Functional role of rat prelimbic-infralimbic cortices in spatial memory: evidence for their involvement in attention and behavioural flexibility

Behavioural Brain Research 109 (2000) 113 – 128 www.elsevier.com/locate/bbr

Research report

Functional role of rat prelimbic-infralimbic cortices in spatial memory: evidence for their involvement in attention and behavioural flexibility Benoıˆt Delatour 1, Pascale Gisquet-Verrier * Laboratoire de Neurobiologie de l’Apprentissage, de la Me´moire et de la Communication (NAMC), CNRS-UMR 8620, Uni6ersite´ Paris-Sud, Baˆt. 446, 91405 Orsay Cedex, France Received 31 May 1999; received in revised form 18 November 1999; accepted 18 November 1999

Abstract The involvement of the medial prefrontal cortex (mPFC), and more particularly the prelimbic and infralimbic cortices (PL-IL area), in spatial memory remains controversial. The present study investigates the effects of neurotoxic lesions restricted to the PL-IL area of the mPFC in rats trained in two different spatial tasks. In experiment 1, PL-IL lesioned rats showed normal acquisition of a delayed non-matching to position task. They were also able to plan their responses for a prospective strategy but were transiently disrupted when the initial delay was extended. In experiment 2, rats were trained to locate one baited box among 13 identical boxes distributed on a circular arena. Lesioned rats performed normally when trained from a single start position but were severely disrupted when four start positions were used. A probe trial showed this deficit was not due to failure to learn the goal location. The addition of a proximal cue signalling the goal box helped lesioned rats to directly open the goal box, but did not compensate for greater distances that they travelled to reach it. Results from both experiments indicate that the PL-IL area is directly involved neither in allocentric spatial representations nor prospective memory and is not specifically involved in working memory. This area seems more likely to be involved in both attentional processes and behavioural flexibility that may be important for processing information for working memory as well as for spatial memory. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Prefrontal cortex; Planning of response; Prospective memory; Working memory; Delayed non-matching to position task

1. Introduction The role of the prefrontal cortex in the organization of spatially directed behaviours has been shown in both primates and humans. Defining the function of this cortical area has come from both classical neuropsychological and clinical evidence [43,60,64] as well as from numerous studies using electrophysiological and brain * Corresponding author. Tel.: +33-1-6915-4979; fax: + 33-1-69157726. E-mail address: [email protected] (P. GisquetVerrier) 1 Dr B. Delatour is now at the Laboratoire de Neuropathologie R. Escourolle, Hoˆpital La Salpeˆtrie`re, 47 Bd de l’Hoˆpital, 75651 Paris, Cedex 13, France.

imagery approaches [18,19,49,68]. In rats, the medial prefrontal cortex (mPFC) is also considered to be one of the most important structures implicated in the processing of spatial information. The main evidence supporting this argument comes from lesion studies where damage to the mPFC results in an impairment in performance in various spatial tasks such as spatial alternation [7,27,42,46,47,63,67,69], spatial reversal [2,14,35–37,48,70], elimination in the radial maze [1,25], and navigation in the Morris water maze [16,34,38–40,44,65,66]. As most (if not all) of the disruptive effects observed with prefrontal lesions are also observed following damage to the hippocampal formation, it has been proposed that these two anatomically interconnected

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structures could form an integrated ‘spatial mapping system’ [1,38,48]. Recent evidence, however, suggests that as opposed to the hippocampus, the mPFC should be viewed as being directly involved in not the building up of spatial cognitive maps but more so in the ability to select and plan spatial-directed responses that are possibly related to an involvement of this structure in behavioural flexibility [8,9]. It must be also noted that the mPFC is not an homogeneous structure and can be divided into a number of subregions along its dorso-ventral axis. The dorsal part comprises the medial precentral and the dorsal anterior cingulate cortices whereas the ventral part comprises the prelimbic and infralimbic cortices. While the dorsal part of the mPFC can be considered as a premotor cortex, the ventral part has important connections with the limbic circuitry [5,61]. Over the last few years there has been an increasing interest in understanding the functional role of each of these different mPFC subregions more precisely [12,13,22, 31,55,57–59]. In this sense, the prelimbic cortex (PL) and the infralimbic cortex (IL) have been the focus of attention because of their specific anatomical relationship. These areas receive the highest density of direct projections from the ventral hippocampal CA1 field and from the subiculum [5,26]. In addition, it has been shown that learning can modulate the synaptic strength and efficacy of the hippocampo-prelimbic pathway [15]. As the ventral mPFC (PL-IL area) is one of the main output structures of the hippocampal system, this prefrontal region might be expected to be involved in memory processes, and more particularly in spatial memory. Such an assumption has gained some support from the literature. For example, lesions restricted to the PL-IL area have been shown to reproduce disruptive effects in spatial delayed alternation that have been generally reported with larger mPFC lesions [3,12]. PL-IL lesions have also been shown to disrupt other delayed spatial response tasks in which rats were required to locate either a baited arm or a series of baited arms in a radial arm maze, based on previously delivered information [12,59]. In accordance with the results from primates [20], Ragozzino et al. [55] have recently proposed that the rat prefrontal cortex is a brain region specialized in working memory. They further suggest that PL-IL is an area more specifically involved in allocentric spatial working memory. On the other hand, however, Seamans et al. [59] have suggested that PL is not crucial to maintaining specific information over a time delay but is involved in planning prospective search behaviour in spatial tasks [17]. Despite the different points of view, it must be emphasised that Seamans et al. [59], as well as Raggozino et al. [55] claim that the PL-IL area plays a restrictive role in the organization of spatial delayed responses.

