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Brain Research Bulletin 70 (2006) 356–367

NMDA receptor activity in learning spatial procedural strategiesII. The influence of cerebellar lesions

Francesca Federico a,d, Maria G. Leggio a,b, Paola Neri b,Laura Mandolesi a,c, Laura Petrosini a,b,∗

a Department of Psychology, University of Rome “La Sapienza”, IRCCS S. Lucia, Via dei Marsi 78, 00185 Rome, Italyb Department of Psychology, University of Rome “La Sapienza”, Rome, Italy

c University of Naples “Parthenope”, Naples, Italyd University of Siena, Siena, Italy

Received 13 January 2006; received in revised form 12 June 2006; accepted 15 June 2006Available online 5 July 2006

bstract

Experimental data support the involvement of cerebellar circuits in the acquisition of spatial procedural competences. Since the ability to acquireew procedural competences is lost when cerebellar regions are lesioned or when NMDA receptor activity is blocked, we analyzed whether theearning of explorative strategies is affected by blocking NMDA receptor activity in the presence of cerebellar lesions. To this aim, the NMDAeceptor antagonist (CGS 19755, 7 mg/kg) was administered i.p. to un-lesioned rats, or rats subjected to total ablation of the cerebellum or to hemi-erebellectomy. CGS 19755 and cerebellectomy both produced water maze behavior characterized by circling. Administration of CGS 19755 didot modify the Morris Water Maze (MWM) peripheral circling behavior of cerebellectomized animals. Circling was the dominant strategy ofemicerebellectomized animals in the absence of drugs. However, increasingly compulsive circling was observed under the action of CGS 19755.

ircling was not observed if the drug-treated animals (un-lesioned or lesioned) had been previously trained. In conclusion, the NMDA antagonistaused severe impairment in the acquisition of spatial procedures, thus mimicking the consequences of cerebellar ablation on spatial proceduralearning. Based on the present findings, we hypothesize that cerebellar NMDA receptor activity is involved in the acquisition of procedural spatialompetence.

2006 Published by Elsevier Inc.

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eywords: Spatial function; Procedural learning; Cerebellum; Navigational str

. Introduction

In the spatial function, essential knowledge regards whereifferent places in the environment are, where the subject is andow to reach a specific place in the environment. The neuralasis underlying these different aspects of spatial cognition haseen studied extensively. Experimental, clinical and neuroimag-ng studies demonstrate that hippocampal formation provideshe allocentric representation of localizations, i.e., the so-called

cognitive map” [1,2,8,9,22,54,60,61,70,82,83]. The parietalortex has been implicated in providing a complementarygocentric representation of the environment centered on parts

DOI of original article:10.1016/j.brainresbull.2006.06.006.∗ Corresponding author. Tel.: +39 06 49917522; fax: +39 06 49917522.

E-mail address: laura.petrosini@uniroma1.it (L. Petrosini).

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361-9230/$ – see front matter © 2006 Published by Elsevier Inc.oi:10.1016/j.brainresbull.2006.06.005

s; Rats

f the body [69,75]. Other brain regions, such as the striatumnd cerebellar regions, appear to be involved in the naviga-ional system guiding the subject from one place to another17,28,59,63,66,72,79]. The experimental literature indicateshe contribution of NMDA receptors to spatial learning throughhe disruption of place learning following pharmacologicallockade of the NMDA receptor-dependent long-term poten-iation (LTP) at the hippocampal level [21,55–57]. However,nder the action of NMDA antagonists, rats readily learn tond a hidden platform if they are first non-spatially pre-trained5,12,13,74]. Furthermore, it was reported that rats with alockade of NMDA-LTP in the hippocampal areas are ableo learn behavioral explorative strategies, suggesting that

ippocampal LTP is not essential for spatial procedural learning33]. In the companion paper, we demonstrate that rats subjectedo hippocampal lesions and not treated with the drug persistedn foraging around the pool, whereas animals subjected to hip-

earch Bulletin 70 (2006) 356–367 357

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Table 1Experimental groups, number of subjects (N), presence of lesion, testing proce-dure and presence of drug

Group name N MWMtraining

Lesion CGS MWMtesting

Control 8 − − − +No-lesion/CGS 6 − − + +Train/no-lesion/CGS

8 + − + +

Cb 5 − Cerebellar − +Train/Cb 6 + Cerebellar − +Cb/CGS 5 − Cerebellar + +Train/Cb/CGS 6 + Cerebellar + +HH

