Neurobiology of Learning and Memory 93 (2010) 495–505

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Neurobiology of Learning and Memory

journal homepage: www.elsevier .com/ locate/ynlme

Directional learning, but no spatial mapping by rats performing a navigationaltask in an inverted orientation

Stephane Valerio 1, Benjamin J. Clark 1, Jeremy H.M. Chan, Carlton P. Frost, Mark J. Harris, Jeffrey S. Taube *

Department of Psychological and Brain Sciences, Dartmouth College, 6207 Moore Hall, Hanover, NH 03755, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 14 October 2009Revised 1 January 2010Accepted 20 January 2010Available online 28 January 2010

Keywords:Spatial navigationVestibular systemHead direction cellsSpatial task

1074-7427/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.nlm.2010.01.007

* Corresponding author. Fax: +1 603 646 1419.E-mail address: jeffrey.taube@dartmouth.edu (J.S.

1 These authors contributed equally to this study.

Previous studies have identified neurons throughout the rat limbic system that fire as a function of theanimal’s head direction (HD). This HD signal is particularly robust when rats locomote in the horizontaland vertical planes, but is severely attenuated when locomoting upside-down (Calton & Taube, 2005).Given the hypothesis that the HD signal represents an animal’s sense of directional heading, we evaluatedwhether rats could accurately navigate in an inverted (upside-down) orientation. The task required theanimals to find an escape hole while locomoting inverted on a circular platform suspended from the ceil-ing. In Experiment 1, Long-Evans rats were trained to navigate to the escape hole by locomoting fromeither one or four start points. Interestingly, no animals from the 4-start point group reached criterion,even after 29 days of training. Animals in the 1-start point group reached criterion after about six trainingsessions. In Experiment 2, probe tests revealed that animals navigating from either 1- or 2-start pointsutilized distal visual landmarks for accurate orientation. However, subsequent probe tests revealed thattheir performance was markedly attenuated when navigating to the escape hole from a novel start point.This absence of flexibility while navigating upside-down was confirmed in Experiment 3 where we showthat the rats do not learn to reach a place, but instead learn separate trajectories to the target hole(s).Based on these results we argue that inverted navigation primarily involves a simple directional strategybased on visual landmarks.

� 2010 Elsevier Inc. All rights reserved.

1. Introduction

The different strategies used by humans and animals to navigateaccurately in their environment have been studied extensively byneuroscientists (Aguirre, Zarahn, & D’Esposito, 1998; Hartley, Magu-ire, Spiers, & Burgess, 2003; Maguire et al., 1998; O’Keefe & Nadel,1978). The theories postulated to account for successful navigationhave been shaped, in part, by the discovery of different types of spa-tially tuned neurons that code for location (place cells), directionalorientation (head direction cells), and grid of the environment (gridcells) using an allocentric frame of reference (Ekstrom et al., 2003;Hafting, Fyhn, Molden, Moser, & Moser, 2005; O’Keefe & Dostrovsky,1971; Taube, Muller, & Ranck, 1990). Indeed, these findings, com-bined with animal lesion studies showing that disruption of brainareas containing these neurons prevents animals from solving allo-centric spatial tasks (Morris, Garrud, Rawlins, & O’Keefe, 1982), ledto the idea that these neurons, primarily found in the limbic system,were the neural substrate of a flexible representation of space or cog-nitive map (O’Keefe & Nadel, 1978). On the other hand, when facing a

ll rights reserved.

Taube).

spatial problem, rats do not always rely on a cognitive map-basedstrategy, but may also: (1) use a specific landmark situated close tothe goal (beacon navigation), (2) use a small number of distal cuesthat provides them the direction to find the goal (direction strategy),or (3) simply learn a sequence of turns (e.g., left–right–left) and thususe a body-centered (egocentric) frame of reference (response strat-egy) (McDonald & White, 1993; Packard & McGaugh, 1996; Skinneret al., 2003).

Most studies on mammalian navigation have involved two-dimensional terrestrial environments. However, many species liveand move about in a three-dimensional (3-D) environment – themost obvious species include marine and arboreal mammals, aswell as rodents that live in subterranean environments. But hu-mans also frequently navigate in 3-D environments, particularlyair pilots and astronauts, many of whom frequently encounterperiods of disorientation that often lead to motion sickness, whichcan have serious consequences during flight (Oman, 2007). Thus,determining which strategies and reference frames are used duringnavigation in a 3-D environment is crucial for understanding howanimals live and orient in a 3-D world.

Previous experiments have studied how head direction (HD)cells respond when a rat locomotes in a vertical plane or upside-down in 1-g (Calton & Taube, 2005; Stackman, Tullman, & Taube,2000) and in 0-g (Taube, Stackman, Calton, & Oman, 2004). In these

496 S. Valerio et al. / Neurobiology of Learning and Memory 93 (2010) 495–505

experiments, it was observed that the vertical plane was treated asan extension of the floor and that HD cells maintained their direc-tion-specific firing when the rat locomoted on the wall. However,when the rats locomoted upside-down on the ceiling, HD cellsshowed a dramatic change in their activity, including a completeloss of directional-specific firing in two-thirds of the cells, and amarked reduction in directional activity in the remaining cells (Cal-ton & Taube, 2005). Furthermore, in cells that maintained somesemblance of directional firing, there was frequently a shift in theirpreferred firing direction. However, even with this distorted direc-tional signal, rats performed quite well in the task – climbing a ver-tical wall to a ceiling, then traversing the ceiling inverted, andfinally down another wall in order to reach a reward compartment.Observations of the rats appeared to indicate that they knew whichdirection to go in order to obtain the reward. This accuracy in thetask was attributed to the low navigational demand and may nothave required a functional HD system. To date, there are no exper-iments that have tested the extent to which rats are capable ofaccurate navigation in an inverted orientation. In the present studywe devised a more demanding spatial paradigm in order to exam-ine whether rats are capable of accurate navigation in an invertedorientation, and if so, what spatial strategies they use to performthe task.

2. General methods

2.1. Subjects

Experimentally naive female Long-evans rats served as subjectsfor all three experiments. The animals were housed singly in clearplastic cages in a room maintained on 12 h light/dark cycle. Waterand food were available ad libitum in the home cage. All procedureswere conducted under an institutionally approved animal care anduse protocol.