Our own results do not support this latter position as we have shown that PL-IL lesions induced delay-dependent disruptions of performance in a non-spatial task, i.e. a tone-light conditional task tested in an operant chamber [13]. Furthermore, it was found that PL-IL lesions did not unequivocally affect spatial working memory, as we have always observed that classical spatial working memory tested on the radial maze was preserved [11,12]. It has also been shown that damage to the PL-IL area may induce some performance disruptions in spatial tasks such as the Morris water maze that do not require working memory [16,21,34]. All these results have led us to suggest that the PL-IL area is involved in the planning of forthcoming behavioural responses, principally by relying upon behavioural flexibility and attention, an hypothesis that may account for deficits in delayed-response as well as impairments in complex spatial tasks [13]. The aim of the present paper is to elucidate two major points concerning the role of the PL-IL area. First, to determine whether or not the PL-IL area is involved in the processing of spatial information and to specify its specific role in the building up of cognitive maps and/or the use of spatial information. Second, to determine which hypothesis, spatial working memory, prospective planning of behavioural response or attentional and behavioural flexibility deficits account for the effects of PL-IL lesions. To this end, we studied the effects of neurotoxin-induced PL-IL lesions in rats trained in two different spatial tasks that either involved a working memory component or did not involve one. In the first experiment, rats were trained in a delayed non-matching to position task, allowing us to investigate the role of the PL-IL area (1) on the processing of spatial information, (2) on prospective memory, by forcing rats to plan their responses from the start arm, and (3) on working memory, by extending the delay separating the sample information and the response. In the second experiment, rats were trained in a navigation task, either from a single start position (experiment 2a) or from four different start positions (experiment 2b). In this task no obvious working memory was required and the role of the PL-IL area was investigated in terms of processing of allocentric spatial information under different constraints of behavioural flexibility and attention.

2. Experiment 1: delayed non-matching to position task We have previously shown that rats with PL-IL lesions are impaired in a spatial delayed alternation task on a Y-maze [12]. As the location of the goal arm on a given trial was defined by the position of the arm just previously visited, this task was considered to be a working memory task. The type of impairment demon-

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strated by PL-IL lesioned rats in this task remained unclear because the alternation rule may have been solved based on egocentric cues (going left or right) and/or allocentric cues (distal spatial cues). Ragozzino and his collaborators [55] recently proposed that the PL-IL area is specifically involved in working memory for spatial allocentric locations. In order to investigate this, we tested the effects of PL-IL lesions in rats trained in a delayed non-matching to position task which can only be solved based on allocentric information. In this experiment, rats were trained on a crossmaze with a two-run procedure, each trial consisting of a forced run in which rats were required to visit a particular arm of the maze, and a choice run where rats were required to visit the opposite arm. As the use of egocentric cues was made irrelevant by using two diametrically opposite start positions, this task could be considered a pure allocentric spatial working memory task in which rats were tested for delayed responses guided by spatial distant cues. Once the non-matching rule was established, the effects of PL-IL lesions were investigated when rats were forced to prospectively plan their responses from the start position. This was done by making spatial distal cues unavailable at the time of choice, with the use of an opaque cylinder placed in the choice area. Finally, the involvement of PL-IL area in working memory processes was further investigated by extending the delay between the forced run and the choice run from 10 to 40 s.

2.1. Method 2.1.1. Subjects In the present study, 23 male Sprague – Dawley rats (50 – 57 days old), weighing 250 g on arrival in the laboratory, were used. Rats were housed in pairs in wire-mesh cages and maintained on a 12:12-h day/night cycle in a temperature-controlled room (21°C), with free access to food and water. 2.1.2. Surgery Rats underwent surgery 1 week after their arrival being randomly assigned to either a cortical lesion or sham operation group. Rats assigned to the PL-IL lesion group (n=13) were injected with atropine (i.p. injection, 0.3 ml), and anaesthetised 15 min later with pentobarbital (50 mg/kg, i.p.) before being mounted in a stereotaxic frame (Narashige Instruments, model SR6). Two holes were drilled in the skull into which a cannula (180 mm diameter) was lowered to deliver ibotenic acid (Sigma; concentration: 1 mg/1 ml, dissolved in phosphate buffered saline; pH 7.4). Injections were made bilaterally (AP= + 3.5 mm from bregma; ML = 9 0.7 mm from the midline suture and DV = − 3.5 mm from the dura) and ibotenic acid (0.5 ml) was injected over a period of 4 min, using a microinjector.

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After each injection, the cannula was left in place for 10 min. At the end of the surgery, the incision was sutured and sulfamides were applied locally. Sham-operated rats (n= 10) underwent the same surgical procedure without penetration of the cannula.

2.1.3. Histology After completion of behavioural testing, rats were overdosed with pentobarbital (120 mg/kg) and perfused transcardially with saline, followed by a 10% buffered formalin solution. Brains were post fixed in buffered formalin and cryoprotected by immersion in a 30% sucrose-formalin solution for 48 h. The brains were sectioned (40 mm) on a freezing microtome and every second section was taken and stained with cresyl violet. Evaluation of the lesion size was performed by digitizing the extent of the lesion with a graphic software package (Canvas, Deneba). To be included in the lesioned group, it was necessary that damaged cortical lesion was more than 60% of the PL-IL in each hemisphere, with no or only minor encroachment of the lesions on the dorsomedial prefrontal cortex. 2.1.4. Material The apparatus was a wooden eight-arm maze painted in grey and elevated 70 cm above the floor surface, on a rotating device. Each arm (60× 12 cm) radiated from a central octogonal platform (30 cm diameter), had 2-cm high plastic walls and was equipped with a food cup at the end. During pretraining and training phases, the radial arm maze was used as a cross maze. A cylinder (height 25 cm; diameter 28 cm), placed on the central platform, gave access to a restricted set of three arms arranged in a T-shape (a start arm and two diametrically opposed goal arms; Fig. 1), through three square (10× 10 cm) apertures. Entry to one particular goal arm could be prevented by the use of a metallic door. Two different cylinders were used: (1) a transparent cylinder and (2) an opaque cylinder in which the three apertures had fringed curtains to prevent rats from viewing the external environment when placed on the central platform. The maze was placed in an experimental room providing numerous distal visual cues (two doors, shelves and posters on the walls, two chairs etc...). Chocolate cereal was used as reinforcement. 2.1.5. Procedure After 1 week of postoperative recovery, rats were placed on a food-deprivation schedule to gradually reduce their weight to 85% of their free-feeding weight and they were maintained at this level during the course of behavioural testing. 2.1.5.1. Pre-training. On the first 2 days of pre-training, rats were placed in groups of three or four on the central platform equipped with the transparent cylin-

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Fig. 1. In the delayed non-matching to position task, each trial comprised a forced run (left part) and a choice run (right part), separated by a time delay. During the forced run, rats started from one of two possible positions (S1 or S2) and reached a baited arm selected by the experimenter (for example: G1). During the choice run, rats had to choose the arm opposite to the one visited during the forced run (G2 in our example), independent of the start position (S1 or S2).

der. Rats had access to the two goal arms and to one of the two diametrically opposed start arms for 15 min (Fig. 1). Chocolate cereal was randomly scattered in the food cups as well as on the floor of the goal arms and rats were allowed to explore three arms of the maze freely. On the 2 following days, rats were given four pre-training trials a day, during which they were placed in a start arm and had access to only one goal arm which was baited. Over the eight pretraining trials, S1 and S2 were equally used as start positions and G1 and G2 as goal arms.