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F. Federico et al. / Brain Res

ocampal lesion and treated with systemic administration of theMDA receptor antagonist were compelled to circle throughout

he testing [41]. This indicates that the NMDA antagonist foundsite of action to influence the acquisition of spatial procedural

earning even in hippocampal lesioned animals [41].A number of experimental findings indicate that the cere-

ellar networks are heavily involved in the navigational sys-em [27,35]. Data obtained in mutant mice support cerebellarnvolvement in spatial learning [6,10,15,39,40]. Hemicerebel-ectomized rats are impaired in developing efficient explorationtrategies but not in building a spatial map [47,48,66,68]. Whenxplorative procedural strategies are acquired pre-operatively,he cerebellar lesion does not alter spatial task execution, butampers the acquisition of more efficient strategies [43]. It isoteworthy that explorative strategies can be learned by obser-ation only when cerebellar circuits are intact [29,42]. Even ifhe ultimate definition of the neuronal systems involved in thecquisition of explorative strategies has not yet been achieved5,49,74], a number of experimental data, taken together, sup-ort the involvement of cerebellar circuits in the acquisition ofpatial procedural abilities [15,39,41,67,68].

Consequently, we set out to explore the issue of cerebellarnvolvement in the acquisition of procedural learning by inves-igating in detail the role of cerebellar NMDA receptors whosectivity is supposed to be involved in a number of learning func-ions [16,45]. As the hippocampal regions, the cerebellar cortexnd deep nuclei display a high density of NMDA receptors [77].urther, NMDA-mediated (pre- and post-synaptic) LTP and

ong-term depression (LTD) are reported in cerebellar excitatorynd inhibitory synapses [3,14,20,31,34]. Electrophysiologicalxperiments have described a great variety of phenomena relatedo use-dependent plasticity of the cerebellum, including persis-ent modulation of intrinsic neuronal excitability [31]. It wasypothesized that these cellular phenomena function and inter-ct to endow cerebellar circuits with particular computationalnd mnesic properties [31].

In the present study, cerebellectomized rats were system-cally administered [±]cis-4-phosphono-methyl-2-piperidinearboxylic acid [CGS 19755 (CGS)], a specific potent NMDAeceptor antagonist [7,44]. This procedure was aimed at analyz-ng whether the acquisition of spatial procedural competencesnd the execution of already acquired procedural competencesould be affected by the administration of the NMDA receptor

ntagonist in the presence of cerebellar lesions of differentxtent.

. Materials and methods

.1. Experimental groups

Adult male Wistar rats (200–250 g) were randomly assigned to one of theollowing experimental groups (Table 1):

Control group (n = 6): intact naıve animals (group name: Control);

Un-lesioned drug-treated group (n = 6): intact naıve animals, tested underCGS action (group name: No-lesion/CGS);Trained un-lesioned drug-treated group (n = 8): intact animals trained in theMorris water maze (MWM) and then tested under CGS action (group name:Train/no-lesion/CGS);

tbtlt

Cb 7 − Hemicerebellar − +Cb/CGS 5 − Hemicerebellar + +

Cerebellectomized group (n = 5): naıve animals with complete cerebellar abla-tion (group name: Cb);Trained cerebellectomized group (n = 6): animals with complete cerebellarablation trained in the MWM before the cerebellar lesion (group name:Train/Cb);Cerebellectomized drug-treated group (n = 5) naıve animals with completecerebellar ablation tested under CGS action (group name: Cb/CGS).Trained cerebellectomized drug-treated group (n = 6): animals with completecerebellar ablation trained in the MWM before the cerebellar lesion, and thentested under CGS action (group name: Train/Cb/CGS).Hemicerebellectomized group (n = 5): naıve animals with right hemicerebellarablation (group name: HCb);Hemicerebellectomized drug-treated group (n = 5) naıve animals with righthemicerebellar ablation tested under CGS action (group name: HCb/CGS).

Parts of these findings have appeared previously in abstract form25].

All experiments were carried out according to the European Commu-ity Council Directives of November 24, 1986 (86/609/EEC) and approvedy the Ethical Committee for animal experiments at our University. Allfforts were made to minimize the number of animals used and theiruffering.

.2. Surgery

The rats were anesthetized i.p. with a solution of ketamine (90 mg/kg) andylazine (15 mg/kg). After craniotomy, both cerebellar hemispheres and theermis (for total cerebellectomy) or the right cerebellar hemisphere and rightemivermis (for hemi-cerebellectomy) were ablated by suction. The cavity waslled with sterile gel foam, the wound edges were sutured and the animals werellowed to recover from anesthesia and surgical stress. After at least 20 daysf recovery (minimum latency period: 20 days; maximum latency period: 27ays), animals exhibiting motor symptoms that were stable and consistent withcerebellar lesion were behaviorally tested.

.3. Drug treatment

CGS 19755 (7 mg/kg) was dissolved in saline (0.09% NaCl) solution andnjected i.p. in a volume of 10 ml/kg in drug-treated animals. The drug wasdministered 60 min before MWM testing. Animals not treated with the drugeceived the same amount of saline.

