Retrosplenial cortex lesions impair water maze strategies learning or spatial place learning depending on prior experience of the rat

Behavioural Brain Research 170 (2006) 316–325

Research report

Retrosplenial cortex lesions impair water maze strategies learning or spatial place learning depending on prior experience of the rat Donald P. Cain ∗ , Richard Humpartzoomian, Francis Boon Department of Psychology and Graduate Program in Neuroscience, University of Western Ontario, London, Ont., Canada N6A 5C2 Received 30 November 2005; received in revised form 28 February 2006; accepted 7 March 2006 Available online 18 April 2006

Abstract There has been debate whether lesions strictly limited to retrosplenial (RS) cortex impair spatial navigation, and how robust and reliable any such impairment is. The present study used a detailed behavioral analysis with naive or strategies-pretrained rats given RS lesions and trained in a water maze (WM). Naive RS lesioned rats failed to acquire the required WM strategies throughout training. Strategies-pretrained RS lesioned rats were specifically impaired in spatial place memory without a WM strategies impairment. Additional training overcame the spatial memory impairment. Thus the behavioral consequences of the lesion depend on the specific previous experience of the animal. The use of appropriate training and testing techniques has revealed experience-dependant dissociable impairments in WM strategies learning and in spatial memory, indicating that RS cortex is involved in both forms of learning. © 2006 Elsevier B.V. All rights reserved. Keywords: Navigation; Learning; Memory; Cortex; Rat; Water maze

1. Introduction Evidence indicates that a variety of extrahippocampal structures, including many areas of cortex, are involved in navigation behavior in the water maze (WM). Damage to nearly all regions of cortex including prefrontal, frontal, somatosensory, parietal, visual, temporal, insular or retrosplenial/cingulate cortex impairs performance in the WM task [24,26–29]. These results are consistent with the long-held view that cortex is important for neural changes that underlie learning [30]. Retrosplenial (RS) cortex may be particularly important in spatial behavior because of its reciprocal neuroanatomical connections with visual, auditory and cingulate cortex, and also with the hippocampal formation, suggesting that RS cortex may form a functional link between neocortical areas important for receiving visual and other information, and the hippocampus [46]. Evidence indicates that RS neurons respond to spatial location, orientation and movement in behaving rats and that inactivation of RS cortex alters hippocampal place cell fields [16–19]. These findings are consistent with the idea that RS may process and relay sensory

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information relevant to navigation to the hippocampal formation. Additional evidence from human neuroimaging and lesion studies supports the idea that RS cortex plays an important role in human spatial cognition and navigation [31,52]. Large lesions of the posterior cingulate area that removed RS cortex together with the underlying cingulum bundle and also additional subcortical tissue in some cases, produced clear impairments in WM acquisition [42,50]. However, there has been considerable debate whether damage to RS cortex alone impairs spatial navigation or whether damage to adjacent structures may be related to any impairments found, and whether the additional factors of rat strain and the kind of spatial task used also play a role [2,3,21,22]. This debate has been fueled in part by variability in the degree of behavioral impairment reported with RS lesions in different studies. Impairment in the conventional WM task has often been mild [45] or inconsistent between behavioral measures within the same study. For example, in two detailed studies with RS lesioned rats, lesions strictly limited to RS cortex produced equivocal evidence for a spatial memory impairment in the WM task, with various behavioral measures indicating either impairment, no impairment or even superior performance by lesioned rats relative to controls [21,22]. This raises the question whether RS cortex itself, apart from neighboring tissue, plays an important role in spatial behavior.

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A limitation of the studies discussed above is that none of them separated WM strategies training from spatial training [4,32] and few evaluated the possibility that impairments in WM strategies learning or use might have played a role in the outcomes. These are important points because recent work has shown that naive rats typically behave very differently from rats that are familiar with the general behavioral strategies required to both cope with the stress of the WM task and acquire the spatial information necessary for solving it. These strategies include learning how to swim, learning to suppress thigmotaxic behavior to search for the hidden platform away from the pool wall and learning to recognize and use the hidden platform as refuge [32,47]. A rat must first acquire these specific strategies before it can obtain spatial information about the location of the hidden platform and complete the second component of the task, i.e., learning the location of the hidden platform in relation to distal visual cues [4,32,47]. In studies that have directly compared the performance of naive rats and strategies-pretrained rats, naive rats given an experimental treatment frequently have been impaired due to an inability to acquire and effectively use necessary WM strategies whereas strategies-pretrained rats given the same experimental treatment learned the location of a hidden platform as quickly as controls ([5,7,9,11,13,14,23,34,35,40,41]; but see refs. [10,12,53]). This includes studies involving lesions of prefrontal, parietal or visual cortex, which produced WM acquisition impairments in naive rats but no impairments in strategies-pretrained rats [24]. This implies that many of the treatments may have impaired WM strategies acquisition rather than spatial memory per se. It is important to directly compare the performance of both naive and non-spatially pretrained rats in studies of brain function and navigation behavior. Therefore, we have revisited the topic of spatial navigation in rats with lesions limited to RS cortex. For this purpose, Morris’ non-spatial pretraining [32] was first given to intact rats, followed by bilateral RS lesions, followed by spatial training. Other naive rats received RS lesions followed by spatial training. Non-spatial pretraining teaches rats to swim freely in the WM pool in the absence of distal visual cues, suppressing the thigmotaxis response to search for the hidden platform away from the pool wall, where it is never placed, and to use the hidden platform as a refuge. Training trials were both videotaped and tracked using an overhead camera and digital tracking system for detailed analysis of both WM strategies acquisition and memory for the hidden platform location. Male hooded Long-Evans rats were used because of their superior vision [39], their excellent spatial navigation ability [20] and for comparability with much of the previous WM research. Both the mixed findings in spatial memory after RS lesions discussed above and our recent experience [6,9,10] point to the importance of evaluating the behavioral navigation performance of both intact and lesioned rats in a simple swim-tovisible-platform task to determine the swim paths that the rats normally take in navigating to a goal in the WM. This was done in Experiment 1, which provided information on the most appropriate measures for determining spatial place memory in Experiment 2.