2.1.5.2. Acquisition of the non-matching rule (10 -s delay). Training began on the following day. Rats were trained with the transparent cylinder in order to allow them to guide their responses based on distant cues. Each rat was trained for eight consecutive trials a day, 6 days a week. Each trial comprised two successive runs: (i) a forced run in which the rat was placed in a start arm and had access to only one baited goal arm; (ii) a choice run in which the rat was placed either in the previously used start position or in the opposite position and had free access to both goal arms (Fig. 1). Rats had to go to the arm spatially opposite to the one previously visited in order to get food reinforcement. The two runs were separated by a 10-s delay during which time the rat was placed in a holding cage, under the maze. For half of the choice runs the position of the start arm was opposite to the one used during the forced run; for the remaining trials, the forced and choice runs were delivered from the same start arm

position (Fig. 1). There were eight possible combinations of start-goal locations between the forced and the choice runs. Each of these combinations was used once during the eight trials of the daily session, using a pseudo-random order, that was kept constant for each individual rat on a particular session, but which was modified from day to day. At the end of a session, the percentage of correct choices was calculated for each animal, as well as two types of perserveration indices: a place perseveration index, defined by the number of visits to the preferred arm (G1 or G2) divided by number of visits to the other arm (G2 or G1) and a response perseveration index, defined by the number of the preferred turns (left or right) divided by the number of the other turns (right or left). Rats were trained under these conditions until they reached a criterion of one error or less for three consecutive sessions or until a maximum number of 25 sessions.

2.1.5.3. Prospecti6e memory test (10 -s delay). Following acquisition of the non-matching rule, rats were trained under the same conditions, with the exception that the transparent cylinder was substituted for the opaque cylinder equipped with the fringed curtains. Under these conditions, rats were unable to choose the goal arm from the central platform of the maze and were thus forced to plan their responses from the start arm, i.e. to select prospectively the goal arm they will enter. Rats were trained under these conditions until they reached a criterion of one error or less for two consecutive sessions or until a maximum of ten sessions. 2.1.5.4. Extending the delay from 10 to 40 s. All rats were then retrained for five sessions using the initial training conditions, i.e. with the transparent cylinder. On the following session, the delay between the forced run and the choice run was increased from 10 to 40 s and the number of trials in each daily session was reduced from eight to six. Rats were trained with these conditions for 7 consecutive days. At the end of training, the number of sessions necessary to reach a criterion of one error or less for two consecutive sessions was determined by post-hoc analyses. 2.2. Results 2.2.1. Histology A schematic reconstruction of PL-IL lesions from coronal sections derived from a standard stereotaxic atlas [50] is illustrated in Fig. 2. In most cases, lesions were characterized by a central cavity (in the vicinity of injections sites) surrounded by non-functional necrotic tissue, composed of glial cells and picnotic shaped neurons. Cytoarchitectonic nomenclature for the medial prefrontal cortex advanced by Krettek and Price [41] was used in the description of the cortical lesions in the

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Fig. 2. Reconstruction of PL-IL cortical lesions (adapted from Ref. [50]), at various rostro-caudal levels (+5.2 to +1.7 mm from bregma) from the rats trained in experiments 1 and 2b. Black area: region lesioned for 75 – 100% of rats; hatched area: region lesioned for 50 – 75% of rats; grey area: region lesioned for 25–50% of rats. Lesions were centered around the ventral medial prefrontal cortex, damaging PL and IL cortices. Nomenclature adapted from Krettek and Price [41]: Acb, accumbens nucleus; ACd, dorsal anterior cingulate cortex; ACv, ventral anterior cingulate cortex; Cpu, caudate putamen; dp, dorsal peduncular cortex; IL, infralimbic cortex; LO, lateral orbital cortex; MO/VO, medial orbital and ventral orbital cortices; PL, prelimbic cortex; PrCm, medial precentral cortex; tt, taenia tecta; VLO, ventrolateral orbital cortex.

present study. A total of six rats were discarded from behavioural analyses due to unilateral lesions (n =2) or lesions restricted to the most rostral part of the prelimbic cortex (n=4). The seven remaining rats showed bilateral lesion of the PL (mean damage: 73.19 3.4%) and IL cortices (mean damage: 62.89 5.7%) resulting from an extended lesion throughout the ventral mPFC (global mean damage to the PL-IL area: 71 9 3.2%; minimum: 61.9%; maximum: 82.6%). Some rats also showed some damage to the orbital prefrontal cortex but the dorsomedial areas (anterior cingulate and medial precentral cortices) were almost always preserved in all rats.

2.2.2. Beha6ioural results 2.2.2.1. Acquisition of the non-matching rule (10 -s delay). Due to exaggerated stress reaction, one sham-operated rat and one PL-IL rat were discarded from subsequent analysis. Post-histological behavioural analyses were finally carried out on nine sham-operated rats and six PL-IL lesioned rats.

Rats in both groups gradually improved their performance up to the training criterion that was attained by the tenth session for some rats. An analysis of variance performed on the first ten sessions (Fig. 3A) indicated a significant main effect of Training session (F(9,117)= 5.83; PB 0.001) with no effect of Lesion and no Lesion by Training session interaction (FsB 1). PL-IL lesions did not affect the percentage of rats reaching the training criterion (Fisher test; P\ 0.297; Fig. 3B). An analysis of variance performed on the two perseveration indices throughout the 10-s training phase did not reveal an effect of the lesion (Fs(1,13)B 1.58; NS). The two groups of rats did not differ on the number of sessions required to reach the training criterion (a score of 25 was attributed to those that did not reach the criterion; FB 1), and did not differ on the percentage of correct choices during their last three training sessions either (F(1,13)=2.16; NS; Fig. 3A).