Under CGS 19755 action, control animals displayed slight postural andotor symptomatology, as described elsewhere [24] and summarized in Table 2.hen tested on dry land, intact drug-treated animals exhibited slight limb hypo-

onia and a locomotor pattern characterized by a tendency to collapse on theelly. More severe impairment was evident in complex abilities requiring sub-le balance control and motor coordination [71], such as rearing, ascension on aadder, or suspension on a wire. When tested in the water maze, drug-treated con-rol animals maintained good swimming and climbing abilities; specifically, they

358 F. Federico et al. / Brain Research

Table 2Mean severity of postural and dynamic symptoms in the control and cerebellarlesioned animals under CGS action

Symptoms x Control animals x Cb animals x HCb animals

Body tilt 0.0 0.16 1.09Limb hyperflexion 0.1 1.89 1.61Ankle extrarotation 0.3 1.96 1.34Hypotonia 0.4 1.13 1.12Nystagmus 0.0 0.49 0.40Vestibular drop 0.1 1.55 1.29Rearing 0.1 1.62 1.74Head bobbing 0.0 1.49 1.14Wide base 0.0 1.90 1.50Collapse on the belly 0.2 1.61 1.37Side falls 0.0 1.48 1.15Tremor 0.1 0.57 1.10Steering 0.1 1.50 1.67Ascending a ladder 1.0 1.74 1.79Suspension on a wire 1.0 1.80 1.68Hyperactivity 0.8 0 0

A score ranging from 0 to 2 was assigned to each symptom according to itsdegree of severity. The rating scale is described in details in Federico et al.[24]. One-way ANOVA revealed a highly significant difference among the threegroups (F2,31 = 146.80; p = 0.0000) Post hoc comparisons revealed significantdH

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ifferences between control and lesioned groups (C vs. Cb: p < 0.0001; C vs.Cb: p < 0.0001; Cb vs. HCb: p n.s.).

wam with forelimb inhibition and showed alternate thrusting of the hindlimbs.heir ability to turn in the water was also preserved. The presence of slightypotonia did not impede the animals from completing the 60-s swimmingrials.

In cerebellar lesioned animals systemic CGS administration caused a severedecompensation” syndrome, and symptoms of cerebellar origin reappeared,s described elsewhere [24] and summarized in Table 2. In particular, underGS action totally cerebellectomized animals displayed marked limb hypo-

onia and severely impaired wide-based locomotion. In tasks requiring subtleostural adjustments and motor coordination, drug-treated cerebellectomizednimals exhibited dysmetric movements and asthenia (i.e. diminished mus-le strength). In spite of their critical impairment in dry land tasks, whenested in water the cerebellar drug-treated animals were perfectly able towim in a coordinated manner, showing turning abilities and forelimb inhi-ition similar to those observed in drug-treated controls. Despite markedsthenia, cerebellectomized animals were able to complete 60-s swimmingrials.

Under CGS action the most evident decompensation symptoms of the ani-als hemicerebellectomized on the right side were represented by severely

symmetrical postural and motor behaviors (Table 2). Marked hypotonia ofight limbs was observed as well as wide-based locomotion and body tilt tohe right side. When the animals swam this asymmetrical posture also resultedn body tilt to the right. Interestingly, to keep their noses out of the waternd to keep their balance, hemicerebellectomized animals hyperextended theirails and used them as “counterpoise”. In spite of this, they showed coordi-ated swimming movements with the typical rat motor pattern. The hemicere-ellectomized animals also succeeded in completing the 60-s swimmingrials.

Both groups of cerebellar animals (cerebellectomized or hemicerebellec-omized) climbed the raised platform without difficulty because of its low height2 cm) and stayed on it for 30 s. To offer a stable support, the platform was cov-red with a white carpet that allowed a firm grip.

.4. Testing: MWM apparatus

The rats were placed in a circular plastic pool (diameter 140 cm) filled withater (24 ◦C) 50 cm deep and made opaque by the addition of 2 l of milk. A

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Bulletin 70 (2006) 356–367

hite escape platform (10 cm in diameter) was placed in the middle of oneuadrant, 30 cm from the side walls; it was either submerged 2 cm below oraised 2 cm above the water level. The animals were allowed to swim aroundo find the platform for 60 s. If they were unable to find the platform alone, thexperimenter guided them to it. At the end of each trial the rats were left on thelatform for 30 s.

.5. Experimental protocols

All experimental groups were tested in the MWM according to a 20-trialrotocol for analyzing MWM performances under CGS action that lasted aboutn hour and a half. In the first 10 trials (60 s maximum) the hidden platform wasept in the SE quadrant. Starting points from the pool wall at the cardinal pointsN, S, E, W) were pseudo-randomly varied. The inter-trial interval was 3 min.n the last 10 trials (60 s maximum) the animals were tested with the visiblelatform randomly moved to a new quadrant in each trial and the starting pointept at the north point.

