of the mGlu1 receptor primarily affects learning of new information, but leaves retention of spatial i

elatively unaffected. Blockade of the mGlu5 receptor with MPEP also impaired spatial learning, although only at the highest d10 mg/kg). An ex vivo receptor occupancy study in rats revealed that MPEP occupied central mGlu5 receptors with an ED50 of 2.0 mg/kgne hour after subcutaneous administration. This is 50–150 times higher than the ED50 reported for JNJ16259685 at central mGlu1 recepnd suggests that one reason why the two compounds cause cognitive effects at different doses might be due to differences in ceceptor occupancy, rather than fundamentally different roles of mGlu1 and mGlu5 receptors in the modulation of cognitive functi

2005 Elsevier B.V. All rights reserved.

eywords:Metabotropic glutamate receptor 1; mGlu1; Spatial; Learning; Memory; Retention

. Introduction

Glutamate is the major excitatory neurotransmitter inhe brain. It mediates its functions through two types ofeceptors, ionotropic glutamate (iGlu) receptors, such as theMDA, AMPA and kainate receptors, and metabotropic glu-

amate (mGlu) receptors[22]. The mGlu receptors formfamily of eight subtypes, which are divided into three

roups (Group I: mGlu1 and mGlu5; Group II: mGlu 2

∗ Corresponding author. Tel.: +32 14 60 7373; fax: +32 14 60 3753.E-mail address:tsteckle@prdbe.jnj.com (T. Steckler).

1 Present address: Department of Psychiatry and Neuropsychology, Maas-richt University, Maastricht, The Netherlands.

and mGlu3 and Group III: mGlu4, mGlu6, mGlu7 amGlu8) [8,9,12]. Over recent years, the mGlu recepthave received considerable attention as potential targeanxiolytic, antidepressant, antipsychotic and antinocicedrugs[5,6,14,18,20,21,25–27,30]. More specifically, mGlureceptors have been proposed to be involved in the medof anxiety-related processes[7,15,29,32].

However, mGlu1 receptors have also been suggestplay a role in the modulation of cognitive processes, bon results obtained with mouse mutants[1,10] or usingeither non-selective mGlu1/mGlu5 antagonists and/or cpounds that were locally administered into the hippocamarea[4,8,19,23,24,31]. Hippocampal long-term potentiati(LTP) was impaired in knockout mice lacking the mG

166-4328/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.bbr.2005.05.010

T. Steckler et al. / Behavioural Brain Research 164 (2005) 52–60 53

receptor[1,10], and more recently it was demonstrated thatpharmacological mGlu1 receptor blockade also impairs hip-pocampal LTP[23]. All this suggests a prominent role of themGlu1 receptor in learning and memory processes. However,a more detailed investigation of the different types and stagesof memory affected by mGlu1 receptor blockade has onlyrecently begun.

In the present study we examined the effects of theselective mGlu1 receptor antagonist 3,4-dihydro-2H-pyrano[2,3]�-quinolin-7-yl)(cis-4-methoxycyclohexyl)met-hanone (JNJ16259685, also named R199912) on spatialand non-spatial learning (acquisition) and spatial memory(retention and re-acquisition) in a water maze in mice.JNJ16259685 is a selective mGlu1 antagonist with anaffinity of 0.34 nM (Ki value) and antagonist potency of3.24 nM (IC50 value) for rat mGlu1 receptor[17]. Aftersubcutaneous administration, the compound has beenshown to occupy central mGlu1 receptors with an ED50of 0.014–0.04 mg/kg, indicating its good brain penetration[17]. In contrast, JNJ16259685 only has low activity at themGlu5 receptor and inhibits the glutamate-induced rise inintracellular Ca2+ concentrations at the rat mGlu5 receptoronly at high concentrations (IC50 value of 1.3�M) [17].Further, the effects of the mGlu5 receptor antagonist MPEPwere investigated. MPEP blocks the mGlu5 receptor witha potency of 36 nM (IC value) but has no affinity fort wnt yedm atiall thep tionwp itivee vivom derc forJ

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�-cyclodextrin acidified with tartaric acid. Because of poor oralbioavailability of JNJ16259685[29], the compounds were adminis-tered subcutaneously (s.c.), 30 min before the start of a session/probetrial, at a dose range from 0.63 to 10 mg/kg.