2.2.2.2. Prospecti6e memory test (10 -s delay). The substitution of the transparent cylinder by the opaque

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Fig. 3. Experiment 1: performance of sham-operated (Sham) and PL-IL lesioned rats (PL-IL) in the delayed non-matching to position task. (A) Mean percentage of correct responses ( 9 S.E.M.) obtained when rats were trained with a 10-s delay; a 10-s delay and a prospective strategy; a 10-s delay under the initial training procedure; and a 40-s delay with the initial training conditions. L1, L2, L3 sessions correspond to the three criterion sessions with a 10-s delay condition; L1 and L2 sessions correspond to the two criterion sessions with the prospective strategy. PL-IL lesioned rats demonstrated a significantly lower percent of correct choices than sham rats only when they were trained with a 40-s delay. (B) Percentage of rats reaching training criterion under the three different training conditions (10 s, 10 s using a prospective strategy, and 40 s). A clear impairment was noted with the 40-s delay condition due to the PL-IL lesions.

cylinder equipped with fringed curtains dramatically reduced the percentage of correct choices (F(1,13)= 95.91; PB 0.001; Fig. 3A). This was reduced to chance level with no significant Lesion by Training session interaction (F B1). Percentage of correct choices, however, rapidly increased and rats reached the training criterion from the fifth session. An analysis of variance performed on these first five sessions indicated a significant effect of Training session (F(4,52) = 3.93; PB 0.01) with no effect of Lesion (F(1,13) = 1.61; NS) and no Lesion by Training session interaction (F B 1). Analysis of variance performed on the perseveration indices

obtained for each rat throughout the prospective training phase did not indicate any effect of the lesion (Fs(1,13)B 1.26; NS). As indicated in Fig. 3B, there was no significant effect of PL-IL lesions on the percentage of rats reaching the training criterion (Fisher test; P\ 0.294). Only four sham-operated rats (out of nine) and four PL-IL rats (out of six) were able to reach the training criterion within the limit of ten sessions. Both groups of rats, however, exhibited a percent correct choices that was significantly above the random score during the two last training sessions (Sham: t(8)= 3.354; P B0.01; PL-

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IL: t(5)=3.207; PB 0.025; Fig. 3A) with no between group difference (FB 1).

2.2.2.3. Extending the delay from 10 to 40 s. Rats were then retrained under the initial conditions with the transparent cylinder for five successive sessions during which they showed a high level of performance (ranging from 75 to 90% of correct choices) and no effect of Lesion (F B1). During the last retraining session, the percent of correct choices was similar to those obtained during the last initial acquisition session (F(1,13)= 2.75; NS). Extending the delay between the two runs from 10 to 40 s disrupted performance in both groups (F(1,13)=7.74; PB0.05; Fig. 3A) with no interaction between Lesion and Training session factors (FsB 1). PL-IL lesions, however, disrupted the performance of rats when trained with a 40-s delay (Fig. 3). Analyses of variance performed on the percent correct choices during the seven training sessions with a 40-s delay, indicated a significant main effect of Lesion (F(1,13) =4.89; PB 0.05) due to poor performance of PL-IL lesioned rats, with no effect of Training session (F B1) and no Lesion by Training session interaction (F B1). It must be noted, however, that when restricted to the two last training sessions, analyses did not show a significant effect of Lesion (F(1,13) =1.326; NS), suggesting that the disruption demonstrated by PL-IL lesioned rats was progressively overcome. An analysis of variance performed on perseveration indices throughout the 40-s training phase did not indicate any effect of Lesion (F B 1). When trained with a 40-s inter-run interval, eight out of the nine sham-operated rats and only two of the six PL-IL rats were able to reach the post-hoc criterion (Fig. 3B). Analyses performed on the percentage of rats reaching criterion confirmed a significant disruptive effect of the PL-IL lesions (Fisher test; P B 0.05). 2.3. Summary The present findings indicate that PL-IL lesions did not affect acquisition of a delayed non-matching to position task which requires the use of allocentric spatial cues. This result confirms our previous results [12] and further indicates that the PL-IL area is not necessary for processing allocentric spatial discriminations. PL-IL lesions did not affect the ability of rats to plan spatial responses prospectively and finally, PL-IL lesions did not induced any perseverative tendencies. In contrast, these lesions disrupted the performance when the delay between the two runs was increased from 10 to 40 s. Lesioned rats demonstrated a lower percentage of correct choices when compared with control rats and did not reach the training criterion to the same extent as the controls. These data confirm that rats with lesion in the PL-IL area were more susceptible to making

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errors when a longer delay was introduced [12,13], however, the disruptive effect in the present experiment was only transient, as both groups of rats showed similar performance by the last two 40-s delay sessions.

3. Experiment 2: spatial navigation tasks The results in the first experiment show that rats with PL-IL lesions were able to discriminate between two distinct locations using spatial distal cues. This suggests that PL-IL area does not play an important role in the building up of allocentric representations necessary for spatial guidance. Nevertheless, some studies have reported a disruptive effect of PL-IL lesions, when rats were trained in complex spatial tasks involving or not involving a working memory component [21,59]. This might suggest that the PL-IL area is particularly required for structuring accurate spatial representations necessary to link multiple spatial locations, but not for guiding responses based on simple spatial representations. Disruptive effects of PL-IL lesions in complex spatial tasks might also suggest that the PL-IL area is engaged in the planning of spatial trajectories which requires attention and a high level of behavioural flexibility [21]. To investigate each of these possibilities, rats were trained in a spatial task where they had to reach a particular box among 13 similar ones that were placed on a circular arena. In experiment 2a, rats were trained under a regimen requiring a low level of behavioural flexibility and attention, using single start position. Under these conditions, spatial navigation task can be performed by using a constant relationship between the start position and distal cues, i.e. using a fixed route that is possibly based on simple stimulus-response associations. In experiment 2b, four different start positions were used, forcing rats to direct their behaviour by using complex and flexible spatial representations. Accuracy of spatial representations was further tested during a probe trial at the end of training. Finally, rats were trained with a black lid covering the goal box in order to investigate the effects of an additional discriminative cue on their performance.

3.1. Method 3.1.1. Subjects and surgical/histological procedures In experiment 2a, 18 male Sprague–Dawley rats were used. They were kept under conditions similar to those described for the first experiment. They were assigned to one of two groups: sham-operated rats (n=9) and PL-IL lesioned rats (n= 9). The same surgical procedure as that described previously was used. In experiment 2b, rats were the same as those used in experiment 1 (sham-operated rats, n= 10, and PL-IL lesioned rats, n= 13).

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Fig. 4. In the navigation task, only one goal box (always located in position 3) was baited. Rats were trained to reach this box either from one start position (‘South’; experiment 2a) or from the four different start positions (experiment 2b). During the probe trial (experiment 2b), time spent in the sectors surrounding the six numbered boxes (constituting a central hexagon in the apparatus) was recorded.