2.2. Apparatus

The ‘‘inverted maze” apparatus consisted of a 190-cm diametercircular wire mesh that was suspended �50 cm from the ceiling,with four evenly spaced holes that formed four possible escape ex-its from the maze (Fig. 1). Each one of these 15-cm holes could beclosed with a piece of wood. The wire mesh was composed of me-tal screening with square size of 0.5 � 0.5 cm. The maze was in aroom containing many salient visual cues including posters, a TV,a door, a blackboard, two windows along one wall, and a desk witha computer on it.

Fig. 1. The test room and the inverted maze apparatus. The apparatus consisted of a 190four evenly spaced holes that formed four possible escape exits from the maze. Each oncomposed of metal screening with square size of 0.5 � 0.5 cm.

2.3. Behavioral procedure

Prior to the commencement of training all rats were handled3 min per day for 3 days. The basic task involved the rat beingplaced in an inverted position, either on the periphery of the mazeor at the maze center (see below), and because rats contain a nat-ural proclivity to remain in an upright position they were highlymotivated to find a hole to escape through in order to enable them-selves to return to an upright position on top of the circular board.For all experiments three of the four holes were blocked with apiece of wood – thus, only one hole was open to escape through.Although the rat could see the small depression made by the fourholes, it was not possible for it to determine which hole was thecorrect one to escape through without being close to it becauseof the small angle between its head and the wire mesh surface. Thispoint is firmly demonstrated by noting the great difficulty rats hadin finding the correct hole in the four start position condition (seeresults below). Throughout all the experiments there was no at-tempt to disorient the rat when bringing it into the room or whenplacing it on the inverted maze.

2.3.1. PretrainingBefore being trained to solve the spatial task, the rats were

trained to climb short distances in an inverted position on the in-verted maze. More specifically, pretraining consisted of eight trialsper day where the rats were released on the maze (upside-down)in close proximity to one of the four escape holes. This procedureenabled them to learn how to locomote inverted on the mazeand to understand how to exit from the maze by passing throughthe hole and returning to a normal upright position on top of themaze. The target hole and the release direction of the rats werevaried each trial. Depending on the improvement of the rats, theexperimenter progressively increased the distance between the re-lease point and the hole until the animals were able to reach thetarget hole when released 20 cm away from it. When animals wereable to climb to the open hole from that distance, the animals wereassigned to a testing group and the training phase started. The ratsthat could not complete this pretraining phase after 8 days wereconsidered ‘‘bad climbers” and excluded from the experiments.

2.3.2. TrainingAt the start of training the rats’ cages were brought into the maze

room on a cart and placed 2 m away from the maze on one side ofthe room. A typical training trial consisted of releasing the rat fromone of the four start points (north, south, west and east) along theperimeter of the maze and allowing it to locomote inverted to thecorrect uncovered escape hole. In cases when the rats could not

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Fig. 2. Experimental protocol for the one (1SP), two (2SP), and four (4SP) start points training conditions. (A) 1SP condition, where the rat is always released from the sameentry point. (B) 2SP-Same place-Opposite turn: the rats had to learn to reach a single target hole from two different start points (e.g., south and east) that required twoopposite turns (right from south, and left from east). (C) 2SP-Different place-Opposite turn: the rats were also released in the maze from two start points, but in this conditionthey had to reach one of two distinct target holes (different place) depending on their release point in the maze. The start points were selected such that the rats had to learntwo opposite turns, similar to the conditions in B. (D) 2SP-Different place-Same turn: the rats were trained to reach two different escape holes (different place) from two startpoints, but in this case, both escape holes were in the same relative position from the start point. In this condition, learning a particular egocentric turn (e.g., always turn right)could be a successful strategy for the rats. (E) 4SP condition, where the rat is released from one of four different entry points.

S. Valerio et al. / Neurobiology of Learning and Memory 93 (2010) 495–505 497

reach the hole after 30 s, they were led to it by the experimenter andgiven a score of 30 s. We could not allow for more search time be-cause the rats frequently became tired while suspended and wouldinevitably fall off the maze after a minute. Moreover, we observedthat well-trained rats were proficient at navigating to the escapehole in less than 30 s, regardless of whether the W, E, S, or N startpoint was used (see Fig. 1 right panel). At the end of each trial therat was returned to its home cage while the other rats were givena training trial. Each training session consisted of four trials and ani-mals were given one training session per day. The latency to find thetarget hole was timed by the experimenter standing at differentlocations around the edge of the circular platform. All training trialswere videotaped via a video camera placed on the floor below themaze and aimed upward at the inverted maze.

2.3.3. CriterionIn all three experiments, the animals were considered to have

learned the task when they accurately reached the target hole(in <30 s) on three out of four trials for two consecutive days.

2.4. Statistical analysis

In Experiment 1, an ANOVA with repeated measures for the fac-tor session was performed to analyze the performance of animalstrained from four start points (4SP). In all other cases learning com-parisons were performed on the mean group value (±SEM) for thenumber of training days necessary to reach criterion, which wereanalysed using Student’s t-test. All tests comparing the perfor-mance of animals in a specific testing situation with their perfor-mance in their last regular training session were analysed usinga two-way ANOVA (session � group), with repeated measures forthe factor session.

3. Experiment 1

The aim of the first experiment was to test whether, given themarked disruption of the head direction signal during invertedlocomotion, the rats were able to learn a spatial task that requiredthem to navigate accurately upside-down. This task resembled theclassic Morris water maze task (Morris et al., 1982) in that the ratshad to locate a spatial location relative to room cues, either fromone or four different entry points, in order to avoid an aversivestimulus, which in this case was the continuous state of being inan inverted position.

3.1. Method

Two groups of animals were trained to two different versions ofthe task: (1) 11 animals were trained to find the target hole from

four start points (4SP, Fig. 2E) and (2) 10 animals were trained fromone start point (1SP, Fig. 2A). For each animal, the target hole waskept constant during the entire training procedure.

Following training on the inverted hole board escape task, the4SP group was trained in a Morris water maze task. The pool(180 cm diameter, 50 cm high) was positioned 14 cm above thefloor. The water, which was �23 cm in depth, was made opaquewith white powder and a 13 cm diameter plastic platform was lo-cated in one quadrant of the pool, such that its top was submerged�1 cm beneath the surface. The platform’s location was fixed for alltrials. Each trial was videotaped from an overhead camera. For eachtrial we measured the time it took for them to find and climb ontothe platform (escape latency). Animals received four trials per dayuntil they reached the learning criterion, which was defined asfinding the platform in less than 10 s in three out of four trialson two consecutive days.