2.3. Behavioural apparatus

A circular swimming pool (diameter 100 cm, white plastic) wasfilled with water (about 25◦C), rendered opaque by the addition of anon-toxic dye. Eight start boxes (10× 10 cm, also filled with water)fitted with sliding doors were fixed at the outside of the maze. Amouse could enter the maze when the door was raised[2]. Mice hadto find a submerged transparent plexiglass platform (diameter 9 cm,1 cm below water level) with a fixed position in the pool. Ten cmabove the water level at the position of the submerged platform aplastic white cone (height 3 cm, lower diameter 1.5 cm) was hangingupside down. A second cone was hanging 10 cm above the waterlevel exactly opposite from the one above the submerged platform.The circular area (diameter 9 cm) underneath that cone served as areference area (dummy platform area). Performance was recordedvia a video camera connected to a video tracking system (Ethovision,Noldus, The Netherlands).

2.4. Spatial acquisition

Mice were trained over five sessions, with 10 trials per dailysession. A trial lasted maximally 30 s. The start positions were ran-domized over two positions (equidistant to the escape platform andt nshipb con-s thep ocen-t ed ina

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50he mGlu1 receptor[11]. MPEP has recently been shoo induce mild cognitive impairment in operant delaatching to position, but to leave water maze sp

earning in rats relatively unaffected except duringrobe trial, where a small but significant effect on retenas seen at the highest dose tested (30 mg/kg)[3]. To putotential compound-related differences to cause cognffects better into context, we also performed an exGlu5 receptor occupancy study with MPEP in rats un

onditions comparable to those previously reportedNJ16259685.

. Material and methods

.1. Animals

The present study was approved by the Animal Ethics Boa&J PRD, Belgium. Male C57BL/6 mice (body weights 22–2anvier, France) were used, 9–10 animals per drug dose/vroup. The animals were individually housed in individually ve

ated type 2 cages and had free access to food and water. Theept under a normal 12/12 h light/dark cycle (lights on from 6.08.00 h). For ex vivo receptor occupancy, male Wistar rats weig20–240 g (Charles River, Sulzfeld, Germany) were used.

.2. Drug treatment

For behavioural testing and ex vivo receptor occupaNJ16259685 and 2-methyl-6-(phenylethynyl)pyridine (MPocris-Cookson, Essex, UK) were dissolved in 10% hydroxypro

he dummy reference area) to ensure that the spatial relatioetween the start position and the platform position did notistently reward turns into one direction, i.e., mice had to findlatform on the basis of spatial cues surrounding the maze (all

ric orientation). At the end of the last session, mice were testprobe trial (60 s) without platform.

.5. Less demanding spatial acquisition

In the second experiment, all conditions were identical to thexperiment, except that only one fixed starting position was uhis makes the task conceptually easier to perform and animase both spatial (allocentric) and non-spatial (egocentric) strat

.6. Spatial retention and spatial re-acquisition

In experiment three, mice were first trained for six sessions,lar to the first experiment but only receiving saline injections po training. This was followed by a probe trial 24 h after all animad learned the spatial task. Thirty minutes before the probehich lasted 30 s, animals received treatment with JNJ162596ssess the effects on retention. In the following two days, aneceived two sessions (10 trials per session) in which the escaporm and the dummy reference positions were reversed and anad to re-learn this position.