Three weeks later the drug-treated animals were again tested in the MWM,his time in the absence of drugs. The results of this testing will not be reportedn detail since they did not provide any additional information to the presenttudy, since lesioned animals exhibited only minor differences in the presencer absence of the drug.

.5.1. MWM trainingThe animals belonging to the trained groups (Train/no-lesion/CGS, Train/Cb

nd Train/Cb/CGS) were trained to find the hidden platform in the SE quadrantor 5 consecutive days, 10 trials a day. The starting points at the four cardinaloints were pseudo-randomly varied. In the Train/no-lesion/CGS group, theraining was performed 1 week before MWM testing under CGS action, whilen the cerebellectomized animals it was performed a week before the cerebellaresion. Trained cerebellar animals were then tested in the MWM under CGSction 20 days after the lesion.

.6. Behavioral parameters

Swim paths were monitored by a video camera mounted on the ceiling.he resulting video signal was relayed to a monitor and videotape, and ton image analyzer (Ethovision, Noldus). In addition to latencies, successfulscapes, swimming velocity, percentage of path length spent in 20-cm wideuter annuli, swimming trajectories were analyzed in detail to categorize thexploration strategies put into action. Thus, as in the procedure described in anarlier paper [30], by considering spatial and temporal distribution of swim-ing trajectories, path length, percentage of time spent in inner or in outer

nnuli, and percentage of time spent in the different quadrants and headings,xploration behavior was classified in five main categories: circling, swimmingn the peripheral tank areas, with inversion of swimming direction and counter-lockwise and clockwise turnings in the peripheral sectors of the pool; extendedearching, swimming around the pool in all quadrants, visiting the same areasore than once; restricted searching, swimming in some pool quadrants, not

isiting some tank areas at all; restricted circling, swimming in some pool quad-ants, reaching the platform by means of a roughly semicircular trajectory; directnding, swimming towards the platform without any foraging around the pool.he categorization of swimming trajectories drawn by the image analyzer wasade by three different researchers who were unaware of group assignment.hey attributed the dominant behavior in each trial to a category. The cate-orization was considered reliable only when their judgments were consistent.urthermore, when considered appropriate, the crossings in the pool center werenalyzed in selected groups.

.7. Histology

At the end of behavioral testing, all lesioned animals were deeply anes-

hetized and perfused intracardially with saline followed by 10% bufferedormalin. The extent of cerebellar lesions was determined from Nissl-stained0 �m-thick frozen sections.

Cerebellar animals were included in the present study only if cerebel-ar ablation was complete or a hemi-cerebellectomy resulted in total ablation

F. Federico et al. / Brain Research Bulletin 70 (2006) 356–367 359

Fig. 1. Histological sections of representative lesioned animals and diagrammatic reconstruction in coronal sections of the rat brain illustrating the smallest (blacks nd het ificat

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hading) and the largest (grey plus black shading) cerebellar (upper side, Cb) ahe bregma according to the stereotaxic atlas of Paxinos and Watson [64]. Magn

f deep nuclei and complete sparing of extra-cerebellar structures (exceptor the dorsal cap of the Deiters’ nuclei which, in some cases, was slightlyamaged) (Fig. 1). Following this criterion, three animals that should haveeen totally cerebellectomized and one hemi-cerebellectomized animal werexcluded from all analyses because of incorrectness of the cerebellar abla-ions. The final number of animals in each experimental group is reported inection 2.1 and in Table 1. Variability in the extent of floccular and parafloc-ular lesions was considered not relevant, since in all cases these structuresere functionally disconnected due to ablation of cerebellar peduncles and deepuclei.

.8. Statistical analysis

Metric unit results of animals in the different experimental groups wererst tested for homoscedasticity of variance and then compared using one-

ay or two-way “p × q” analyses of variance (ANOVAs) with repeated mea-

ures, followed by multiple post hoc comparisons using Duncan’s tests. Whenhe condition of homoscedasticity of variance and the normal distributionf raw data were not obeyed, χ2 non-parametric Friedman ANOVAs weremployed.

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micerebellar (lower side, HCb) lesions. The numbers refer to millimeters fromion 1×.