3.2. Results and discussion

The rats trained from one start point reached criterion after amean of 5.9 ± 0.57 (SEM) training sessions. The mean latency toreach the escape hole on their last training day was 16.2 ± 1.41 s,range 9–22 s. In contrast, none of the 11 rats trained in the 4SPcondition could reach criterion even after 29 training sessions,although there was a small improvement over time. As shown inFig. 3A, these rats improved their mean latency to the target holewith training days, and the ANOVA confirms this slight improve-ment over training sessions; F(28, 280) = 3.864, p < 0.0001, butnone of the rats met criterion. Although statistically there was animprovement over time, the graph in Fig. 3 clearly indicates thatthis improvement was minimal, particularly after training Day 6where subsequent learning is virtually absent.

Part of the overall small improvement is due to correct re-sponses on some trials when the start point was at one of thetwo locations adjacent to the target hole. On the last 3 days of test-ing, the 4SP rats needed an average of 20.9 ± 1.6 s to reach the tar-get hole when released from the nearer start points. However, theyapparently could not use this knowledge to improve their perfor-mance from the farther start points, as the average time to reachthe escape hole was 29.7 ± 0.3 s, which is almost the entire 30 slength of the trial. This latency difference could be explained bythe longer distances the rats had to walk when the escape holewas on the opposite side of the platform from the release point.Furthermore, because it was difficult to match the distances therats had to locomote in the two versions of the task (1SP vs.4SP), we cannot exclude the possibility that this distance factorcontributed to the observed learning deficit (i.e., longer escapelatencies) in the 4SP version of the task. However, analysis of head-

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Fig. 3. Escape latencies for the 4SP and 1SP conditions in Experiment 1. (A) Mean escape latencies for the 29 training sessions (four trials per session) for the animals trainedto reach the escape hole from 4-start points. (B) Mean escape latencies for the animals trained to reach the escape hole from a single start point (1SP condition). Because thetwo groups did not reach criterion in the same amount of time, the graph plots the individual learning curve for each animal.

498 S. Valerio et al. / Neurobiology of Learning and Memory 93 (2010) 495–505

ing trajectory (measured after the rat had walked a body lengthfrom their release point), revealed that the poor performance therats showed from the farther start points was more likely due toan initial poor directional heading. Indeed, when released fromthe two closer start points, the rats directional heading only devi-ated 12.9 ± 3.7� away from the correct direction to the target hole,but deviated by 53.1 ± 4.9� when released from the two fartherstart points. Thus, 4SP animals were modestly able to learn howto reach the escape hole from the two close start points, but evenafter 29 training sessions, this knowledge did not appear to be suf-ficiently flexible to transfer to the two other more difficult startpoints that were distant to the escape hole.

In order to verify that these animals were able to learn a morenormal spatial task under similar room conditions, we trainedthem in a comparable version of the standard water maze task.The results indicate that these animals display normal perfor-mance in this task and readily acquire the task. On average, theanimals only required 3.9 ± 0.5 sessions to reach the learning crite-rion in finding and escaping onto the submerged platform. Thisfinding indicates that their poor performance in the 4SP invertedmaze cannot be attributed to: (1) a general learning deficit, (2)an inability to see the surrounding visual landmark cues, or (3)the stressful characteristic of the task, because they showed nor-mal performances in the water maze task. Moreover, the fact thatthe rats were able to learn the inverted escape task in the 1SP ver-sion of the task also supports this view.

In summary, although we cannot exclude the possibility thatdistance played a role in the difficulty the animals had in learningthe 4SP condition, it is noteworthy that the spatial knowledge theyhad was not sufficiently flexible to enable them to reach criterion.Thus, our hypothesis is that the learning deficit is due to an inabil-ity to form a flexible, map-like representation of the environmentwhen in an inverted position. To test this hypothesis, in Experi-ments 2 and 3 we tested whether the deficit was due to the in-creased spatial demand of the 4SP condition.

4. Experiment 2

The aim of the second experiment was to test whether the ratshad a flexible representation of the environment when navigatingin the inverted maze. Experiment 1 showed that they could solvethe task when they were released from a unique (single) start point.What type of strategy did they use in this situation? Did they learnthe location using a simple response strategy (i.e., turn right), ordid they learn the location using a more cognitive map-like repre-sentation? We also wanted to determine whether the rats couldlearn a more demanding version of the task by using two start points.Using this procedure and several probe trials enabled us to deter-mine what strategies and cues the rats were using to solve the task.

4.1. Method

4.1.1. TrainingTwo new groups of rats were trained to reach the escape hole in

the inverted maze, either from one start point (1SP, n = 7), or fromtwo start points (2SP, n = 11). To directly compare these two learn-ing conditions, and to reduce the non-specific difficulties of thetask, we trained the 2SP rats from the two closest start points oftheir assigned hole (e.g., Fig. 2B). After reaching criterion the ratswere submitted to a series of probe tests. In between each of theseprobe tests the rats were trained again to criterion. Analysis of thepercentage of correct trial revealed no differences between groupsfor any of the criterion performance scores on days precedingprobes (p > 0.05).

4.1.2. Probe testsAs in the training sessions, the rats had 30 s to find the hole, but

when they failed to find it, they were removed from the maze, re-turned to their home cages (no correction procedure), and the trialwas scored 30 s.

4.1.2.1. Center probe. The rats were released from the center of themaze facing one of the four cardinal points (four trials). The targethole was open and the rats had 30 s to reach it. The experimenterscored the latency they needed to reach the hole when releasedfrom this novel start point. This test enabled us to determinewhether the rats had learned the task using a cognitive map-likestrategy - if so, then their performance should be good when re-leased from the novel center point. In contrast, if they had learneda more rigid strategy (response or directional strategy), then theyshould be impaired on this probe.

4.1.2.2. Olfactory probe. In order to test whether rats used olfactorycues to find the target hole, they were submitted to a four trial testafter the maze had been rotated 90� and cleaned with alcohol. Ifthey were using olfactory cues to locate the correct target hole,then their behavior should be altered by the suppression of thesecues during the maze rotation trial.