.7. Behavioural measures

During acquisition, the total distance swum and the relativepent in the margin (outer 9 cm ring of the maze) were meashe mean distance and mean relative time spent in the marginalculated per block of 10 daily trials. Finding the submerged

54 T. Steckler et al. / Behavioural Brain Research 164 (2005) 52–60

form prior to crossing the dummy reference area was considered acorrect choice, whereas if a mouse crossed the reference area firstit was counted as an incorrect choice. If a mouse did not find theplatform nor crossed the reference area during a trial it was countedas an error of omission. A correct choice was given the value of +1,an incorrect choice was given the value of−1, while an error ofomission (no choice) was given a value of zero. From these data itwas possible to calculate an accuracy index per day as an additionalmeasure of spatial performance, with accuracy index = sum of allchoice values/number of trials. Chance performance would result inan accuracy score of 0, whereas optimal performance would be 1.In addition, the total number of errors of omission was calculatedper day. During the probe trials, the time spent in each quadrant, thetotal distance swum and the number of crossings over the formerplatform position were measured.

2.8. Ex vivo mGlu5 receptor occupancy

Rats were treated by subcutaneous administration of vehicle orMPEP at five dosages ranging from 0.16 to 40 mg/kg (three animalsper dose). The animals were decapitated 1 h after drug administra-tion. Brains were immediately removed and rapidly frozen in dryice-cooled 2-methylbutane (−40◦C). Coronal sections at the levelof the striatum (20�m thick) were cut with a Leica CM3050 cryostatmicrotome (van Hopplynus, Belgium) and thaw-mounted on adhe-sive microscope slides (Superfrost Plus, Menzel-Glaser, Germany).The sections were stored at−20◦C until use. After thawing, sec-tions were dried under a cold stream air. Three adjacent brain slicesf wereu ed forn sec-t5CS after17 ry-i lysisw ,Pv ccu-p g theG

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ment× session interaction (F(12, 128) = 1.80,P= 0.055).Visual inspection of the data (Fig. 1A) suggested that vehicle-treated animals reached higher accuracy level at the end of theacquisition phase, but this effect did not reach significance(F(3, 32) = 2.83,P= 0.054). The number of errors of omis-sion decreased over sessions (F(4, 128) = 22.18,P< 0.001;data not shown) and a tendency for a session× treatmentinteraction effect was also seen for this measure (F(12,128) = 1.69,P= 0.075). Furthermore, an overall treatmenteffect was found in the number of errors of omission(F(3, 32) = 3.27,P< 0.05). Post-hoc analysis revealed thatvehicle-treated mice made less errors of omission than allthe groups treated with JNJ16259685 (LSD post-hoc tests,P’s < 0.05). An effect of treatment was also observed in theoverall distance swum to reach the escape platform dur-ing training (F(3, 32) = 3.56,P< 0.05; Fig. 1B). The latterwas caused by the vehicle-treated mice swimming a shorterdistance than the JNJ16259685-treated groups (LSD post-hoc tests,P’s < 0.05), which did not differ from each other.In addition, the mice reduced the distance swum to theplatform during acquisition (F(4, 128) = 14.74,P< 0.001)and the session× treatment interaction effect indicated thatrate in reduction was different between the experimentalgroups (F(12, 128) = 4.07,P< 0.001). The relative time spentin the margin of the maze decreased over sessions (F(4,128) = 13.72,P< 0.001; data not shown), but the experi-mP

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rom the same animal were collected per slide. Two brain slicessed to measure the total binding, and the third one was uson-specific binding. Total binding was measured by incubating

ions with 1 nM [3H] MPEP (Tocris-Cookson, Essex, UK) in 400�l0 mM Tris–HCl buffer (pH 7.4), containing 1.2 mM MgCl2, 2 mMaCl2. Non-specific binding was determined by addition of 200�MIB 1757 to the incubation buffer. The reaction was stopped0 min by washing the slides (3× 5 min) with Tris–HCl buffer (pH.4) at 4◦C, followed by a rapid dip in cold distilled water and d

ng under a stream of cold air. Quantitative autoradiography anaas performed after 1 h acquisition with the�-imager (BioSpacearis, France) according to our standard protocol[16]. The ED50

alue (dose of compound producing 50% of mGlu5 receptor oancy) was calculated by nonlinear regression analysis, usinraphPad Prism program (San Diego, CA, USA).