. Results

.1. Un-lesioned groups

Detailed descriptions of MWM behavior of intact animalsn the presence or in the absence of the drug (Control, No-esion/CGS, Train/no-lesion/CGS) are provided in the com-anion paper [41]. In brief, Control animals exhibited steadyearning of spatial procedures and localizatory competence thatrogressively led to reduced latency in reaching the platformFig. 2) and to efficient strategies (Fig. 3). Their mean swim-ing velocity was 29.06 ± 1.98 cm/s and mean percentage of

eripheral swimming 34.20 ± 7.05. Drug-treated naıve animals

No-lesion/CGS group) circled at the pool periphery for thehole duration of the testing (Figs. 2 and 3), displaying a

wimming velocity of 27.29 ± 2.36 and a mean percentagef peripheral swimming of 96.43 ± 13.1. Train/no-lesion/CGS

360 F. Federico et al. / Brain Research Bulletin 70 (2006) 356–367

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ig. 2. Mean (±S.E.M.) escapes latencies by the various experimental groups in the SE quadrant. Starting points at the pool wall from the cardinal points (Nisible platform was randomly moved to a new quadrant in each trial and the staresented in detail in Table 1.

nimals maintained all spatial competences acquired duringraining (Figs. 2 and 3). Their mean swimming velocity was7.21 ± 3.60 cm/s and mean percentage of peripheral swimming4.61 ± 9.43.

.2. Totally cerebellectomized groups

As described in Section 2, in spite of the presence of posturalnd motor symptoms on dry land, cerebellectomized animalsere able to swim with a coordinated and stable pattern. From

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WM trials. In the first 10 trials (60 s maximum) the hidden platform was kept, W) were pseudo-randomly varied. In the last 10 trials (60 s maximum), the

point kept at the north point. The characteristics of the experimental groups are

navigational point of view, when the platform was hidden theb group exclusively used a wall-hugging strategy. The animals

hifted slightly to a restricted circling strategy when the plat-orm was visible (Fig. 2). Their mean swimming velocity was8.08 ± 8.09 cm/s and mean percentage of peripheral swimming5.22 ± 7.09. When the percentages of explorative behaviors

erformed by Cb and No-lesion/CGS groups were compared, theeneral pattern of their explorative strategies appeared similarFig. 2; Table 3). The statistically different comparisons betweenhese groups concerned circling (F1,9 = 10.88; p = 0.0092) and

F. Federico et al. / Brain Research Bulletin 70 (2006) 356–367 361

Fig. 3. Mean (±S.E.M.) percentages of specific explorative strategies dominant in each experimental group in the first 10 place trials. The lower figures represent thet positiS the hir r traje* oups a

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ypical explorative patterns of the five categories. The black circles indicate theearching for the hidden platform around the pool; RS, restricted searching forestricted circling at pool periphery, reaching the platform through a semicirculap < 0.05; **p < 0.005; ***p < 0.0005. The characteristics of the experimental gr

xtended searching (F1,9 = 9.29; p = 0.0381), more frequentlynd even if with very low percentages, performed by the un-drug-reated cerebellectomized and drug-treated un-lesioned animals,espectively.

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on of the hidden platform. C, circling strategy at pool periphery; ES, extendeddden platform in some pool quadrants, not visiting some tank areas at all; RC,ctory; F, finding the platform directly without any exploration around the pool.re presented in detail in Table 1.

Interesting results were obtained by pre-operatively traininghe animals that would be cerebellectomized (Train/Cb group).n spite of the cerebellar lesion, the Train/Cb group put intoction very efficient strategies, such as restricted searching and

362 F. Federico et al. / Brain Research

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Bulletin 70 (2006) 356–367

nding, and rarely circled in the peripheral section of the pool.his behavior resulted in a high number of successful findingsith low latencies (Fig. 2). Their mean swimming velocity was6.08 ± 8.01 cm/s and mean percentage of peripheral swimming4.09 ± 10.08. When the percentages of strategies performed byrain/Cb and Cb animals were compared, significant differencesere found for almost all strategies (Fig. 3; Table 3), demonstrat-

ng that the pre-operatively acquired procedural competencesere fully maintained following the cerebellar lesion.By analyzing the CGS effect on the cerebellar animals’

ehavior, it was observed that the Cb/CGS group performancesere not significantly different from those of the Cb group

Figs. 2 and 3; Table 3). The Cb/CGS group mean swimmingelocity was 26.98 ± 8.76 cm/s and mean percentage of periph-ral swimming 96.48 ± 9.19. When the percentages of explo-ative behaviors of Cb/CGS and Cb groups were compared,ifferences barely reaching the threshold of significance under-ining the behavioral similarity of the two groups (Fig. 3).

CGS action was irrelevant on performances of trained cere-ellectomized animals (Train/Cb/CGS) that, in spite of therug action, persisted in using efficient preoperatively acquiredxplorative strategies. Their mean swimming velocity was4.46 ± 11.19 cm/s and mean percentage of peripheral swim-ing 38.19 ± 5.69. Not significant difference in the explorative

trategies of the Train/Cb/CGS animals and the Train/Cb ani-als was found (Figs. 2 and 3; Table 3). The findings on trained

nimals allowed us to exclude that the circling observed inhe drug-treated groups was due to motor impairment causedirectly by the drug.