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2. Experiment 1—visible platform task Impairments in swimming to a visible platform have been found in rats with brain lesions outside the visual system ([9,10]; Fig. 1 in ref. [33]; [44]). Detailed behavioral analysis of performance in a simple swim-to-visible-platform task by such rats has revealed impairments in generating efficient swims to a refuge even when the refuge is clearly visible and the visual system is intact [9,10]. Therefore, naive rats with RS lesions were tested in a simple swim-to-visible-platform task to evaluate basic swimming and navigation behavior in lesioned rats and document any impairments in navigating to a visible platform. 2.1.1. Material and methods 2.1.1.1. Subjects and experimental groups Adult male Long-Evans hooded rats weighing approximately 300 g and housed individually with ad libitum food and water were used. Behavioral testing was in the light phase of a 12-h light:12-h dark cycle for comparability with previous WM research. Rats were naive to swimming and all experimental procedures prior to surgery and were randomly selected to receive lesions. Rats recovered for 14 days before testing. Procedures were in accordance with guidelines of the Canadian Council on Animal Care and approved by the University of Western Ontario. 2.1.1.2. Surgery Rats received bilateral lesions of retrosplenial cortex (RS Lesion group, n = 8) or Sham surgery (Sham Control group, n = 6). Rats first received atropine pretreatment followed by sodium pentobarbital anesthesia (60 mg/kg i.p.) and placement in a stereotaxic apparatus. Body temperature was maintained at normothermia using a heating pad. Four burr holes were drilled through the skull over each hemisphere, two at AP 1.8 relative to bregma and ML ± 1.5 and two at AP 7.3 and ML ± 1.5. Small rongeurs were then used to remove the bone between the holes, which created a longitudinal window approximately 2 mm wide over the RS cortex in each hemisphere and which allowed a strip of skull approximately 1.5 mm wide to remain in place over the longitudinal fissure to prevent damage to the blood vessels in the fissure. This strip of bone over the fissure led to sparing of some RS cortex bordering the fissure in most rats but this done to prevent severe bleeding and resultant unintended damage that might result from this bleeding. The dura was then pierced and carefully removed using a sterile hooked 30-ga syringe needle. RS lesions were created by gently scraping the surface of RS cortex with the needle to disrupt the blood vessels that supply the cortex, followed by removal of blood and the outer layers of cortex using gentle suction applied strictly to the cortex surface using a small glass pipette; the lesion did not extend into underlying white matter of either the cingulum bundle or corpus callosum. This approach necessarily damages a portion of area OC2M, which overlies area RSA. We did not expect a spatial memory impairment from this damage because our earlier large visual cortex lesions, which involved most of visual cortex and included most or all of area OC2M failed to impair spatial memory in strategiespretrained rats [24]. This lesion technique was used because damage to blood vessels and cortex surface reliably results in loss of cortex without damaging underlying white matter [21]. This technique also allows good control of the areal extent of damage without the risk of spread of a neurotoxic agent [25]. 2.1.1.3. Apparatus The WM was a white circular pool (1.5 m diameter) located in the center of a large room with numerous visual cues (doors, cabinets, posters on the walls, etc.). The refuge was a visible platform (15 cm × 15 cm) that protruded 2 cm above the surface of the water (29 ± 1 ◦ C) and was marked by a cylindrical object 3 cm in diameter and 10 cm tall. Both the platform sides (as viewed by the rat from water level) and the object were painted in alternating black and white stripes for maximum visibility. The platform was placed close to the wall of the pool, with a 9 cm gap between the wall and the closest edge of the visible platform to prevent rats from simply bumping into the platform while swimming