.9. Behavioural data analysis

During acquisition, data were analyzed using a two-way ANOsession× treatment), with repeated measures on session. Probata were analyzed with a one samplet-test (difference from chanc

evel) or one-way ANOVA (treatment). In case of significant effen the ANOVA, data were further analyzed in detail with the leignificant difference (LSD) post-hoc test (P< 0.05).

. Results

.1. Effects of mGlu1 receptor blockade on spatialcquisition

Mice increased accuracy over daily sessions (F(4,28) = 5.48,P< 0.001) and there was a tendency for a tr

ental groups did not differ from each other (F’s < 2.21,’s > 0.05).

During the probe trial, which was given immediately ahe last training session, only vehicle-treated mice sho

preference above chance level for the quadrant whe escape platform was located during training (t(8) = 2.34< 0.05;Fig. 1C), while the relative time spent in this qua

ant did not differ between treatment groups (F(3, 35) = 1.57> 0.05). However, there was a treatment effect in the numf crossings above the location of the former platform p

ion (F(3, 35) = 3.61,P< 0.05) and post-hoc analysis showhat all three JNJ16259685-treated groups crossed ther platform position less frequent than the vehicle-treice (P’s < 0.05;Fig. 1D). The total distance swum duri

he probe trial, which can be considered as an indirecture of speed, i.e. locomotor activity, was not affectedGlu1 receptor blockade (F(3, 35) = 0.66,P> 0.05; data no

hown).

.2. Effects of mGlu1 receptor blockade on lessemanding spatial acquisition

The accuracy index (Fig. 2A) and number of errors omission increased and decreased, respectively, oveions (bothF’s > 16.45,P’s < 0.001). For both measuresreatment effect was apparent (bothF’s > 3.78,P’s < 0.05).ost-hoc analysis showed that the vehicle-treated grou

ormed at higher accuracy level and made less erromission when compared to the JNJ16259685-treated gP’s < 0.05), but animals treated with the mGlu1 antago

T. Steckler et al. / Behavioural Brain Research 164 (2005) 52–60 55

Fig. 1. Effects of JNJ16259685 on spatial acquisition in C57BL/6 mice. Measures are shown as means + S.E.M. (A) The rate in the increase of the accuracyindex tended to be different between the experimental groups. (B) The distance swum to the platform during training showed that only the vehicle-treated grouplearned to locate the platform. (C) During the probe trial only vehicle-treated mice spent relative time in the training quadrant above chance level (25% dottedline); *p< 0.05. (D) During the probe trial all JNJ16259685-treated groups made less platform crossings than the vehicle-treated group;#p< 0.05.

did not differ from each other. An effect of mGlu1 blockadewas also found in the overall distance swum to the escapeplatform (F(3, 32) = 9.40,P< 0.001;Fig. 2B). Post-hoc anal-ysis revealed that the vehicle-treated group swum a smallerdistance than the JNJ16259685-treated groups (P’s < 0.05),which again did not differ from each other. The distanceswum to the platform also decreased over sessions (F(4,128) = 16.07,P< 0.001) and the rate of decrease tended to bedifferent between the experimental groups (F(12, 128) = 1.65,P= 0.087). The relative time the mice spent in the margin ofthe maze decreased over sessions (F(4, 128) = 2.75,P< 0.05;data not shown), but did not differ between groups (F’s < 0.92,P’s > 0.05).