Rats in both experiments underwent food deprivation as described in experiment 1.

3.1.2. Material The experimental apparatus consisted of a 120-cm diameter circular platform (beige colour) surrounded by a 10-cm high transparent plastic wall. There were 13 white circular boxes (diameter: 3 cm) glued on the platform. Their positions in the arena defined a regular geometrical pattern illustrated in Fig. 4. Each box was covered by a white lid; a black lid was used in the second phase of experiment 2b. The arena was mounted on a rotating pedestal, 70 cm above the floor, and placed in the same experimental room as that described in the first experiment. Sucrose pellets (45-mg food pellets, Campden Instrument, Loughborough, UK) were used as food reinforcement. 3.1.3. Beha6ioural procedure 3.1.3.1. Pre-training. On the 1st day, rats were placed in a plastic box (25× 25 × 35 cm) in which they were habituated to eat the sucrose pellets placed in an opened circular box glued to the floor. On the following day, rats were replaced under similar conditions and were trained to open the circular boxes in order to get the food reinforcement. Rats were trained for four trials a day for 2 consecutive days. At the end of this training period, all rats were able to open the circular box in less than 10 s and eat the sucrose pellets. On the last pre-training day, rats were placed in groups of three or four on the arena and were allowed to freely explore the non-baited test apparatus for 15 min. 3.1.3.2. Training. Experiment 2a: spatial navigation — one start position: During training, only one of the 13

boxes was baited. This goal box was always located in position 3, in the inner hexagon (Fig. 4). At the beginning of each session, the rat was placed on the platform, facing the plastic wall from a single start position (South position; Fig. 4) and the latency to open the goal box was recorded. At the end of the trial, the rat was replaced in a holding cage placed under the apparatus for  10 s and the maze was rotated to prevent rats using olfactory cues. Thus the goal box was different from trial to trial but its location relative to the external distal cues remained the same. The arena was rapidly wiped between rats. Rats were trained for four consecutive trials a day for 12 consecutive days. Each day, the number of trials for which only the baited box was opened, was noted and defined as the number of hits (ranging from 0 to 4). Experiment 2b: spatial navigation — four start positions: (a) Acquisition. The same behavioural procedures as those described in experiment 2a were used in the present experiment, with the exception that there were four different start positions (Fig. 4) used alternatively during the four daily trials. These start positions were used in a pseudo-random order that remained constant for all rats for a given session but varied between sessions. Rats were trained under these conditions for 14 consecutive days. In addition to the response latencies and the number of hits, the distance covered to reach the goal box was recorded for each trial from the seventh training session (in-house software, only available at that time). We chose to analyse this variable because previous work has shown path length to be a sensitive measure for such a dry-land version of the water maze task [32,57]. (b) Probe trial. After the end of the 14th session, a probe trial was given to test memory of the goal box location. Rats were placed on the arena in the ‘South’ start position for a 1-min period of free exploration with non-baited boxes. The time spent in each sector surrounding the six central hexagon boxes was recorded (Fig. 4). (c) Addition of a proximal visual cue. During the last six training sessions, rats were trained as previously, but the goal box was further indicated by a black lid, acting as a proximal cue.

3.2. Results 3.2.1. Histology 3.2.1.1. Experiment 2a. Cortical lesions observed in rats trained in experiment 2a were very similar in terms of location to those described in the first experiment. The only difference was in lesion size, which was more extensive in these rats, compared with those in experiment 1. A total of four rats were discarded from behavioural analyses as the lesions were extensive (en-

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croachment of the lesion on the dorsomedial prefrontal cortex). For the remaining rats, damage of the PL and of the IL was almost complete (mean = 85.5% for the PL and mean=91.9% for the IL). In all, the PL-IL area was almost completely damaged (global mean damage: 86.893.4%; minimum: 83.7%; maximum: 100%) but the dorsomedial prefrontal cortex was mostly spared in all rats. Statistical analyses were finally carried out on nine sham-operated rats and five PL-IL lesioned rats which reached the histological criteria previously described.

3.2.1.2. Experiment 2b. Histological analyses have been previously described (Fig. 2), as the rats used in experiment 2b were the same as those used in experiment 1. Behavioural analyses were carried out on ten sham-operated rats and seven PL-IL lesioned rats. 3.2.2. Beha6ioural results 3.2.2.1. Experiment 2a: spatial na6igation — one start position. Response latencies: The latencies rapidly decreased from the first session to the end of training. Analyses of variance performed on these latencies indicate a significant effect of Training session (F(11,132)=7.257; P B 0.001) with no effect of Lesion and no Lesion by Training session interaction (FsB 1). Hits: as depicted in Fig. 5, there was no between group difference with the number of hits, which progressively increased across the 12 training sessions. Analysis of variance performed on this measure showed a significant effect of Training session (F(11,132)= 10.879; P B0.001), with no effect of Lesion (F(1,12)= 1.622; NS) and no Lesion by Training session interaction (F(11,132)= 1.11; NS).

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3.2.2.2. Experiment 2b: Spatial na6igation — four start positions. (a) Acquisition: (i) response latencies. The latencies rapidly decreased across training sessions (F(13,195)= 22.99; PB0.001). Although PL-IL lesioned rats showed slightly higher response latencies than sham-operated rats (mean response latencies from the first to the 14th session: sham-operated: 11.3 s; PL-IL lesioned rats: 13.5 s), analyses revealed no significant effect of Lesion and no Lesion by Training session interaction (FsB 1). (ii) Distances (Fig. 6A) Analyses on the distance covered to reach the goal box were only performed from the seventh training session. These analysis showed a significant main effect of Lesion (F(1,15)= 5.44; PB 0.05), indicating that PL-IL lesioned rats covered greater distances than control rats before opening the goal box. There was, however, a progressive decrease in the distance travelled (F(7,105)=4.6; PB 0.001), with no significant Lesion by Training session interaction (FB 1). (iii) Hits (Fig. 6B). Analyses of variance performed on the number of hits showed a significant main effect of lesion (F(1,15)= 5.7; P B0.05). In addition, there was a significant effect of Training session showing that rats gradually improved their performance during the 14 training sessions (F(13,195)= 8.96; PB 0.001), with no significant Lesion by Training session interaction (F(13,195)= 1.16; NS) (b) Probe trial: During the probe trial (Fig. 7) there was no between group difference on the time spent outside the inner hexagonal area where the goal box was located (F(1,15)= 2.72; NS). Analyses of variance performed on the time spent in each sector of the inner hexagon showed a significant main effect of Sector (F(5,75)= 20.9; PB 0.001), with no effect of Lesion (F(1,15)= 2.822; NS) and no interaction between these

Fig. 5. Experiment 2a: Mean number of hits (9 S.E.M.) performed by sham-operated (Sham) and PL-IL lesioned rats (PL-IL) when trained from a single start position in the navigation task. No difference was observed between the two groups of rats.