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thigmotaxically (see Fig. 2). The same white polypropylene pellets that were used to render the surface of the water opaque in Experiment 2 were used in this experiment to make swimming conditions comparable [15]. Rats were placed under a heat lamp between trials to maintain core body temperature. The signal from a video camera recessed into the ceiling above the center of the pool was sent to a VCR and a tracking system (Poly-Track, San Diego Instruments) that produced videotaped records and a digital file of each swim trial. These were later objectively analyzed during the detailed behavioral analysis. 2.1.1.4. Training Visible platform training began 14 days after surgery and took place over 3 days (10 trials per day; 5 min intertrial interval). On days 1 and 2, rats were released one at a time into the water immediately adjacent to and facing the pool wall opposite the visible platform (see Fig. 2). The distance from the start point to the closest edge of the visible platform was 128 cm. After the rat climbed onto the visible platform it remained there for 15 s. The experimenter was not visible to the rats while they were swimming, and the rats were monitored on a video monitor situated outside the WM room. Given the occurrence of impairments in the lesioned group (see below) a third day of training was given with the procedure modified to reduce the difficulty of the task. The start of each trial was moved 30 cm closer to the visible platform and the rat was placed into the water facing the visible platform. On day 3, the distance from the start to the closest edge of the visible platform was 98 cm.

2.1.2. Histological and data analysis Within 48 h after completion of behavioral testing the rats were transcardially perfused with formalin-saline under deep anesthesia. Brains were removed from the skull and placed in a cryostat at 20 ◦ C for coronal sectioning at 40 thickness. Every fifth slice was mounted on glass, stained with gallocyanin for Nissl material and coverslipped. Brain sections were viewed using a Leitz light microscope with a computer-based digital imaging system. Light and digitized images were evaluated for measures of damage defined as cell loss and inconsistent and paled density, with reference to the brain atlas of Paxinos and Watson [37]. Behavioral data were analyzed using one-way and repeated measures ANOVA with Newman–Keuls post hoc tests. Only significant results are reported.

Fig. 1. Photomicrographs of coronal brain sections showing representative damage at three anterior/posterior levels of RS cortex. (A) Approximate level of Plate 37, −4.8 relative to Bregma (Paxinos and Watson). (B) Approximate level of Plate 43, −6.3 relative to Bregma. (C) Approximate level of Plate 45, −6.8 relative to Bregma. Damage is largely limited to RS cortex with the exception of overlying and adjacent portions of area OC2 that were necessarily damaged in creating the lesion. Portions of deep RS cortex under the midline strip of skull were spared in many rats.

2.1.3. Results As shown in Fig. 1, the RS lesions were largely selective for areas RSA and RSG bilaterally and spared the underlying cingulum bundle as well as the corpus callosum (Paxinos and Watson). The damage involved most of RS cortex from approximately AP −1.8 to −7.3 with the exception of some deep portions of RSG under the midline strip of skull. The presubiculum and subiculum were spared, and there was no damage to the hippocampal formation. Posterior RS cortex beyond approximately −7.3 also was spared. Overlying and adjacent portions of OC2 were necessarily damaged in creating the lesion in all rats, as described above in Section 2.1.1, but the balance of occipital cortex was undamaged. Thus, the lesions appear to be restricted largely to RS cortex and overlying area OC2 and are comparable to the RS lesions of Harker and Whishaw [21]. They are much less extensive than conventional suction ablation lesions of posterior cingulate cortex [42,50] and are also smaller than neurotoxic lesions that damaged the ventralmost portions of RS cortex and contiguous portions of the hippocampal formation [1].

All rats displayed normal swimming behavior, with the expected forelimb inhibition and alternate thrusting of the hindlimbs, and climbed onto and remained on the platform when they contacted it. As shown in Fig. 2, which displays every swim path for all rats in each group for each day, lesioned rats swam more in the periphery than controls and therefore generated less efficient swims than controls. On day 1, only 5% of swims by the RS Lesion group were classified as direct swims [21,22]. The corresponding score for the Sham Control group was 28%. Sham Control rats displayed a mix of both periphery and direct swims on all days, an outcome similar to our previous findings [6,9,10]. Digital files of swim paths were objectively analyzed for three performance measures: swim time from release to contact with the visible platform, time swum in the pool periphery and heading errors. The periphery was defined as the outer 50% of the water surface, where the hidden platform was never placed during spatial training in Experiment 2 [14]. A heading error was scored if a rat swam away from the visible platform

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Fig. 2. Composite displays of digitized swim paths to the visible platform for all rats on specific days of training in Experiment 1. All swim paths for all rats within a group on a given day were overlain on an outline of the pool as seen from above. Paths from the RS Lesion and Control groups on days 1–3 are shown from left to right. The positions of the release points and the visible platform are shown at the bottom. Note that the start point was moved closer to the visible platform on day 3. There was a 9 cm gap between the pool wall and the closest edge of the visible platform to prevent swimming along the wall and running into the platform. Five percent of swims by the RS Lesion group were direct swims on day 1, whereas 28% of swims by the Sham Control group were direct swims on day 1.