In the probe trial, which took place immediately afterthe last training session, only vehicle-treated mice spenttime in the training quadrant above chance level (t(8) = 2.78,P< 0.05;Fig. 2C). Comparison between groups showed thatthe relative time spent in the training quadrant was dif-ferent between the experimental groups (F(3, 35) = 2.94,P< 0.05). However, post-hoc analysis showed that only thegroup treated with a dose of 2.5 mg/kg JNJ16259685 spentless relative time in the training quadrant than the vehicle-treated group (Fig. 2C). There was a tendency in the number

of platform crossings between groups, which just failed toreach significance (F(3, 35) = 2.82,P< 0.055;Fig. 2D).

3.3. Effects of mGlu1 receptor blockade on spatialretention (probe trial) and spatial re-acquisition(reversal learning)

During spatial learning, all mice increased their accu-racy index over daily sessions (F(5, 235) = 16.93,P< 0.001;data not shown) and reduced the distance swum to the plat-form (F(5, 235) = 108.40,P< 0.001; data not shown). Inaddition, the relative time spent in the margin as well asthe number of errors of omission decreased over sessions(F’s > 7.81,P’s < 0.001; data not shown). Of note, the experi-mental groups, which were matched at the end of the trainingstage, performed at comparable level (data not shown).

When animals were treated before the probe trial, whichwas given 24 h after the last session, only the vehicle-treatedmice spent time in the training quadrant above chance level(t(9) = 3.25,P< 0.05;Fig. 3A). Moreover, the relative timespent in the training quadrant tended to be above chance levelin mice treated with 1.25 mg/kg JNJ16259685 (t(9) = 2.07,P= 0.069). When compared between groups, no difference

56 T. Steckler et al. / Behavioural Brain Research 164 (2005) 52–60

Fig. 2. Effects of JNJ16259685 on less demanding spatial acquisition in C57BL/6 mice. Measures are shown as means + S.E.M. (A) mGlu1 receptor blockadeimpaired accuracy. (B) Distance swum to the platform: the vehicle-treated group learned to locate the platform, while all JNJ16259685-treated miceshowedimpaired acquisition. (C) Relative time spent in the training quadrant during the probe trial: only vehicle-treated mice performed above chance level (25%dotted line);*p< 0.05. (D) The number of platform crossings only tended to be different between groups (p= 0.055).

was seen in this measure (F(4, 47) = 0.95,P> 0.05), in thenumber of platform crossings (F(4, 47) = 0.72,P> 0.05;Fig. 3B) or in the total distance swum during the probe trial(F(4, 47) = 1.70,P> 0.05, data not shown).

Spatial reversal learning was tested in the following twodays after the probe trial. The accuracy index increased overthe two reversal sessions (F(1, 43) = 7.42,P< 0.01) and atreatment effect was seen for this measure (F(4, 43) = 4.01,P< 0.01), but no significant treatment× session interaction

was found (F(4, 43) = 1.47,P> 0.05). Post-hoc analysis ofthe treatment effect showed that the vehicle-treated animalsreached a higher accuracy index than groups treated with0.63, 1.25 or 5 mg/kg of the mGlu1 antagonist (Fig. 4A).The number of errors of omission and the relative timespent in the margin were not affected by JNJ16259685 dur-ing this stage of the experiment (F’s < 0.58,P’s > 0.05; datanot shown). There was a decrease in the distance swum tothe new platform position over the two reversal sessions

Fig. 3. Effects of JNJ16259685 on spatial retention in C57BL/6 mice. Measures are shown as means + S.E.M. (A) During the probe trial, only the vehicle-treatedmice spent significantly more time in the training quadrant (chance level 25%; dotted line);*p< 0.05,*p= 0.069. (B) The number of platform crossings duringthe probe trial of the JNJ16259685-treated groups did not differ between groups.

T. Steckler et al. / Behavioural Brain Research 164 (2005) 52–60 57

Fig. 4. Effects of JNJ16259685 on spatial re-acquisition in C57BL/6 mice. Measures are shown as means + S.E.M. (A) mGlu1 receptor blockade impairedaccuracy during the two reversal sessions. (B) The decrease in distance swum was also affected by mGlu1 receptor blockade.