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Fig. 6. Experiment 2b: Performance of sham-operated (Sham) and PL-IL lesioned rats (PL-IL) when trained from four start positions in the navigation task. Rats were first trained in a spatial version of the task (left panel) and then in a spatial-cued version (right panel). (A) Averaged distances in centimetres ( 9S.E.M.) covered to reach the goal box. In both training conditions, PL-IL lesioned rats demonstrated longer path lengths than Sham rats when trained in the spatial as well as the spatial-cued version of the task. (B) Mean number of hits ( 9S.E.M.). PL-IL lesioned rats demonstrated significantly fewer number of hits when trained in the spatial version of the task. This effect disappeared when the location of the goal box was further indicated by a discriminative cue (spatial-cued).

two factors (FB1). Complementary analyses indicated that rats in both groups spent significantly longer periods of time in the sector surrounding the goal box (sector B3 \ in Fig. 7) than in any of the others sectors (Fs \30; Ps B0.001). (c) Addition of a proximal visual cue: (i) response latencies. Analyses of variance performed between the 14th and 15th session indicated that signalling the goal box with a discriminative black lid did not significantly modify the response latencies (F B1) and there was no interaction between Lesion and Training session factors (F B 1). During the six following training sessions re-

sponses latencies, however, significantly decreased (F(5,75)= 4.06; P B0.005), with no effect of Lesion (F(1,15)= 2.84; NS) and no Training session by Lesion interaction (FB 1). (ii) Distances (Fig. 6A). Statistical analysis performed on the 14th and the 15th sessions revealed that the introduction of a proximal cue did not alter the distances covered (FB 1) and there was no significant interaction between Lesion and Training session factors (F(1,15)=2.56; NS). Analyses performed on the six following training sessions showed a significant main effect of Lesion (F(1,15)= 6.69; P B 0.05), indicating

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that PL-IL lesioned rats still covered a greater distance than sham-operated rats. The progressive decrease in distances just failed to reach statistical significance (F(5,75)= 2.33; P= 0.0505) and there was no Lesion by Training session interaction during this training period (F B 1). Complementary analysis aimed at comparing the learning rates with and without the additional proximal cue were performed between spatial training period (sessions 7– 14) and the spatial-cued training period (sessions 15– 20). An analysis of variance on individual linear regression slopes calculated for each of these two training periods indicated no effect of the Training period (F(1,15) = 1.27; NS), no effect of Lesion (F(1,15)= 1.42; NS) and no interaction between these two factors (FB1), confirming that the proximal cue did not critically affect distances covered to reach the goal box in either group. (iii) Hits. As depicted in Fig. 6B, the number of hits was not significantly modified between the 14th and the 15th training sessions (F(1,15) = 2.53; NS) and there was no Lesion by Training session interaction (FB 1). During the six consecutive training sessions with the black lid, a significant improvement in the number of hits was observed (F(5,75) =8.31; P B 0.001) with no lesion effect and no Lesion by Training session interaction (FsB 1). Analysis of individual linear regression slopes calculated for the spatial training period (sessions 1 – 14) and the spatial-cued training period (sessions 15 to 20) showed a significant main effect of training period (F(1,15)= 6.78; PB 0.025), indicating that the introduction of the proximal cue modified the time course of the

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hit performance. Complementary analyses showed, however, that only the lesioned rats benefited from the additional proximal cue, as PL-IL lesioned rats but not control rats showed a significant increase in their learning slopes between the two training periods (PL-IL rats: 0.085 vs. 0.282; F(1,6)= 5.94; P= 0.0507; Sham rats: 0.125 vs. 0.197; F(1,9)= 1.6; NS).

3.3. Summary The results from experiment 2a indicate that lesioned rats were perfectly able to reach a fixed location in a complex spatial environment when a single start position was used. This suggests that the PL-IL area is not necessary for rats to learn simple goal-directed task and confirms previous results of our own [12]. In contrast, lesions of the PL-IL area disrupted performance when the fixed location had to be reached from four different start positions (experiment 2b), confirming previous results from rats trained in a water maze with multiple start positions [21]. PL-IL lesioned rats made fewer hits and travelled longer distances to reach the goal box, but were able to locate the goal box during the probe trial. The deficit due to PL-IL lesions therefore seems to be more likely due to difficulties in response adjustments when training requires a high level of behavioural flexibility rather than an inability to form spatial representations [21,23]. When further training was given with an additional proximal cue, signalling the location of the goal box, PL-IL lesioned rats rapidly improved their performance in terms of the number of hits they made, showing no difference to control rats. They still, however, travelled a longer distance to reach the goal box. This suggests that the additional cue only partially overcame the behavioural deficit demonstrated in rats with PL-IL lesions, as they still had difficulties in selecting the shortest path to reach the goal box.

4. Discussion The main aim of this study was to investigate the involvement of the PL-IL area in the building up and use of spatial allocentric representations. The second aim was to test the validity of concurrent functional hypotheses concerning the role of the PL-IL area in spatial working memory, in planning of prospectively organized spatial responses and in behavioural flexibility and attentional processes. Fig. 7. Experiment 2b: Mean time in seconds ( 9 S.E.M.) spent around each of the six boxes which constituted the inner hexagon of the arena during the probe trial, delivered at the end of session 14, for sham-operated (Sham) and PL-IL lesioned rats (PL-IL). Rats in both groups spent significantly longer periods of time in the sector surrounding the goal box with no group difference.