Hemicerebellectomized groups. The similarity of effectslicited by cerebellectomy and the NMDA receptor antagonistn navigational strategies made it necessary to analyze the CGSffects in a cerebellar lesion model in which no additional effectsetween factors could be observed. This issue was addressed bynalyzing the CGS effects in hemicerebellectomized animals.

In the absence of the drug, hemicerebellectomized animalsisplayed a mean swimming velocity of 22.03 ± 6.38 cm/s andmean percentage of peripheral swimming of 73.11 ± 22.75.hey exhibited a marked tendency to circle at the pool periphery

Fig. 3). This behavior was repetitive, but not compulsive. Thus,he animals were able to interrupt their circling to traverse theool center more than once during a trial. Once they had crossedhe pool, they regressed to peripheral circling without displayingny foraging. This behavior did not evolve into more efficientxplorative strategies as the sessions went by (one-way ANOVA:9,54 = 0.18; p n.s.).

When CGS effect on hemicerebellectomized animals’ behav-or was analyzed, influence of the drug treatment was detected.Cb/CGS animals constantly circled at the pool periphery and,nlike the HCb group, did not make any attempt to traversehe tank. Their mean swimming velocity was 22.04 ± 5.92 cm/snd mean percentage of peripheral swimming 82. 93 ± 17.59.y comparing the total number of crossings across the pool

n both hemicerebellectomized groups by means of non-arametric Friedman’s ANOVA, a significant difference wasound (χ2 = 16.05; p = 0.003). The data demonstrate that in thebsence of the drug hemicerebellectomized animals displayed a

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ess compelling circling strategy than they had displayed underhe effect of the drug.

When the HCb/CGS group performances were comparedith those of the Cb/CGS group, not significant difference was

ound for latencies in platform reaching (Table 3) or for navi-ational strategies put into action, regardless of whether or nothe platform was visible (Fig. 3). When the HCb/CGS grouperformances were compared with those of the No-lesion/CGSroup, not significant difference was found for either platformisibility condition (Figs. 2 and 3; Table 3).

. Discussion

.1. Motor effects evoked by cerebellar ablations

The first step in analyzing the cognitive impairment evokedy cerebellar lesions is to evaluate how much the motor symp-oms that inevitably occur in the presence of a cerebellar lesionctually interfere with it. Indeed, motor symptomatology mayide cognitive impairments and may elicit a behavioral patternn which motor components are blurred with cognitive ones,hus impeding separate evaluations. Furthermore, the difficultyn maintaining symmetrical posture and coordinated locomo-ion may disrupt the motivational aspects of cerebellar animals’erformances. However, while cerebellar lesions provoke motorymptoms that may confuse dry land performances, these symp-oms did not severely affect swimming performances, as widelyeported in the literature [46,52,67]. Furthermore, cerebellarblations do not alter forelimb movement inhibition, hindlimbhrusting or turning abilities [46,52,67]. Asthenic symptomso not overtly affect swimming tasks, provided the trials lasto longer than 60 s, because swimming functions are underultisystem control, so that extra-cerebellar circuits can effi-

iently operate to provide vicarious support [46,52,67]. Conse-uently, testing cerebellar animals on cognitive tasks that requirewimming performances is advantageous for analyzing spatialrocedural abilities not influenced by motor impairment or moti-ational failures.

As in the situation evoked by cerebellar lesions, the NMDAeceptor antagonist elicited motor impairments on dry land testsut did not alter swimming abilities. The CGS dosage wasarefully chosen to ensure that it would have a homogeneousnfluence on procedural abilities, without affecting swimmingnd climbing motor abilities, and would make it possible to ana-yze navigational strategies that were not confused by motorymptoms. The distinction between the acquisition of procedu-al competences and the execution of motor abilities was a keyeature in interpreting the findings of the present research.

.2. NMDA antagonist effects on navigational strategies inontrol and cerebellectomized animals

In naıve intact animals the NMDA antagonist provoked

eripheral circling as the dominant strategy, without any use-ul searching. Similar behavior was observed in naıve cere-ellectomized animals that exhibited persistent circling in theeripheral sectors of the pool. Furthermore, in neither group of