4.1.2.3. Reversal probe. To make sure that the rats could not see thecorrect escape hole from the start point, they were submitted to atraining session (four trials) with a new target hole located in theopposite quadrant of the maze and released from new startingpoints that remained in the same relative position to the escapehole for both the 1SP and 2SP conditions. Good performance onthe reversal probe would suggest that they could see the correcttarget hole from their start position and/or were using a simple re-sponse strategy.

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4.1.2.4. Blind start probe. In this four trial test, the rats were given aregular training session (same hole and start point), except thatduring the last two trials the experimenter used his lab coat toshield the rats from viewing visual cues within the room until theywere released on the maze. This probe tested the extent to whichrats used spatial information they derived from room cues beforebeing placed upside-down on the maze periphery.

4.1.2.5. Curtain probe. Finally, in order to verify that distal visualcues were essential for performing the task accurately, we sup-pressed these cues by hanging a black floor-to-ceiling curtainaround the maze. To completely eliminate any visual cues, this testwas performed under infrared light. The rats were given four reg-ular training trials in these conditions.

4.2. Results and discussion

4.2.1. TrainingBoth 1SP and 2SP groups reached criterion. As shown in Fig. 4A

the rats trained from one start point were much faster to learn thetask than those trained from two start points with the mean num-ber of training sessions to reach criterion: 1SP = 5 ± 0.7 sessions;2SP = 13.3 ± 1.6 sessions. Statistical analysis confirms that the1SP group needed significantly fewer training sessions to learnthe task [t(16) = �3.899, p < 0.01]. It is noteworthy that the 2SP ratsrequired more than twice as many training sessions as the 1SP ratsto reach criterion. This difference suggests that when learning toreach the target hole from a second start point, the animals donot benefit from knowing how to reach it from one start point.

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Fig. 4. Results from Experiment 2. (A) Mean number of sessions to criterion for the an(2SP). (B) Mean escape latency that 1SP and 2SP animals needed to reach the escape hocompared with the mean escape latency on their last regular training session (criterion)the maze was cleaned with alcohol and rotated 90� (olfactory probe), compared with tescape latency that 1SP and 2SP animals needed to reach a new target hole (reversal pr(criterion). (E) Mean escape latency that 1SP and 2SP needed to reach the new targetlatency that 1SP and 2SP animals needed to reach the escape hole when the rats were shmaze (blindfolded trials), compared with the mean escape latency on the first two regu2SP animals needed to reach the escape hole when deprived of distal visual cues (curtsession (criterion).

One possible explanation for this deficiency is that the 2SP ratswere required to build a more complex representation of the envi-ronment to encode the target hole location compared to the 1SPrats, which could have used a simple response strategy. Thus, sev-eral probe tests were conducted to determine what kind of spatialstrategy each group used to solve the task.

4.2.2. Center probeThis probe determined whether the animals used a flexible,

map-like representation of the environment that permits them toreach the target hole from a new start point – in this case the cen-ter of the maze. Fig. 4B shows that the performance of both groupswas severely altered in this testing condition. Indeed, there is aclear increase in the mean latency to reach the target hole duringthe probe session (mean = 28.38 ± 0.43 s) compared with the meanlatency observed on the last regular training session (mean =15.60 ± 0.58 s). A two-way ANOVA with repeated measures con-firms this strong session effect; F(1, 16) = 85.342, p < 0.0001. Thisanalysis also reveals a global group effect [F(1, 16) = 5.088,p < 0.05], reflecting the fact that the 1SP group is on average fasterto reach the hole than the 2SP group. But more importantly for ourquestion of whether the animals used a map-like representation,there is no session � group interaction (p > 0.05). This result indi-cates that both groups were similarly affected by this testing situ-ation, where both groups displayed poor performances whenreleased from a new start point. This absence of flexibility may ac-count for the poor performance we observed in the 4SP conditionin Experiment 1. However, the question now arises as to whatstrategy they used when performing the 1SP and 2SP versions ofthe task.

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imals trained to reach the escape hole from 1-start point (1SP) and 2-start pointsle when released on the maze from a novel starting location (center of the maze),. (C) Mean escape latency that 1SP and 2SP animals needed to reach the hole afterhe mean escape latency on their last regular training session (criterion). (D) Meanobe), compared with the mean escape latency on their last regular training session

hole over the course of the reversal session (four probe trials). (F) Mean escapeielded from viewing visual cues within the room before they were released on the

lar trials of the same session (regular trials). (G) Mean escape latency that 1SP andain probe), compared with the mean escape latency on their last regular training

500 S. Valerio et al. / Neurobiology of Learning and Memory 93 (2010) 495–505

4.2.3. Olfactory probeOne possibility is that the rats navigated to the escape hole

using surface cues (e.g., olfactory or surface texture). In order totest this possibility, we cleaned the maze with alcohol and rotatedit 90�. As shown in Fig. 4C, this procedure did not have any effecton the performance of the animals. Indeed, analyses did not revealany effect for session (p > 0.05) or an interaction between ses-sion � group (p > 0.05). As in the center probe, the only differenceis a global group effect [F(1, 16) = 11.875, p < 0.01] showing thatthe 1SP group was faster to reach the escape hole. This resultclearly shows that the rats did not simply learn to use olfactoryor surface cues to reach the escape hole, since the suppression ofthese cues did not alter their performance.

4.2.4. Reversal probeA second possibility for how the rats learned the task could be

that they were able to see the target hole. To test this hypothesis,they were given a training session with a new target hole, andreleased from new starting points that remained in the same rela-tive position to the escape hole. Fig. 4D shows an increase in thelatencies to reach the hole in this condition compared with theirlast regular training session for both the 1SP and 2SP conditions.The analysis again indicates a global effect for group [F(1, 14) =9.019, p < 0.01], but more importantly, confirms the session effect[F(1, 14) = 60.029, p < 0.0001], with no interaction for session �group (p > 0.05). These results suggest that the rats were not ableto see the target hole, nor could they be using a simple responsestrategy because otherwise their performance would not have beendisrupted at all on the reversal probe. It is also noteworthy thateven when the performance of 1SP animals was disrupted by thesetesting conditions, they still showed a small improvement in per-formance (mean latency: 19.0 ± 2.4 s) over the course of the rever-sal session (Fig. 4E). The analysis reveals that the latency for the 1SPgroup is significantly different from 30 s [t(6) = �4.64, p < 0.01],while the latency for the 2SP group (27.0 ± 4.5 s) is not significantlydifferent from the 30 s maximum allowable time. In addition, theimprovement in performance during this test session (two-wayANOVA) reveals a group effect F(1, 14) = 8.85, p < 0.01, a global trialeffect F(3, 42) = 3.02, p < 0.05, and interestingly, a strong trend forinteraction F(3, 42) = 2.23, p = 0.09. Taken together, these resultsindicate that the 1SP group may have used a response strategy tofind the hole in this new set of conditions. However, using aresponse strategy cannot be the sole explanation for the improvedperformance in the 1SP group, because if their behavior was basedentirely on a response strategy, then their performance should nothave declined at all when moving both the target hole and therelease point together, but Fig. 4E shows that there was a significantdecrement in performance on the first reversal trial.