(increase in the distance between the rat and the platform) at any point in a swim. If a heading error was scored and the rat then closed the distance between itself and the visible platform and again swam away from the platform, a second heading error was scored. As shown in Fig. 3, the data indicated impairment on all measures in the RS Lesion group on days 1 and 2. Repeated measures ANOVA confirmed this impression by revealing significant effects on all measures for days 1 and 2 (swim time per trial, Group: F(1,2) = 11.3, p < .006; Trials: F(1,12) = 21.5, p < .001; time in periphery per trial, Group: F(1,2) = 25.2, p < .0001; Trials: F(1,12) = 22.8, p < .001; heading errors, Trials: F(1,12) = 29.8, p < .0001, Interaction: F(1,12) = 7.1, p < .02). The basis for the significant interaction in heading errors was an impairment in the RS Lesion group relative to Sham Controls on day 1 and improvement to levels of Sham Control performance on day 2 (see Fig. 3). On day 3, when the start point was moved closer to and facing the visible platform the RS Lesion group was impaired on two measures: swim time per trial (one-way ANOVA, F(1,12) = 6.1, p < .04) and heading errors (F(1,12) = 6.0, p < .03). An important aspect of the RS lesion rats’ impairment was their tendency to swim directly to the wall upon release (see Fig. 2). 2.1.4. Discussion Despite the utter simplicity of the task, and with no impairment in swimming behavior as such, the RS Lesion group

was impaired on all measures on days 1 and 2 and continued to be impaired on day 3 after the difficulty of the task was reduced. These impairments are similar to impairments seen in rats with fimbria-fornix, hippocampal or medial thalamus lesions tested in this task ([10]; Fig. 1 in ref. [33]). In previous studies of rats given either cingulate cortex lesions or diazepam, impairments were not found in a visible platform task carried out in a WM pool [7,42]. However, in both cases the rats had been given extensive spatial training in a conventional hidden platform WM task before being tested in the visible platform task. Naive rats given the same dose of diazepam and tested in a visible platform task were significantly impaired [7], a result comparable to results obtained in the present experiment with RS lesions. It appears that training in a conventional WM task might serve as a form of strategies pretraining for subsequent testing in a visible platform task, and can obscure impairments that might be manifested in naive rats tested in a visible platform task. The present results demonstrate that lesions limited to RS cortex can impair navigation from a stable start point to a stable visible refuge in naive rats. Visual impairment was not likely a factor because (1) swims ended at the visible platform with no local searching for the platform, suggesting that rats could visually detect the platform, (2) no lesioned rat was ever seen to walk off the edge of the transport cart or bump into objects or other rats, (3) damage was restricted to RS cortex, and apart from damage to overlying and adjacent portions of area OC2 there was no damage to any main visual area of the brain and (4) pretrained RS lesioned rats

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results imply that rats normally generate a variety of efficient swim trajectories in navigating within a pool, some of which are direct swims as previously defined [21,22], and some of which approximate an arc of a circle, as shown in Fig. 2. These facts have been taken into consideration in defining a direct and circle swim measure of spatial memory [9,24] that is used in Experiment 2, along with pretrained control groups for separation of WM strategies training and spatial training [4,32,34]. 3. Experiment 2—spatial learning task Experiment 2 examined the effect of RS lesions on WM acquisition with a hidden platform. Some groups consisted of rats given WM strategies pretraining prior to the lesion to separate the strategies learning and spatial learning components of the task [4,32,34]. 3.1.1. Material and methods 3.1.1.1. Subjects and experimental groups The same lesions used in Experiment 1 were given to naive adult male LongEvans rats for Experiment 2. All rats were naive to experimentation and no rats from Experiment 1 were used in Experiment 2. General housing and testing conditions were as in Experiment 1. Rats were randomly allocated to groups that were given pretraining [32] or no pretraining followed by lesions or Sham Lesions. Groups were: Naive RS Lesion (n = 9), Pretrained RS Lesion (n = 10), Naive Sham RS Lesion (n = 6), Pretrained Sham RS Lesion (n = 6). 3.1.1.2. Apparatus The same pool and testing room used in Experiment 1 was used. A uniformly white hidden platform (11 cm × 11 cm) with serrations on top for gripping was used for both pretraining and spatial training, with the water level adjusted to 2 cm above the top surface of the platform. The center of the hidden platform was 37.5 cm away from the pool wall, midway between the geometric center of the pool and the pool wall. Floating white polypropylene pellets prevented rats from seeing the platform directly [15]. Data were recorded and analyzed using the VCR and Poly-Track system.