4.1. PL-IL and the processing of allocentric spatial information Our results provide evidence suggesting that the PLIL area is not directly involved in processing allocentric

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information. We have previously shown that the integrity of the PL-IL area is not required to learn a spatial delayed alternation task in a Y maze, when the alternation rule may require egocentric and/or allocentric cues [12]. The present experiments show similar results when the behaviour was only possibly guided by allocentric cues (experiment 1). Results obtained in experiment 2a further indicate that lesioned rats were also able to perform more subtle spatial discriminations as they learned the location of a single baited box among several others, providing that they were trained from a single start position. Taken together, these results suggest that PL-IL lesions do not dramatically affect spatial representations, confirming previous results of our own [12]. This result is also in agreement with several other reports which have shown that even greater damage to the prefrontal cortex, invading variable extents of the PL-IL area, largely preserves the ability to remember spatial locations [29,52,54], even when responses explicitly require the use of allocentric maps [9,30,51,56,57]. The fact that PL-IL lesioned rats were impaired when trained to reach a baited box on a circular arena from four different start positions may be viewed as supporting the view that the cognitive representations of lesioned rats are not accurate enough to guide their behaviour in complex tasks [21,59]. The probe trial given at the end of the initial acquisition phase, however, showed that lesioned rats performed as well as control rats, spending significantly longer periods of time in the sector surrounding the goal box than in any of the other sectors. This then suggests that PL-IL lesioned rats built accurate spatial representations during the course of training. Such a contention is further supported by results showing that rats with PL lesions were also able to react normally to spatial configuration changes [23] and to correctly locate the position of a previously hidden platform in a water maze [21], or the correct hole in a cheeseboard task [57]. Recent studies suggest that this region probably does not directly participate in the neural network subserving spatial representations as there is no strong evidence for ‘place cells’ in the PL area [28,53]. In all, the present results add to the evidence suggesting that the PL-IL area is not directly involved in the building up of allocentric cognitive maps. Nevertheless, according to the present results, as well as to others [21,56,57], it seems that this region might play a role in the use of the spatial representations under particular experimental constraints.

4.2. PL-IL area and (spatial) working memory Raggozino et al. [55] recently claimed that the PL-IL area subserves working memory restricted to allocentric spatial information. The present results partially

support this assumption. Results from experiment 1 indicate that while lesions of the PL-IL area did not affect the acquisition of a delayed non-matching to position task, they impaired the performance when the delay between the forced run and the choice run was subsequently increased from 10 to 40 s. Such a disruption of performance might be analysed as resulting from an increase in the working memory load and might be considered as supporting involvement of the PL-IL area in spatial allocentric working memory. However, the hypothesis of involvement of PL-IL restricted to spatial working memory is unable to account for all the deficits induced by lesions in this area. First, such an hypothesis appears to be too limited as PL-IL lesions may also lead to delay-dependent disruption of performance in a non-spatial conditional discrimination task performed in an operant chamber [13], while they have no effect in a standard elimination task in a radial maze, which is considered to be a spatial working memory task [11,12]. Second, experiment 2b indicates that PL-IL lesions also affected the acquisition of a spatial allocentric task, with no obvious working memory component. In addition, the fact that the delay-dependent disruptive effects of PL-IL lesions (experiment 1) were only transient does not strongly support an involvement of the PL-IL area in the maintenance of specific information over a time delay (see also Ref. [59] for a similar position). This is further supported by the fact that no prefrontal units have shown unambiguous spatially dependent delay activity [28]. Recently, Ragozzino et al. [56] indicated that PL-IL inactivation did not impair the acquisition of a spatial working memory task, contradicting previous results of their own obtained with quinolinic acidlesioned rats in a different behavioural task [55], thus questioning the involvement of this area in spatial working memory. In all, these data suggest that the PL-IL area is engaged during some spatial (and nonspatial) working memory tasks, but it does not seem that working memory processes can fully account for the functional role of this area.

4.3. PL-IL area and prospecti6e memory In our view, prospective memory for rodents involves memory for actions to come that must be executed in the absence of any direct external cues. In experiment 1, rats had to choose between two arms using cues that were accessible only from the start position. This task was used to test the possible involvement of the PL-IL area in prospective memory, previously suggested by Seamans and his collaborators [59]. While it is difficult to determine which strategy was initially used by rats during the acquisition of the spatial delayed task, it should be noted that the performance in both groups of rats dropped to chance level

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when the opaque cylinder was introduced. Performance, however, progressively recovered, with no between group difference when rats were required to use prospective memory, suggesting that PL-IL is not required to plan a spatial response prospectively. Only two previous studies proposed an involvement of the PL- IL in prospective memory. Kesner [30] suggested that the ability to remember spatial locations in a radial maze can be based upon a retrospective encoding (memory of previously visited arms), and upon a prospective encoding (memory of the remaining nonvisited arms) and that damage to the prefrontal cortex specifically disrupted the prospective encoding while preserving the retrospective encoding [6]. As lesions in this study were restricted to the dorsal part of the mPFC, largely sparing the ventral part, these results do not provide definitive evidence to suggest the PL-IL area may be involved in prospective memory. More recently, Seamans and his colleagues [59], using a spatial delayed non-matching task in a radial arm maze, showed that transient inactivation of the PL cortex disrupted performance only when it was effective before the delayed response but not when it was effective during the initial sampling of information. These results indicate that the PL is not required to maintain specific information over a time delay but is involved either in the retrieval or in the use of this information when responses have to be initiated (i.e. prospective planning). Thus, Seamans and his colleagues did not directly address whether the PL is involved in prospective planning, but only suggested it as a possible explanation to account for their results. In summary, the literature does not provide any conclusive evidence of the involvement of the PL-IL area in prospective memory and the present results do not support such an hypothesis.

4.4. PL-IL area, beha6ioural flexibility and attentional processes The results shown in experiment 2b indicate that rats with PL-IL lesions were disrupted in spatial navigation when trained from four different start positions. Lesioned rats had a tendency to go less directly to open the goal box and covered greater distances to reach it. The fact that lesioned rats performed correctly when trained from a single start position (experiment 2a) and were able to recognize the precise location of the goal box, using distal environmental cues, as indicated by the probe trial (experiment 2b), shows that these lesions preserve the accuracy of spatial representations. The disruption of performance demonstrated by lesioned rats might thus be viewed as resulting from difficulties for adequately planning spatially guided responses which occur only when a high level of flexibility is required [21]. Adding a discriminative cue that further