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Bulletin 70 (2006) 356–367 363

nimals was the development of efficient navigational strategiesbserved as the sessions went by. As suggested, in the pres-nce of other brain lesions, e.g., after medial caudate-putamenesions [81], the persistence of peripheral circling could be aompulsive thigmotaxic behavior, acting as a “behavioral trap”,n which drug-treated or totally cerebellectomized rats were con-trained to unremitting aimless circling. However, when swim-ing trajectories were analyzed neither group of animals, even

f they swam in roughly circular paths, ever displayed a position-esponse bias. Conversely they exhibited frequent reversals ofwimming direction, alternating counter-clockwise and clock-ise turns, different swimming speeds during the same trialr occasional stops with motionless floating for a few secondsefore resuming peripheral swimming. Thus, the circling exhib-ted by cerebellar, as well as by intact drug-treated animals,as not a stereotyped constrained behavior. Conversely, it was

he only behavior, however expensive and ineffective, that botherebellectomized and intact drug-treated animals were able tout into action because of their inability to develop useful newearching strategies. As a consequence, it can be advanced thathe circling evoked by the NMDA receptor antagonist or byhe cerebellectomy was provoked by impaired acquisition ofhe sequence of spatial procedures that characterizes the explo-ation of a new environment. In fact, the procedural strategiesre learned from the least efficient (circling) to the most tunedne (finding), because they are arranged in a sort of proceduralhain formed by steps that have to be acquired in sequence.

Further evidence that circling must be related to the inabil-ty to explore the environment derives from results obtainedith trained animals. In fact, when procedural components had

lready been gained by training, neither the NMDA antagonistor the cerebellectomy provoked peripheral circling. This obser-ation allowed excluding that either the NMDA antagonist orhe cerebellar lesion affected the execution of the previouslycquired explorative strategies and impeded any experience-ependent tuning of navigational strategies, as demonstrated byhe flattened latency curves exhibited by intact drug-treated anderebellectomized animals (Fig. 2). Note that this “freezing”ffect under CGS action is similar to that previously describedn hemicerebellectomized animals that had been pre-operativelyrained [43]. Thus, the NMDA antagonist appears to produce

spatial procedural deficiency analogous to the one evokedy cerebellar lesions, thus raising doubts about the previouslydvanced hypothesis [5,13,74] that the effect of the NMDAntagonist on MWM performances could be due to a senso-imotor impairment.

In spite of the close similarity between effects evoked byhe NMDA receptor antagonist and by the cerebellar ablation,t is important to underline a substantial difference between un-esioned drug-treated and cerebellectomized animals not treatedith the drug. Given that the acquisition of efficient naviga-

ional strategies is the pre-requisite of any localizatory learning26,48], deficient spatial procedures masked any localizatory

ompetence [66], thus rendering the two groups very similarrom the behavioral point of view. However, it is reasonable tossume that in the un-lesioned drug-treated group the localiza-ory learning should have been concomitantly impeded because

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f the block of hippocampal LTP elicited by the NMDA antag-nist, as reported in the literature [5,74]. Conversely, cerebel-ectomized animals not treated with the drug should preservehe possibility of gaining localizatory competencies, given theirntact hippocampal functioning. However, their potential spa-ial competence was not detectable because of the proceduralmpairment that blocked the animals into the circling behavior.

In sum, the NMDA receptor antagonist appears to reproducehe consequences of the surgical cerebellar ablation on spatialrocedural learning. In the unitary theory of cerebellar functions a procedural machine [23,37,51,76,78], we suggest that theavigational system for the acquisition of the complete chainf spatial procedures spontaneously put into action when a newnvironment has to be explored might require functional cere-ellar NMDA receptors. If so, the NMDA receptor antagonisthould affect spatial procedural learning by provoking a sort ofharmacological cerebellectomy. Then, cerebellectomized ani-als not treated with the drug and intact drug-treated animals

hould behave in the same manner, since cerebellar NMDAeceptors are out of function in both groups of animals. Thisas the case (Cb and No lesion/CGS groups). In the absence of

he cerebellar circuits, whether or not NMDA receptor activitys blocked should make no difference in the acquisition of newrocedural competencies. This was the case (Cb and Cb/CGSroup performances). By blocking NMDA receptor activity, thelready acquired procedural competences should be maintained,ecause cerebellar activity is not required for recalling spa-ial competences. This was the case (Train/no-lesion/CGS andrain/Cb/CGS groups). Finally, if the cerebellar NMDA func-

ion is needed to shift towards more effective searching strategieshen, in the absence of cerebellar NMDA receptors, animalshould remain blocked at the first step of the spatial procedu-al chain. This was the case (No-lesion/CGS and Cb groups’erformances).

We are aware that even if the procedural deficits provoked byhe NMDA antagonist and the cerebellectomy are very similar,e cannot automatically assume that the procedural impair-ent produced by CGS administration is elicited by blocking

erebellar NMDA receptor activity. We would have to demon-trate that the NMDA antagonist induces procedural impair-ents simultaneously to block cerebellar LTD in vivo, as has

een demonstrated for hippocampal circuits [21]; but, up untilow this experimental demonstration has been unfeasible. How-ver, recent evidence obtained by using a different experimentalpproach [10] strongly supports our position. Indeed, L7-PKCIransgenic mice that lack parallel fiber-Purkinje cell LTD dis-layed a severe impairment in acquiring the procedural com-onents of spatial navigation, while the declarative localizatoryomponent was unaffected. Namely, these mutant mice showedsignificantly larger amount of circling behavior in the MWM

ompared to wild-type animals and the absence of a deficit whenhe procedural demands of the task were strongly reduced, as inhe starmaze. These findings, which suggest that parallel fiber-

urkinje cell LTD is involved in the acquisition of the proceduralomponent of navigation, support the proposal that the NMDAntagonist affected the acquisition of navigational strategies bycting on the cerebellar NMDA receptors. If this were true, in the