Fig. 3. Group mean swim time to the visible platform per trial (A), time swum in the periphery per trial (B) and heading errors per 10 trials (C) on each day in Experiment 1. A heading error was counted when a rat swam away from the visible platform at any point in a trial. On days 1 and 2 rats were started at and facing the wall. On day 3, rats were started away from the wall, closer to and facing the visible platform (see Fig. 2). Heading errors occurred frequently among the RS Lesion group on day 3 because they swam directly toward the wall immediately upon release. Plots and points that differ statistically from Sham Controls are indicated by an asterisk. Error bars indicate ±S.E.M. in all figures.

effectively used distal visual cues to learn and remember the hidden platform position in Experiment 2. The swim paths in Fig. 2 indicate that, in addition to producing direct swims to a refuge (strictly defined as being contained entirely within a virtual 18-cm wide alley from the start point to the refuge [21,22]), naive intact control rats frequently produced curved swims that extended into the periphery of the pool even when swims were started from the same place on each trial and always ended at a stable visible refuge. The data also indicate that these rats improved in task-relevant navigation behaviors from days 1 to 2 even for this simplest version of the WM. These

3.1.1.3. Pretraining Pretraining was carried out prior to surgery, with three trials per day on each of 4 days for a total of 12 pretraining trials, with a 5 min intertrial interval [23,32]. For pretraining thick black curtains were attached to a circular track mounted to the ceiling and were drawn completely around the pool, eliminating all distal visual cues. The visible portion of the ceiling above the pool was uniform and provided no directional cues. The hidden platform was moved to a new quadrant after every pretraining trial. A rat was introduced into the pool and swam until it found the hidden platform or 120 s elapsed, at which time it was placed on the platform where it remained for 30 s. Swimming was observed on a television monitor, and search times were recorded. The acquisition of WM behavioral strategies during pretraining has been documented [23,32,38] and retention of the learned strategies is known to persist for at least the duration of the interval between the end of strategies pretraining and the beginning of spatial training used in the present experiment [10]. 3.1.1.4. Surgery Rats received RS lesions or Sham surgery 24–48 h after the completion of pretraining, if given. The surgical procedures were as described in Experiment 1. Spatial training began 14 days after surgery. 3.1.1.5. Spatial training For spatial training the curtains were removed from around the pool, allowing all rats to make use of the distal visual cues in the room. Spatial training on day 1 consisted of 10 trials with the hidden platform in the center of the

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southeast quadrant (intertrial interval approximately 5 min). A rat was individually introduced into the water facing the pool wall at north, south, east or west, and swam for a maximum of 60 s or until it found and climbed onto the hidden platform. The order of start points was pseudorandomized with the provision that summed swim distances from the start points to the hidden platform in each two-trial block were approximately equal. If the hidden platform was not found within 60 s the rat was placed on it by hand and allowed to remain on it for 15 s. Swimming behavior was monitored on the video monitor, and the experimenter was not visible to the rat at any time while it was swimming.

3.1.3.1. Pretraining Mean search time for the pretrained RS lesion rats to find the hidden platform decreased from 52.4 ± 8.9 s on the first pretraining trial to 20.2 ± 7.8 s on the last pretraining trial. As pretraining progressed all rats learned to swim away from the pool wall and use the platform as refuge. Thus, the pretrained rats acquired appropriate WM behavioral strategies in a manner comparable to earlier results [23,32,38].

3.1.1.6. Reversal training On day 2 similar training was given, but the hidden platform was now in the opposite (northwest) quadrant of the pool.

3.1.3.2. Spatial training All groups displayed normal swimming behavior and had similar swim speeds (p > .05, data not shown). As shown in Fig. 4, the Naive RS Lesion group was impaired relative to Pooled RS Controls on all measures during spatial training on day 1 (search time, Group: F(2,9) = 5.5, p < .01; Trials: F(9,252) = 8.6, p < .0001; time in periphery, Group: F(2,9) = 3.5, p < .05; Trials: F(9,252) = 12.9, p < .0001; percent direct and circle swims, F(2,28) = 3.4, p < .05; Naive RS versus Pooled RS Control, p < .05 all measures). In contrast, the pretrained RS Lesion group was not impaired on the search time and periphery time measures but was impaired on the percent direct and circle swim measure (pretrained RS lesion versus Pooled RS Control, p < .05; Fig. 4B). Very few platform contact errors were made by any group and no group differences were found (p > .05).

3.1.1.7. Behavioral analysis Digitized swim paths of spatial training trials and videotapes of swims were objectively analyzed for: (1) swim time from release to contact with the hidden platform, (2) time swum in the pool periphery, (3) use of hidden platform as a refuge, (4) direct and circle swims and (5) swim speed. The pool periphery was the outer 50% of the surface area, as defined in Experiment 1. The hidden platform was never located in the pool periphery. Time swum in the pool periphery was used as one measure of WM strategies acquisition and use [6] because a necessary WM strategy is to search for the hidden platform away from the pool wall [32,47]. Swim paths were objectively analyzed by the Poly-Track system to obtain search time and time in the periphery. Use of the hidden platform as a refuge was a second WM strategy that was evaluated by blind scoring of videotaped spatial training trials for two kinds of error, platform deflections and platform swimovers. A platform deflection occurred when a rat contacted the hidden platform during swimming, failed to mount the platform and swam away from the platform; a swimover occurred when a rat contacted and mounted the hidden platform, and immediately swam off the far side of the platform in one continuous motion (for photos of these behaviors see ref. [8]; Fig. 2). A direct swim remained within an 18 cm-wide virtual alley from the start to the platform [26]. A circle swim approximated an arc of a circle from the start to the platform without exceeding 360◦ of circling or crossing over itself [9]. Direct and circle swims were analyzed because these are the most efficient swims and the most stringent measures of spatial memory available for the WM [26,49], and because Experiment 1 showed that control rats normally navigate to a goal using either direct or circle swims. Swim speed was obtained by dividing the total distance swum in a training session by the total search time.