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specified the goal box location partially improved performance in PL-IL lesioned rats. More precisely, these rats rapidly increased the number of hits they made, demonstrating that under these conditions they were able to directly select the correct goal box. The effects of the proximal cue on behavioural accuracy was further confirmed by a significant increase in the slope of the learning curve of the hits observed in PL-IL rats once the cue was introduced. The addition of a proximal cue, however, did not completely overcome the disruption in performance observed in lesioned rats as they still covered greater distances to reach the goal box than control rats. This suggests that difficulties in selecting the optimal path from each start position were not attenuated by the introduction of the discriminative cue. Therefore, PL-IL lesions might be seen as inducing a dual effect on navigation behaviour: (1) an effect on the selection of the correct goal box that can be compensated for by the use of an additional proximal cue, suggesting some form of attentional deficit and (2) an effect on the selection of the shortest path according to each start position that persisted even when attention was driven to the goal box by a proximal cue and thereby suggesting it was more likely that PL lesioned rats had some difficulties in behavioural flexibility. In recent years, there has been accumulating evidence that the ventromedial prefrontal cortex is involved in both attention and in behavioural flexibility. The involvement of the PL-IL area in attentional processes is supported by recent results of Bussey and collaborators [4] who showed that rats with medial prefrontal lesions (mainly involving the PL area) were severely disrupted in reversal learning, only when stimuli were difficult to discriminate [45]. In addition, it has recently been shown that the integrity of PL-IL is necessary to detect subtle variations in brightness, suggesting that this area might be involved in sustained attention [24]. The hypothesis of an involvement of the mPFC in behavioural flexibility is also well documented (see Ref. [8] for a review). For example, damage to the prefrontal cortex has been shown to disrupt reversal learning (see Ref. [33] for a review) and to affect the ability to shift from a spatial to a visual strategy in order to locate a platform in a water maze [9]. A similar analysis might be proposed to explain the deficit demonstrated by mPFC lesioned rats when they had to spontaneously switch from an allocentric to an egocentric orientation strategy [10]. Some evidence suggests that these behavioural adjustments rely on a specific part of the mPFC, i.e. on the PL-IL area. For example, a direct correlation has been reported between a deficit in reversal learning and the extent of PL-IL damage [70]. The present results indicate that PL-IL lesions induce impairments in the ability to adequately plan trajectories from different start positions, replicating previous findings observed with water maze training [21]. Deficits in behavioural

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flexibility induced by PL-IL lesions, however, do not appear to result from an increase of perseverative tendencies (experiment 1; [12]) or from a decrease in inhibition [13]. It seems more likely that PL-IL lesions induced difficulties in the ability to select and plan flexible responses. Such a contention is supported by results showing that transient inactivation of the PL disrupted the ability of rats to switch between spatial win-shift and random foraging strategies [59], as well as by our previous results showing that PL-IL lesioned rats were severely disrupted when switching from a fixed-goal location to a variable-goal location strategy in a radial arm maze task [12]. Two recent studies using PL-IL inactivation clearly have demonstrated the role of the PL-IL area in the ability to switch to new behaviour-guiding strategies, giving support to the involvement of this area in behavioural flexibility [56,57].

4.5. What are the precise roles for the prelimbic-infralimbic cortices? The main aim of this paper was to investigate the roles of the prelimbic-infralimbic cortices in the processing of spatial information. The present results show that, despite its strong connections with the hippocampal formation, the PL-IL area is not involved in the building up of accurate allocentric representations. The second aim of this paper was to determine which functional hypothesis among those already proposed can account for the present results as well as previous results concerning the ventromedial part of the prefrontal cortex. First, the present results show that lesioned rats are able to plan a response based upon spatial cues prospectively, strongly suggesting that the PL-IL area is not involved in prospective planning (but see Ref. [17,59]). Second, the present results do not support the hypothesis for an involvement of the PL-IL restricted to spatial allocentric working memory as proposed by Ragozzino and his collaborators [55]. We have presented evidence showing that even a more general hypothesis postulating an involvement of PL-IL in working memory processes cannot account for all the results derived from lesion studies because performance disruption can be obtained in non-working memory tasks (experiment 2b, [21]), while the acquisition of some working memory tasks are not affected by the lesions [12,56]. The present results show a performance disruption due to PL-IL lesions under two different circumstances: (1) in a delayed-response task, when the delay between the information to be remembered and the response was increased and (2) in a navigation task when the response changed from trial to trial, as a function of the starting position. We proposed that these deficits collectively result from a disruption in attention and in behavioural flexibility. We have al-

ready presented arguments supporting that such an explanation may account for the performance deficit demonstrated by PL-IL lesioned rats in the navigation task where it seems that both the attentional and the behavioural flexibility impairments may be possibly dissociated. We have already proposed that delayed-response tasks can be considered as a particular set of tasks involving strategy switching that may be required to bring into action the ventromedial prefrontal cortex in order to provide adapted behavioural adjustment [13]. It is generally considered that extending the delay only increases the working memory load, but it can be alternatively proposed that increasing the delay implicates the development of a new strategy that requires the involvement of the PL-IL area. As recent evidence strongly supports the notion of a role of this area in strategy switching [56,57], such an hypothesis may account for the performance disruptions repeatedly obtained in delayed-tasks when the delays are progressively increased [3,12,13]. Accordingly, different effects of PL-IL lesions might be expected in delayed-response tasks, depending on the use of a progressive or a mixed delay procedure. This is the subject of our ongoing experiments. The involvement of PL-IL in both attention and behavioural flexibility suggested by the present results, as well as by others [12,13,56,57] supports the contention that this cortical region acts as a ‘supervisory attentional system’ [62] required for programming, verifying and regulating activity in situations involving novelty or planned initiative. Such an assumption would certainly strengthen the functional homologies often stressed between human, monkey and rodent prefrontal functions. Acknowledgements Dr B. Delatour was supported by a research fellowship in Neurobiology from Laboratoires Lilly. References [1] Becker JT, Walker JA, Olton DS. Neuroanatomical bases of spatial memory. Brain Res 1980;200:307 – 21. [2] Becker JT, Olton DS, Anderson CA, Breitinger ERP. Cognitive mapping in rats: the role of the hippocampal and frontal systems in retention and reversal. Behav Brain Res 1981;3:1 – 22. [3] Brito GNO, Brito LSO. Septohippocampal system and the prelimbic sector of frontal cortex: a neuropsychological battery analysis in the rat. Behav Brain Res 1990;36:127 – 46. [4] Bussey TJ, Muir JL, Everitt BJ, Robbins TW. Triple dissociation of anterior cingulate, posterior cingulate, and medial prefrontal cortices on visual discrimination tasks using a touchscreen testing procedure for the rat. Behav Neurosci 1997;111:920–36. [5] Conde´ F, Maire-Lepoivre E, Audinat E, Cre´pel F. Afferent connections to the medial prefrontal cortex of the rat: II. Cortical and sub-cortical afferents. J Comp Neurol 1995;352:567–93.

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