4.2.5. Blind start probeAnother strategy the animals may have used is to orient them-

selves before being placed upside-down on the inverted maze byassociating a particular set of visual cues at the start point with atrajectory to the target hole. If this process is true, then blindfold-ing them until they are on the maze (inverted) should disrupt theirperformance. On the day of the blind start probe the animals weregiven four trials, with the first two trials identical to the trainingprocedures on preceding days and the last two trials conductedwith the rats visually shielded on the trip from their home cageto the release point on the maze. Fig. 4F plots the escape latencyfor the first two regular trials of this probe session (regular trials)compared with the performances for the last two trials when theywere blindfolded (blindfold trials). The plot shows that blindfold-ing the rats before their inverted trial did not disrupt their perfor-mance. The only statistical effect is a group effect, but no blockeffect, or interaction between these two factors (p > 0.05). This re-

sult indicates that the rats did not use visual information availablebefore their inverted trial either at their cage or in transit to themaze in order to perform the task. Thus, it appears that bothgroups have all the information they need to perform the taskwhile inverted at the release point.

4.2.6. Curtain probeThe results from the above probes suggest that the rats in the

1SP and 2SP conditions are using a combination of distal room cuesand trajectory learning in order to perform the task. To testwhether visual information about the distal room cues was neces-sary, we suppressed all distal visual cues by enclosing the maze ina floor-to-ceiling curtain and conducted a regular training sessionunder these conditions. Fig. 4G shows that the performances ofboth 1SP and 2SP groups were severely disrupted in this testingcondition, with a clear increase in the mean latency to reach thehole. In addition to the group effect [F(1, 15) = 5.67, p < 0.05], theanalysis reveals a clear session effect [F(1, 15) = 23.595, p < 0.001],and no interaction between these factors. These results indicatethat viewing the distal room cues was critically important for accu-rate performance. It is important to note, however, that the noveltyof the testing conditions (i.e., the darkness) may have been stress-ful for the animals. But this possibility is unlikely when consideredwith the findings that the rats’ performances were unaffected byalterations to the environment in other probe tests (blind startand olfactory probe), as well as the observation made by othersthat rodents demonstrate fewer stress-related behaviors in dark-ness (Alstott & Timberlake, 2009; Clark, Hamilton, & Whishaw,2006; Eilam, 2004; Nasello, Machado, Bastos, & Felicio, 1998).

4.2.7. SummaryTaken together, these results suggest that both groups are able

to orient themselves relative to distal room cues and learn to per-form the spatial task. The results from the center probe also indi-cates that the spatial information they used was not a flexible,map-like representation of the environment, since the rats werenot able to reach the target hole when released from a novel startpoint. Experiment 3 was designed to confirm this finding and dem-onstrate the rats used a directional strategy based on visual land-marks to solve the task, which is similar to the strategy othershave suggested for the Morris water task (Hamilton et al., 2008)and appetitive spatial tasks in an open field and on a T maze (Skin-ner et al., 2003). In essence, the animals learn to associate a smallset of distal visual cues with a start point, and then simply learn atrajectory from the start point to the target hole.

A second issue we wanted to address in Experiment 3 iswhether both 1SP and 2SP groups were using this directional strat-egy. Interestingly, when training the 1SP and 2SP groups we no-ticed two differences between the two learning conditions: (1)the large difference in the mean number of sessions to reach crite-rion (�5 vs. 13 for 1SP and 2SP, respectively) and (2) the group ef-fect in the latency to reach the hole that was significant for alltesting conditions. In addition, we also noticed that 1SP animalsshowed a small improvement in the reversal test, which suggeststhat even if they used visual cues, they may also have encodedan egocentric strategy.

5. Experiment 3

Experiment 3 was designed to test two hypotheses. First, 2SPrats did not learn the location of the target hole relative to distalspatial cues, but rather learned distinct trajectories (directionalstrategy) based on simple associations between its start pointand the set of distal visual cues it viewed from the start point.The second hypothesis was that even if 1SP and 2SP groups both

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showed comparable behavior in different probes (center probe,curtain probe), several differences (learning speed, reversal probe)suggest that they could also be using other strategies to solve thistask. As mentioned above, we hypothesized that 2SP rats hadlearned a directional strategy, but that 1SP rats were at timeslearning a response strategy.

5.1. Method

5.1.1. TrainingNew groups of rats were handled, pretrained, and assigned to

one of three training conditions (Fig. 2B–D). The first group(n = 8) was trained to reach a target hole from two start points ina similar way as in Experiment 2 (Fig. 2B: 2SP-Same place-Oppo-site turn). The second group (n = 8) was also released in the mazefrom two start points, but in this condition the rats had to reachone of two distinct target holes depending on their release pointin the maze (Fig. 2C: 2SP-Different place-Opposite turn). In thiscondition they had to make a left turn from one start positionand a right turn form the other start position. The third group(n = 8) was also trained to reach two different target holes, but inthis case the holes were in the same relative position to their startpoint (e.g., always to the right) (Fig. 2D: 2SP-Different place-Sameturn). The rationale was that if the rats had to learn about the loca-tions of the target holes, then it would be more difficult for them tolearn the task when they had to encode two places (Fig. 2C), thanwhen they only had one location to learn (Fig. 2B). On the otherhand, if they simply learn two trajectories (directional strategy),then both conditions would necessitate the same amount of train-ing to reach criterion. Finally, our last prediction was that if theanimals showed a tendency to learn a response strategy in the in-verted maze, then our third group (Fig. 2D), should reach criterionearlier than animals that had to learn opposite turns (Fig. 2C).