3.1.2. Histological and data analysis Histological and data analyses were performed as in Experiment 1, with the addition of paired or unpaired t-tests in portions of the data analysis. Data from different days of testing were analyzed separately because, as expected from previous WM research with brain lesioned rats [24] there was heterogeneity of variance in some measures between days. Also, brain mechanisms underlying initial acquisition of a place response and subsequent reversal place learning are reported to be different [48], suggesting that initial acquisition and reversal learning constitute different tasks in the WM. Preliminary analysis indicated that the control subgroups did not differ on any measure. Therefore, the Sham RS Lesion Control groups were collapsed to form the Pooled RS Control group (n = 12). 3.1.3. Results Histological results were comparable to those reported in Experiment 1, and included damage to the same structures reported earlier for the RS Lesion group (see Fig. 1).

3.1.3.3. Reversal training If groups learned and remembered the hidden platform location during acquisition, they should search in the old platform location, with longer search times at the start of reversal training than at the end of spatial training. To evaluate this possibility the last spatial training trial on day 1 and the first reversal training trial on day 2 were plotted separately in Fig. 4A and group mean times for these trials were compared. Only the Pooled RS Control group displayed an increase in search time on the first trial of day 2 relative to the last trial on day 1 (t = 2.9, p < .01). To further analyze these data the change in search time between the last trial on day 1 and the first trial on day 2 was calculated for the Pooled RS Control group (+25.9 ± 5.6 s) and the pretrained RS Lesion group (+5.8 ± 5.3 s). The mean search time increase from the last to first trials was significant for the Pooled RS Control group (t = 4.6, p < .0007, paired t-test), but was not significant for the Pretrained RS Lesion group (p > .05). The group mean search time changes also differed between the groups (t = 2.6, p < .018, unpaired t-test). Taken together these results are consistent with the conclusion that both lesioned groups were impaired in memory for the hidden platform location on day 1. During reversal training only the Naive RS Lesion group was impaired on the search time measure (search time, Group: F(2,9) = 5.0, p < .02; Trials: F(9,252) = 4.7, p < .0001, Naive RS Lesion versus Pooled RS Control, p < .05; Fig. 4A). Analysis of the time in the periphery and direct and circle swim measures revealed non-significant trends for group differences (.05 < p < .10). Based on findings reported previously of impairment in naive lesioned or drug treated rats [5,12] these trends were further examined by comparing the performance of the Naive RS Lesion and Pooled RS Control groups. These anal-

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In sum, the Naive RS Lesion group was impaired on all measures throughout training and displayed no evidence of improvement after the first two spatial training trials. The pretrained RS Lesion group was impaired on spatial memory on day 1 in the absence of impairment on WM strategies, but performed as well as controls during reversal training on day 2. 3.1.4. Discussion The outcome of Experiment 1 led us to include pretrained rats in Experiment 2 for the separation of WM strategies learning and spatial place learning, and to employ a method to measure memory for the hidden platform location that was based on navigation behaviors that were generated by both naive and RS lesioned rats navigating to a visible refuge in the same WM pool. Using this approach, a main finding of Experiment 2 was that RS lesions produced a reliable spatial memory impairment that was not secondary to a WM strategies impairment. This conclusion was supported by the fact that the pretrained RS Lesion group was impaired in spatial memory on day 1 despite using the required WM strategies as evaluated by both the periphery time and platform contact error measures. On day 2, the Pretrained RS Lesion group performed as well as controls on all measures, indicating that although RS damage impaired spatial learning it did not completely prevent it. In contrast to previous findings with RS lesions, which were described as mild [45], the spatial memory impairment measured with the direct and circle swim measure was robust. 3.1.5. Role of RS cortex in spatial behavior

Fig. 4. RS Lesion and Pooled RS Lesion Control rats in the WM task, Experiment 2. Mean hidden platform search time (A), mean time swum in the pool periphery (B) and percent of swims that were either direct or circle (C). The Naive RS Lesion group failed to improve after the first block of training trials and was impaired on all measures throughout training. The pretrained RS Lesion group was impaired on the direct and circle swim measure on day 1 without an impairment in WM strategies, indicating a specific spatial memory impairment. (A) Last = last trial on day 1 of acquisition; first = first trial on day 2, reversal. Only the Pooled Control group exhibited a significant increase in search time from last to first (see text). On day 2, the Pretrained RS Lesion group was unimpaired. The Naive RS Lesion group was impaired in learning WM strategies (see text). (*) Significantly different from Pooled RS Controls; (+) day 2 first trial significantly different from day 1 last trial. Plots within a day that differ significantly from other plots are indicated by symbols associated with the first and last symbols of the plot.

yses indicated impairment in the Naive RS Lesion group relative to the Pooled RS Control group on both time in the periphery and direct and circle swims (time in the periphery, F(1,9) = 7.3, p < .014; Trials: p > .05; percent direct and circle swims, F(1,19) = 6.2, p < .03; Fig. 4A and B). The Pretrained RS Lesion group did not differ from controls on any measure during reversal training.