(F(1, 43) = 6.39,P< 0.05) and although groups did not differin the overall distance swum to the new platform position(F(4, 43) = 1.94,P> 0.05), a treatment× session interactioneffect was seen (F(4,43) = 3.28,P< 0.05). Since the interac-tion effect indicated differences in the rate of learning, post-hoc analysis was performed on the delta distance swum to

platform (day 1− day 2) as a measure of the rate of learning.It was found that the rate of learning of the 1.25 and 5 mg/kgJNJ16259685 groups was affected when compared with thevehicle group (Fig. 4A). Swim speed did not change over thetwo sessions and did not differ between groups (F’s < 1.25,P’s > 0.05).

FbalM

ig. 5. Effects of MPEP on spatial acquisition in C57BL/6 mice. Measures aretween the experimental groups. Treatment with 10 mg/kg MPEP increasend the time spent in the periphery (D). (E) During the probe trial 0.63 mg/kg

evel 25%; dotted line);*p= 0.089. (F) The 10 mg/kg MPEP-treated group madPEP-treated groups;#p< 0.05.

e shown as means + S.E.M. (A) The increase of the accuracy index did not differd the number of errors of omissions (B), the distance swum to the platform (C)MPEP-treated mice tended to spend more time in the training quadrant (chancee less platform crossings during the probe trial than the vehicle and 0.63 mg/kg

58 T. Steckler et al. / Behavioural Brain Research 164 (2005) 52–60

3.4. Effects of mGlu5 receptor blockade on spatiallearning

Again, there was an effect of session on various perfor-mance measures (accuracy index:F(4, 136) = 5.60,P< 0.001;number of errors of omission:F(4, 136) = 37.63,P< 0.001;distance swum to reach the platform:F(4, 136) = 14.05,P< 0.001; relative time spent in the margin of the maze:F(4,136) = 10.50,P< 0.001;Fig. 5A–D). However, the experi-mental groups did not significantly differ from each otherin this accuracy measure and no interaction effect was seen(F’s < 2.67,P’s > 0.05;Fig. 5A). A treatment× session inter-action effect was found in the total number of errors ofomissions made (F(12, 136) = 2.67,P< 0.01; Fig. 5B), inthe distance swum to reach the platform (F(12, 136) = 2.71,P< 0.01Fig. 5C) and in the relative time spent in the mar-gin of the maze (F(12, 136) = 2.76,P< 0.01; Fig. 5D). Atthe end of acquisition, i.e. session 5, the errors of omis-sion and distance swum were different between groups (bothF’s > 4.62,P’s < 0.05), while the relative time in the marginwas not (F(3, 37) = 1.90,P> 0.05). Post-hoc analysis revealedthat the vehicle, 0.63 and 2.5 mg/kg MPEP groups made thesame number of errors of omission at the end of acquisition,while mice treated with the 10 mg/kg dose of MPEP madesignificantly more omissions. In addition, post-hoc analy-sis revealed that mice receiving 10 mg/kg MPEP swum al atedw h thep

ea-s ringp es-s3 /kgM bovec ,t reat-m l-y EPm micet -t ults( :5m ,P PEP( ateda

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Fig. 6. Ex vivo mGlu5 receptor occupancy of MPEP. Dose response curveof the ability of in vivo MPEP administration to prevent [3H]MPEP bindingto the rat striatum ex vivo.

4. Discussion

Already low doses of the mGlu1 antagonist JNJ1625968(0.63 mg/kg) impaired acquisition in the task with higher spa-tial demand: there was a strong tendency for vehicle-treatedmice to reach higher accuracy level, which just failed sig-nificance, and mGlu1 receptor blockade resulted into moreerrors of omission and longer distances swum. This effectwas further corroborated by the probe trial, during whichmice treated with JNJ1625968 showed no preference for theold platform quadrant and crossed the previous platform loca-tion significantly less frequent. No effect of mGlu1 receptorblockade was seen on thigmotaxic behaviour. Likewise, weobserved impaired performance in this task after administra-tion of the NMDA receptor antagonist MK-801 at a dose of0.05 mg/kg, indicating its sensitivity to glutamatergic manip-ulations (unpublished observation). Comparable behaviouraleffects have been reported after hippocampal lesions in mice[2], although it should be noted that experimental designsdiffered as in the latter study mice were required to performa spatial discrimination between two visible platforms, onebeing stable and the other unstable. Thus, the present data arein line with a hippocampal deficit induced by mGlu1 receptorblockade[1,4,8,10,19,23,24,31].