5.1.2. Probe testsWhen they had reached the learning criterion, the three groups

were given a center probe test where, as previously described, theywere released from the center of the maze – a novel release pointfor them. They were then trained to criterion again and tested withthe curtain surrounding the maze (curtain test). Analysis of the per-centage of correct trial reveals no significant difference between

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Fig. 5. Results from Experiment 3. (A) Mean number of sessions to reach criterion for aOpposite turn), two different escape holes requiring opposite turns (Different place-Op(Different place-Same turn). (B) Mean escape latency the three groups needed to reach thwith the mean escape latency on their last regular training session (criterion). Note that thfrom the 2SP group. (C) Mean escape latency the three groups needed to reach the targetthe mean escape latency on their last regular training session (criterion). (D) Percentage2SP-Different place-Same turn animals (same turn) during the curtain probe.

groups on performance scores for any of the criterion days preced-ing probe trials (p > 0.05).

5.2. Results and discussion

Fig. 5A depicts the number of sessions to reach criterion for thethree groups. Given that we wanted to test the effect of: (1) theplace factor and (2) the effect of the turn factor, planned compari-son tests were conducted to analyze these variables.

5.2.1. 2SP-Same place-Opposite turn vs. 2SP-Different place-Oppositeturn

A planned comparison between these two groups revealed nosignificant difference for the number of sessions the rats neededto reach criterion (t(14) = 2.125, p > 0.05) with the mean numbersessions = 13 ± 1.12 and 10.88 ± 1.37 for the same place and differ-ent place conditions, respectively. This result suggests that 2SP ratsdid not learn to reach a unique location (the escape hole) from twostart points, but instead learned two separate trajectories withoutbenefiting from the fact that these routes lead to the same location.

Fig. 5B demonstrates that both groups showed an increase intheir latency to find the target hole when released from the centerof the maze (center probe). This finding is confirmed by the ANO-VA, which revealed a significant effect for session [F(1, 14) = 258.48,p < 0.0001], a global effect for group [F(1, 14) = 4.968, p < 0.05], butmore importantly, no interaction between these factors (p > 0.05).This result confirms our previous observations in Experiment 2 andindicates that the rats used a rigid representation to solve the task,although from experiment 2 alone, we cannot determine whetherthe strategy was more route-based or response-based.

Finally, when tested with a curtain surrounding the maze, thesetwo groups showed a similar alteration in their performance(Fig. 5C), with an increase in latencies to find the target hole, whichis confirmed by the analysis revealing an effect for session[F(1, 14) = 172.372, p < 0.0001], but no other significant difference(p > 0.05).

Thus, these two training conditions lead to a similar behavioralprofile. As hypothesized, the rats do not benefit from learning to goto the same location, which suggests that they do not encode a‘‘place,” but more likely encode two trajectories to find the escapehole. The results of the center probe confirm the rigid character of

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nimals trained to reach a single escape hole from 2-start points (2SP) (Same place-posite turn), and two different escape holes requiring turns in the same directione escape hole when released from the center of the maze (center probe), comparede values for the Different place-Same turn group are hidden from view by the valueshole when deprived from viewing distal visual cues (curtain probe), compared withof correct trials for 2SP-Different place-Opposite turn animals (opposite turn) and

502 S. Valerio et al. / Neurobiology of Learning and Memory 93 (2010) 495–505

their representation and support the view that 2SP rats use a simpleassociation between a set of visual cues (curtain probe) and a startpoint for orientation in the inverted maze (directional strategy).

5.2.2. 2SP-Different place-Opposite turn vs. 2SP-Different place-Sameturn

The second issue we wanted to address was whether the ratspreferentially use a response strategy (e.g., always turn right) if gi-ven the opportunity, compared to a directional strategy, which theyhave to use in the regular 2SP condition. As suggested by Fig. 5A,these two groups were not significantly different in the numberof sessions they needed to reach criterion (p > 0.05). This resultsuggests that even if a response strategy is possible, the rats donot preferentially use this strategy in the inverted maze.

Fig. 5B plots the latencies for both groups when released fromthe center of the maze, compared to their performance on their lastregular training session. Consistent with what we observed inExperiment 2, the only significant difference revealed by the anal-ysis is a session effect [F(1, 14) = 162.187, p < 0.0001], confirmingthe rigidity of the representation used by both of these groups. Fi-nally, Fig. 5C shows the results for the curtain probe. The analysisreveals a global group effect [F(1, 14) = 6.631, p < 0.05], indicatingthat the 2SP-Same turn group is faster to reach the escape holethan the 2SP-Opposite turn group, a global session effect[F(1, 14) = 89.32, p < 0.0001], but not the expected interaction be-tween these two factors (p > 0.05). This result suggests that bothgroups were similarly affected by the suppression of distal cues.

Although the 2SP-Same turn and 2SP-Opposite turn groups per-formed similarly on the probe tests, it is possible that a significantdifference between the two groups could be artificially masked bythe 30 s cut-off applied to the failed trials, since the mean latencieswere close to this ceiling value. Another way to analyze the curtaintest that avoids this problem is to plot the percentage of successfultrials performed by each group. A two-way ANOVA comparing thepercentage of correct trials on the probe test with their performanceon criterion day reveals a global group effect F(1, 14) = 8.40, p < 0.05,a session effect F(1, 14) = 109.87, p < 0.0001, but also a trend for aninteraction between these factors F(1, 14) = 2.74, p = 0.12. Even ifthis interaction does not reach significance it is interesting to notethat there is a clear difference in the performance between the2SP-Same turn (46.875 ± 7.38 %) and 2SP-Opposite turn animals(18.75 ± 7.83 %), with the 2SP-Same turn animals clearly being moresuccessful without visual cues [t(14) = �2.614, p < 0.05] (Fig. 5D).This analysis is also confirmed by further evaluation of the latenciesdisplayed by the animals in the course of the curtain test session. In-deed, it is noteworthy that 2SP-Same turn animals show better per-formance scores when deprived of visual cues (mean: 21.75 ± 1.67 s)compared to 2SP-Opposite turn animals [mean: 26.90 ± 1.55 s,t(14) = 2.262, p < 0.05)] and from the cut-off value (30 s)[t(7) = �4.95, p < 0.01)]. Thus, even if not very strong, the egocentricstrategy appears to have been utilized by the 2SP-Same turn animalssince they appear to be able to solve the task without visual cues.