These results clarify the role of RS in navigation behavior. In two separate studies navigation ability was evaluated in both naive RS lesioned rats and in spatially trained rats that were then given RS lesions followed by additional spatial training [21,22]. By some behavioral measures the groups were impaired in the conventional WM task but by other measures the groups were unimpaired or even reliably superior to controls. Given the view that RS is important for spatial navigation this is a surprising set of results, especially the superiority of a Naive RS lesioned group relative to controls [21], which would have been expected to be impaired relative to intact control rats. Results from the present study reveal a reliable and specific spatial memory function for RS in the form of impaired direct and circle swims in the Pretrained RS Lesion group. This result was obtained from strategies-pretrained rats in which confounding impairments in WM strategies acquisition and use were excluded, and using a measure of spatial memory that was based on navigation behaviors actually employed by both naive and RS lesioned rats when navigating to a visible refuge in the same WM pool. An advantage of the direct and circle swim measure is that it allows the collection of data on every trial without the need to use unreinforced trials. The documenting of direct and circle swims on every trial can be expected to provide a more complete assessment of memory for the hidden platform position than a single post-training probe trial.

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The results from the Naive RS Lesion group further suggest the novel conclusion that RS also has an important role in the learning of WM strategies. This is supported by the fact that this group was impaired in both WM strategies and spatial memory throughout training, with virtually no improvement in any aspect of performance after the first two spatial training trials. When considered together with the finding that the Pretrained RS Lesion group was capable of learning the hidden platform location by day 2 if they used appropriate WM strategies, the fact that the Naive RS Lesion group could not learn the hidden platform position must have been secondary to their failure to learn and use the required WM strategies. Thus, although RS lesions did not disrupt the use of strategies acquired prior to the lesion, data from the Naive RS Lesion group show that rats were unable to acquire the crucial WM strategy of searching for the hidden platform away from the pool wall, at least through training trial 20. This outcome is consistent with results from Experiment 1 showing impairment in swimming away from the pool wall by naive RS lesioned rats swimming to a visible platform (Figs. 2 and 3). Thus, the results of Experiments 1 and 2 suggest that RS cortex is involved in both acquisition of WM strategies and learning spatial locations in a WM pool, and that the behavioral consequences of the lesion depend on the previous experience of the animal. 3.1.6. Lesions of RS cortex and related brain structures Although comparisons between experiments that were conducted successively should be treated with caution, such comparisons may be worthwhile between recent experiments conducted in our laboratory using the same WM apparatus, testing room, behavioral training and analysis personnel and techniques, as well as the same rat source and strain. Comparison between the current results with RS lesions and results with fimbria-fornix lesions [10] shows that both lesions produced a WM strategies learning impairment in naive rats and a specific spatial memory impairment in pretrained rats. The current results with RS lesions in naive rats are also similar to results with non-fibersparing lesions of medial thalamus in naive rats, in that both lesions produced a WM strategies learning impairment that was not overcome during multiple days of spatial training [10]. However, the current results with RS lesions differed from results with lesions of medial thalamus in that pretrained rats with thalamic lesions learned a hidden platform location as quickly and accurately as controls [10], whereas the present study found that pretrained rats with RS lesions had a spatial learning impairment. This set of results distinguishes the dual role of RS cortex in learning both WM strategies and hidden platform locations from the specific role of medial thalamus in learning only WM strategies. Further contrasts are apparent between the current results with RS lesions and results from large lesions of visual, parietal or prefrontal neocortex, which produced less impairment in both naive and pretrained rats [24] than RS lesions produced in similar groups. In particular, as with medial thalamic lesions, lesions of any of these three areas of neocortex failed to impair any aspect of WM performance when given to pretrained rats [24].