In contrast to hippocampal lesions, which did not affectperformance in animals always starting from the same posi-t redt sin-g lu1r spa-t ts ofJ n ando n theo earn-i roupsrd

onger distance, while vehicle-treated mice or mice treith 0.63 mg/kg MPEP swum the lowest distance to reaclatform.

Only weak statistical effects were found on the mure relative time spent in the training quadrant durobe trial testing immediately after the last training sion: a tendency for a group difference was seen (F(3,7) = 2.83,P= 0.053) and only mice treated with 0.63 mgPEP tended to spent time in the training quadrant a

hance level (t(9) = 1.93, P= 0.089; Fig. 5E). Howeverhe number of platform crossings showed a clear tent effect (F(3, 37) = 6.05,P< 0.01) and post-hoc ana

sis revealed that the mice treated with 10 mg/kg MPade less crossings than the vehicle-treated mice or

reated with 0.63 mg/kg MPEP (Fig. 5F). The total disance swum during the probe trial showed similar resvehicle: 724± 57 cm; 0.63 mg/kg: 817± 88 cm; 2.5 mg/kg89± 64 cm; 10 mg/kg: 434± 46 cm; means± S.E.M.). Thiseasure also revealed a group difference (F(3, 37) = 4.64< 0.01) and mice treated with the highest dose of M

10 mg/kg) swum a shorter distance than the vehicle-trend 0.63 mg/kg MPEP-treated groups.

.5. Ex vivo mGlu5 receptor occupancy

In vivo mGlu5 receptor occupancy was measured bivo autoradiography 1 h after subcutaneous administrf MPEP. It was found that MPEP dose-dependently oied the mGlu5 receptor (seeFig. 6) with an ED50 (95%onfidence limits) of 2.0 mg/kg (1.3–3.0).

ion in the maze[2], mGlu1 receptor blockade also impaihe less spatially demanding water maze task with onele start location. If at all, the deficit induced by mGeceptor blockade was more pronounced than in theially more demanding task: there were significant effecNJ16259685 on accuracy as well as errors of omission the distance swum to reach the escape platform. Other hand, accuracy in the less spatially demanding l

ng task increased and distance swum decreased in the geceiving treatment with JNJ16259685 as well (Fig. 2), whichiffered from the flat learning curve in experiment 1 (Fig. 1),

T. Steckler et al. / Behavioural Brain Research 164 (2005) 52–60 59

demonstrating some residual learning capability under theseconditions. This would suggest that the mice could at leastpartially revert to a non-spatial strategy even though themGlu1 receptor was blocked. However, the impairment wasalso apparent in the probe trial, during which mice treatedwith JNJ16259685 again failed to show a preference for theprevious target quadrant. Again, no effect on thigmotaxiswas seen. These findings would suggest that areas other thanthe hippocampus were also negatively affected by mGlu1receptor blockade, which can be expected, given the widedistribution of this receptor throughout the brain[28]. Alongsimilar lines, it has recently been shown that a metabolicallyless stable analog of JNJ16259685 (JNJ16567083, namedEMQMCM in Ref.[12]) impairs acquisition of another non-spatial paradigm, i.e. passive avoidance[12].