In summary, the third experiment demonstrates that 2SP ani-mals do not learn to reach a place from two different start points,but rather learn two independent trajectories, and do not benefitfrom whether the two places are the same or different. In addition,this experiment suggests that when an egocentric strategy is pos-sible, the animals appear to encode a body-centered strategy, andare able to use this strategy when distal cues are unavailable.

6. Discussion

6.1. Can rats form a flexible, map-like representation when inverted?

Experiment 1 examined whether rats, that presumably have adistorted HD signal when locomoting upside-down (Calton &

Taube, 2005), would be able solve a spatial task that required aflexible, map-like representation of the environment while in aninverted position. The task used by Calton and Taube required arat to traverse a 30 cm wide path that started on the floor, thenclimbing a four foot wire mesh to the ceiling, traversing a four footceiling in an inverted position to another wall, and then climbingdown the four foot wall into a second compartment on the floorto retrieve a food reward. In this task Calton and Taube madetwo apparently incompatible observations: (1) on the one hand,HD signals recorded in the anterior thalamus were clearly dis-rupted when the rat was locomoting upside-down, and (2) onthe other hand, the authors did not notice any behavioral sign ofdisorientation as the rats performed the task. Indeed, the rats wereable to climb and walk on the ceiling of this square ring shuttle boxto reach the reward compartment without any apparent difficulty.To account for the accurate performance, but poor HD cell signals,the authors postulated that the rats may have used a simple asso-ciative strategy to compensate for their supposed loss of allocentricorientation caused by the absence of a strong directional signal.Using a simple associative strategy to perform the task wouldnot have required the rats to remain adequately oriented whenin an inverted position. Indeed, all the rats had to do once they en-tered the vertical plane was simply keep moving straight aheadand they would eventually reach the reward compartment.

The present experiments support their hypothesis and reconcilethe two observations, since we show that even if the rats were ableto solve a spatial task locomoting upside-down, they cannot build aflexible allocentric representation of the environment. We ob-served that the rats used a simple and rigid orientation strategywhere they encode trajectories to find their way to the target hole.In other words, they display either a directional strategy or a re-sponse strategy when it is possible, but in none of our testing con-ditions did the rats show the flexibility associated with placelearning. Experiment 1 showed that the rats could learn to reacha target location when released from one start point, but not whenthey have to find the escape hole from four different start points,even after extensive training (116 trials over 29 days of testing).Experiment 2 tested whether in a less demanding version of thisspatial task (2-start points), the rats would be able to build a flex-ible representation of the environment. The results show that evenif the animals were able to learn this task, they were severely im-paired when required to reach the target hole from a new startpoint, indicating that, as suggested by their incapacity to performthe 4SP version of the task, they have difficulty forming rich, flex-ible spatial representations of their environment when they areupside-down. Further, the different probe trials revealed that theydo not use a beacon strategy (olfactory or surface cues), but insteadrequire distal visual cues. The aim of the third experiment was toclarify this point and test the hypothesis that 2SP animals wereusing a simple directional strategy. The results confirmed the rigidcharacter of their spatial representation, and more importantlyshowed that the rats did not learn to reach a specific location inspace, but simply headed toward a small set of visual cues. In otherwords, they encode a simple association between a start point anda set of cues that indicates the goal location – they learn a direction,but not a place.

A second issue arising from Experiment 2 was the large differ-ence in learning rate between the 1SP and 2SP animals, whereinunder all of the testing conditions 1SP animals had faster escapelatencies than 2SP animals. This finding suggests that 1SP animalsmay pay less attention to distal cues and have an automatic motorresponse when released onto the inverted maze. We also testedwhether the rats tended to use an egocentric strategy to solvethe task. Although we did not observe the expected difference inlearning rate between 2SP-Different place-Same-turn and 2SP-Dif-ferent place-Opposite turn groups, the curtain probe test showed

S. Valerio et al. / Neurobiology of Learning and Memory 93 (2010) 495–505 503

that even when deprived of distal visual cues, the 2SP-Differentplace-Same-turn animals were able to find the target hole moreaccurately than animals that were not given the opportunity tolearn a response (2SP-Different place-Opposite turn). This resultsuggests that under inverted conditions, a direction strategy ismost likely the animals’ first strategy choice, but are capable ofusing an egocentric strategy as well.

As discussed above, HD cells are known to encode the direc-tional heading of the animal in an allocentric frame of reference(Taube, 1995; Taube et al., 1990). This characteristic is shared withhippocampal place cells and entorhinal grid cells (Hafting et al.,2005; O’Keefe, 1976). Numerous studies have demonstrated thatthese allocentric signals, as well as the brain areas where theyare found, support the formation of cognitive maps and the flexiblespatial representations that are then used to solve navigationaltasks (Eichenbaum, Stewart, & Morris, 1990; Steffenach, Witter,Moser, & Moser, 2005; Taube, Kesslak, & Cotman, 1992). Thebehavioral criterion often used to define flexible, or map-like navi-gation, is the ability of animals to perform short-cuts and find newways to a goal in a familiar environment. This type of spatial rep-resentation is classically opposed to response and directional strat-egies, which instead, are characterized by a simple associationbetween a stimulus and a motor response (e.g., a turn for the re-sponse strategy), or on an association between two simple set ofstimuli (a start point and a salient visual cue for the directionalstrategy).