4

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Bulletin 70 (2006) 356–367

bsence of cerebellar NMDA receptors the NMDA antagonistould not have found the action site to affect spatial procedural

earning. Actually, cerebellectomized rats displayed the samexplorative behavior both when the drug was present and whent was absent.

.3. NMDA antagonist effects on navigational strategies inemicerebellectomized animals

Given the marked similarity of MWM behavior evoked byhe NMDA receptor antagonist and total cerebellar ablation,here was no possibility of observing additional effects betweenactors by using the total cerebellectomy experimental model.o overcome this problem, the effects of the NMDA antago-ist were analyzed in the presence of partial cerebellar lesions,amely hemicerebellectomy. This experimental model has beensed repeatedly to study the cerebellar contribution to the spatialunction [43,42,47,48,50,66]. Although hemicerebellectomizednimals not treated with the drug swam mainly in the periph-ral sectors of the pool, they displayed less compulsive circlingehavior than that displayed in the presence of drug. They wereble to interrupt circling behavior to traverse the pool more thannce during the trial, thus showing they were not locked in aim-ess circling. Conversely, the HCb/CGS animals never crossedhe pool and continued to circle ineffectively throughout the trial.vidently, in the presence of a hemicerebellar lesion the NMDAntagonist found a spared region to influence the acquisitionf navigational strategies and further prevented the proceduralearning already severely impaired by the hemicerebellar lesion.ince the only difference between the two models with cerebellarblations was the presence of spared cerebellar areas, it seemedeasonable to attribute to these circuits the additional effects ofhe NMDA receptor antagonist observed in the HCb/CGS group.

A recent study reported that rats profit from non-spatial train-ng performed under CGS action [33]. The authors hypothesizedhat the acquisition of spatial strategies does not require hip-ocampal NMDA-dependent LTP blocked by the drug. Previ-us research demonstrated that cerebellar structures are heavilynvolved in non-spatial training [43,67]. Thus, non-spatial train-ng performed under CGS action should not be effective if theerebellar NMDA receptors are blocked. However, it is impor-ant to note that the drug i.p. dosage used by Hoh and colleaguesas 4 mg/kg. We believe that this dosage was adequate to blockentate and CA1 LTP [12] but was insufficient to fully antago-ize cerebellar NMDA receptors. Conversely, the drug dosagesed in the present study allowed for the diffuse penetrationf broad cerebellar regions, completely antagonizing cerebel-ar NMDA activity. Not by chance, the question about whetherimilar dosages of NMDA antagonist are required to block LTPn the dentate gyrus and elsewhere in the hippocampus [32]r outside the hippocampus [4] has already been investigated11].

.4. Conclusions

Extending the assumption that the learning of navigationaltrategies can occur without hippocampal NMDA-LTP [33], we

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ypothesize that cerebellar NMDA activity could be necessaryor learning spatial procedural components.

Electrophysiological evidence shows that the activation of thelutamatergic mossy fiber system results in LTP or LTD accord-ng to the frequency and duration of stimulation [3,19,38,65].

ossy fiber LTP, such as that in hippocampal area CA1, requiresost-synaptic depolarization, NMDA receptor activation andonsequent Ca2+ influx, and can be blocked by glutamatergiceceptor antagonists. LTP and LTD of the GABAergic synapsesetween Purkinje cells and deep cerebellar nuclei also requireMDA receptor activation and elevation of post-synaptic Ca2+

53,62]. These cellular mechanisms, which are able to modifyynaptic strength, allow for the synapse-specific modification oflarge array of inputs that become central in many processes

f memory and learning [31,58]. Although it is not yet possibleo relate cellular phenomena to behavior through the interme-iate level of cerebellar synapses, an attempt can be made toorrelate cerebellar NMDA receptors and acquisition of spatialrocedures by taking into account cerebellar multifaceted use-ependent plasticity.

It must also be taken into account that variations in theroperties of the NMDA receptors are imparted by specificeceptor subunits, and recent studies have provided perspectivesnto some of the tasks carried out by individual receptor sub-ypes [18]. In this vein, the possibility of using subunit-directedpecific NMDA antagonists [36,73,80] might lead to better dis-ection of the molecular counterparts of the observed effects inpatial learning.

cknowledgement

This work was supported by MIUR grants to LP.

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