Blockade of the mGlu5 receptor by MPEP also impairedspatial learning, although effects were less pronounced thanafter mGlu1 receptor blockade. Only the highest dose tested(10 mg/kg) caused an impairment during acquisition of themice, which might be influenced by a decreased locomotoractivity as found in the probe trial. Nevertheless, this findingis in line with a previous report, where only relatively smalleffects were seen in a rat water maze and in an operant delayedmatching to position task[3]. However, in the latter spatialwater maze task, only retention was affected while the rat’slocomotor activity was not affected.

lu5r r,J anE re-b i.e.,J whichi indtA ffer-e ptoro hyt arel withJ lock-a itivef mina g/kgJ thep s area ea

lasta t sig-n ncel cqui-s ther er ofp ntalg erew nav-

igate in a spatial environment. This finding is in agreementwith data from others[12], showing that passive avoidanceretention was not affected by mGlu1 receptor blockade. Thus,blockade of the mGlu1 receptor appears to primarily impairlearning, but leaves retention of already stored informationrelatively intact.

However, performance was again impaired followingtreatment with JNJ16259685 once there was a requirementto learn new information during spatial reversal, i.e., whenanimals had to re-learn the new platform location.

Of note, JNJ16259685 did not affect the total distanceswum during probe trials, suggesting that the behaviouralimpairments observed in the present study, where the micewere forced to move, were not due to locomotor effects. Thisis of relevance as JNJ16259685 has been shown to reducespontaneous locomotor activity[29].

Both mGlu1 and mGlu5 receptor blockade withJNJ16259685 or MPEP have been suggested to have thera-peutic potential for the treatment of anxiety disorders[29], butour data indicate that cognitive dysfunction can be expectedas side effect. JNJ16259685 already impaired learning atdoses which were below those required to exert anxiolytic-like properties in rats, while MPEP showed anxiolytic-likeeffects at doses which did not cause learning deficits in thepresent study[29]. This could suggest a more favourablesafety window for the latter compound. However, it shouldb alinet itsa

ckadeo f dif-f henm orml ten-t aredt

A

ancew

R

awaecificCell

ionstask

rendentology

In the present study, MPEP occupied central mGeceptors in rats with an ED50 of 2.0 mg/kg. HoweveNJ16259685 occupied central mGlu1 receptors withD50 value of 0.014 mg/kg (thalamus) to 0.04 mg/kg (ceellum) in rats 1 h after subcutaneous treatment,NJ16259685 caused a half maximal response at a doses 50–150 times lower than that required for MPEP to bo central mGlu5 receptors under identical conditions[17].lthough these data do not allow to rule out species dinces, this difference in central mGlu1 and mGlu5 receccupancy by the two compounds could well explain w

he cognitive effects seen after MPEP administrationess pronounced than after mGlu1 receptor blockadeNJ16259685 and do not support the conclusion that bde of mGlu5 receptors would be less detrimental to cogn

unction than blockade of mGlu1 receptors. In fact, 30fter subcutaneous administration of a dose of 0.63 mNJ16259685, i.e., at the time of behavioural testing inresent study, more than 80% of central mGlu1 receptorlready occupied in rats[17], which would explain why wlready observed effects at this low dose.

When treated before the probe trial, 24 h after thecquisition session, only the vehicle-treated mice spenificantly more time in the training quadrant above cha

evel, suggesting that JNJ16259685 not only affected aition, but also retention of spatial information. However,elative time spent in the training quadrant and the numblatform crossings were not different between experimeroups indicating that the deficit was partial, i.e., that thas a residual ability of JNJ16259685-treated mice to

e noted that MPEP also interacts with the noradrenransporter with low affinity, which might contribute tonxiolytic properties[13].

Taken together, these data suggest that selective blof the mGlu1 receptor impaired acquisition processes o

erent spatial demand. A partial dissociation was seen wice were tested for their ability to remember the platf

ocation: although the mGlu1 receptor blockade altered reion processes, these effects were relatively mild compo the clear-cut effects seen during spatial learning.

cknowledgement

The authors thank Eva Luisa van Donkelaar for assistith the data analysis.

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