6.2. HD signal and response strategy

A large number of studies have shown that a response strategy(learning a motor response) and allocentric spatial mapping rely ondifferent memory systems, which interact with each other, but canfunction independently, too (Gold, 2004; McDonald & White, 1993;Poldrack et al., 2001; Squire, 2004). The importance of motor infor-mation in the control of the HD signal has been demonstrated inexperiments, where in the absence of reliable visual cues, motorcues permit an accurate update in the preferred firing directionof HD cells (Stackman, Golob, Bassett, & Taube, 2003). However,one important issue is whether the reverse is also true – doeslearning a motor response necessitate the presence of an accurateHD signal? Although HD cells have been recorded in the dorsal stri-atum of rats (Ragozzino, Leutgeb, & Mizumori, 2001; Wiener,1993), a brain structure known to be critical for motor responselearning (Knowlton, Mangels, & Squire, 1996; Packard & McGaugh,1996; Yin, Ostlund, Knowlton, & Balleine, 2005), the relationshipbetween the striatal HD signal and the limbic HD signal is poorlyunderstood, and there is no evidence to support the view that stri-atal HD cells and limbic HD system operate independently fromone another. Nonetheless, it is possible that the striatum functionsrelatively normally even without a functional HD signal originatingfrom the limbic system. Indeed, several studies in rats have shownthat lesions of the anterodorsal thalamic nucleus (a key structurein the HD limbic network) severely alters the ability of rats to solveallocentric spatial tasks, but, interestingly, has no effect on egocen-tric variants of these tasks (Aggleton, Hunt, Nagle, & Neave, 1996;Sziklas & Petrides, 1999; Warburton, Baird, & Aggleton, 1997;Wolff, Gibb, Cassel, & Dalrymple-Alford, 2008). Taken together,these findings support the view that the thalamic HD signal isindependent of the circuit responsible for the orientation basedmotor response.

6.3. HD signal and directional strategy

At present very few studies have tested whether a directionalstrategy necessitated a functional HD network. Indeed, a direc-tional strategy was first suggested as an alternative for flexible

map-like navigation in Tolman’s classic experiments (Tolman,Ritchie, & Kalish, 1946), but until recently, this spatial strategyhas been neglected by neuroscientists. A few studies, however,have shown that this strategy is altered (disrupted) by hippocam-pal lesions (Stringer, Martin, & Skinner, 2005) and another studyrevealed that rats do not need a HD signal to perform a directionaltask accurately (Hamilton et al., 2009). In the latter study Hamiltonet al. trained rats with lesions of the dorsal tegmental nucleus(DTN) in a directional version of the water maze task. DTN lesionshave been shown to abolish the HD signal in the anterodorsal thal-amus (Bassett, Tullman, & Taube, 2007). Hamilton showed thatDTN lesioned animals were initially impaired in the direction task,but after further training they ultimately performed as well as con-trols. This result indicates that learning to associate a start pointwith distal visual cues, followed by heading in the correct direc-tion, does not rely on an intact limbic HD signal.

Why was the 4SP condition difficult for the inverted rats tolearn? Although we did not record HD cells during the task, basedon previous studies it is likely that the limbic HD signal was dis-rupted when the animals were in an inverted position. This situa-tion made it difficult for the animals to construct a flexible, map-like representation of the environment, and this lead to poor per-formances from multiple start points. There is, of course, the‘chicken and egg’ issue of which came first: did the loss of theHD signal because of altered otolith input lead to the inability toform a cognitive map? Or, did the inability to construct a cognitivemap, because of presumed disorientation when inverted, lead to adisrupted HD signal? A similar set of questions could be posed forthe influence of inverted orientation on place and grid cell signals,although currently there have been no studies that have examinedthe effects of inverted orientation on place or grid cell activity.Although the current study does not shed light on these questions,it might be possible to address it by determining whether rats arecapable of learning the 4SP condition if a more progressive trainingmethod, comparable to what is used in ‘‘transverse patterning” or‘‘transitive learning” paradigms, was used. In these protocols ani-mals usually learn one separate problem at a time and eventuallylearn to string together a number of representations (Eichenbaum,Mathews, & Cohen, 1989). In our case, the rats would be trained toreach criterion from 1-start point, and then to a second one, and soon. After reaching criterion from a number of start points, it is pos-sible that this type of training would help them form a more flex-ible spatial representation of the environment and possibly enablethem to make inferences about a novel start position based ontheir extensive experience. Under this training regimen, it wouldbe interesting to determine if accurate performances when startedfrom novel entry points coincided with the return of an intact HDsignal or if there was a dissociation between the two parameters.Of course, it is possible that even with extensive training the ani-mals may still only learn a set of associations that entail entrypoints plus trajectories, as do fornix-lesioned rats in odor-discrim-ination tasks (Eichenbaum, Fagan, Mathews, & Cohen, 1988;Eichenbaum et al., 1989). Regardless of whether the animals caneventually learn accurate routes from multiple start points, our re-sults demonstrate that the first strategy of choice under the pres-ent inverted conditions is to use a directional strategy.

6.4. Summary

In summary, our study confirms the prediction made by Caltonand Taube (2005), showing that rats locomoting upside-down havedifficulty building a flexible spatial representation of the environ-ment, but are still capable of simple associative strategies (egocen-tric or directional). This finding is consistent with other studiesshowing an independence of these two simple orientation strate-gies from the HD signal (Golob, Stackman, Wong, & Taube, 2001).

504 S. Valerio et al. / Neurobiology of Learning and Memory 93 (2010) 495–505

Interestingly, our findings are consistent with observations in 0-gconditions where astronauts frequently have great difficulty form-ing a three-dimensional representation of their spatial environ-ment and often report a profound sense of disorientation (Oman,2007). Under 0-g conditions, there is an alteration in the normalotolith signal experienced by astronauts and this unfamiliar situa-tion (in an unknown way) impedes the astronaut’s ability to forman accurate representation of their spatial environment. Usingnon-immersive virtual reality tasks that simulate navigationalproblems, several studies have demonstrated that subjects havedifficulty in building an accurate mental representation of theirroute whenever the path requires a body rotation other than inyaw (Aoki, Ohno, & Yamaguchi, 2005; Aoki, Ohno, & Yamguchi,2003; Vidal, Amorim, & Berthoz, 2004). Further, when performingthree-dimensional mental rotation tasks, subjects appear to usesimple strategies to navigate, rather than a flexible cognitivemap-like representation: either remembering the series of movesthey have to make in order to reach the goal, or, by learning eachmodule’s landmark arrangement from a standard viewpoint(Oman, 2007; Oman et al., 2002; Richards et al., 2003). The latterexplanation is similar to the directional strategy used by the ratsin the inverted hole board escape task. Along different lines a sim-ilar pattern of results was also observed in patients with unilateralvestibular loss (Peruch, Borel, Magnan, & Lacour, 2005). In theirstudy they reported that vestibular patients were as accurate ascontrols when they had to reproduce a previously explored route,but showed significantly poorer performance when the task wasmore complex and required them to return to their point of originafter walking a path formed by two legs of a triangle (path comple-tion task). These results are consistent with the view that an intactvestibular system was necessary to form an accurate, map-like rep-resentation of the environment.

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