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In sum, comparisons of the present findings with our earlier findings using the same experimental techniques and rat strain show that the effects of RS lesions are very similar to those of fimbria-fornix lesions whether rats are naive or pretrained, but are similar to effects of medial thalamic lesions only if naive rats are considered. When pretrained rats are considered, only RS lesions cause a specific spatial learning impairment, whereas lesions of medial thalamus, as well as lesions of visual, parietal or prefrontal neocortex, cause no behavioral impairment. The present results with RS lesions demonstrate behaviorally dissociable roles for RS in both WM strategies learning and spatial place learning. 3.1.7. Neuroanatomical relations of RS cortex RS cortex receives direct neuroanatomical inputs from visual areas 17, 18a and 18b, and from parietal and auditory neocortex [36,51]. RS cortex also receives inputs from entorhinal cortex, which itself receives inputs from widespread areas of neocortex [46], and also has reciprocal connections with areas of the hippocampal formation that contain neurons that respond to spatial information and the position of the animal in the environment, such as the subiculuum and postsubiculum [43,46]. Thus, RS is well placed to receive processed visual and other information and to convey it to hippocampal formation for use in generating adaptive navigation behavior. This idea is consistent with the similarity of the impairments seen with RS lesions in the current study and with fimbria-fornix lesions in our earlier study using the same experimental techniques and rat strain [10], as was discussed above. This idea is also consistent with the fact that RS cortex contains head direction cells and other cells responsive to particular combinations of location, head direction and movement [18] and with the fact that inactivation of RS cortex disrupts the stability of hippocampal place cells in behaving rats [19]. RS cortex also receives direct neuroanatomical inputs from a number of nuclei in medial thalamus, including anteromedial, anteroventral, anterodorsal and reuniens [46]. As was mentioned above, lesions of medial thalamus that included these nuclei reliably impaired WM strategies learning in naive rats without impairing spatial learning and memory in pretrained rats [10]. The fact that lesions of either medial thalamus or RS cortex impair WM strategies learning is consistent with the direct neuroanatomical connections demonstrated to exist between them [46]. Further details of the specific contribution of RS cortex to spatial behavior in the context of both neocortical and medial thalamic mechanisms will require additional research. 4. Conclusion In this study, we demonstrate that lesions limited to RS cortex produce dissociable impairments in both learning WM strategies and learning spatial locations in a WM task. We show that the behavioral consequences of the lesion depend on the previous experience of the animal, and that by using appropriate training and testing procedures in a detailed behavioral analysis the two

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impairments are dissociable. These dissociable impairments are comparable to impairments seen in our similar experiments with fimbria-fornix lesions. The impairment in WM strategies learning due to RS cortex lesions is also similar to the impairment seen with lesions of medial thalamus. These findings together with previous findings of spatially responsive cells in RS cortex and of neuroanatomical connections between RS cortex and visual neocortex, medial thalamus and the hippocampal formation suggest that RS cortex participates in an extended circuit involved in multiple aspects of spatial behavior including both behavioral navigation strategies learning and spatial place learning. Acknowledgement Supported by a grant from the Natural Science and Engineering Research Council of Canada to DPC. References [1] Aggleton JP, Neave N, Nagle S, Sahgal A. A comparison of the effects of medial prefrontal, cingulate cortex, and cingulum bundle lesions on tests of spatial memory: evidence of a double dissociation between frontal and cingulum bundle contributions. J Neurosci 1995;15:7270–81. [2] Aggleton JP, Vann SD. Testing the importance of the retrosplenial navigation system: lesion size but not strain matters: a reply to Harker and Whishaw. Neurosci Biobehav Rev 2004;28:525–31. [3] Aggleton JP, Vann SD, Oswald CJ, Good M. Identifying cortical inputs to the rat hippocampus that subserve allocentric spatial processes: a simple problem with a complex answer. Hippocampus 2000;10:466–74. [4] Bannerman DM, Good MA, Butcher SP, Ramsay M, Morris RGM. Distinct components of spatial learning revealed by prior training and NMDA receptor blockade. Nature 1995;378:182–6. [5] Beiko J, Candusso L, Cain DP. The effect of non-spatial water maze pretraining in rats subjected to serotonin depletion and muscarinic receptor antagonism. Behav Brain Res 1997;88:201–11. [6] Beiko J, Lander R, Hampson E, Cain DP. Contribution of sex differences in the acute stress response to sex differences in water maze performance in the rat. Behav Brain Res 2004;151:239–53. [7] Cain DP. Prior non-spatial pretraining eliminates sensorimotor disturbances and impairments in water maze learning caused by diazepam. Psychopharmacology 1997;130:313–9. [8] Cain DP. Testing the NMDA, long-term potentiation, and cholinergic hypotheses of spatial learning. Neurosci Biobehav Rev 1998;22:181–93. [9] Cain DP, Boon F. Detailed behavioral analysis reveals both task strategies and spatial memory impairments in rats given bilateral middle cerebral artery stroke. Brain Res 2003;972:64–74. [10] Cain DP, Boon F, Corcoran ME. Thalamic and hippocampal mechanisms in spatial navigation: a dissociation between brain mechanisms for learning how vs. learning where to navigate. Behav Brain Res 2006;170:241–56. [11] Cain DP, Finlayson C, Boon F, Beiko J. Ethanol impairs behavioral strategy use in naive rats but does not prevent spatial learning in the water maze in pretrained rats. Psychopharmacology 2002;164:1–9. [12] Cain DP, Ighanian K, Boon F. Individual and combined manipulation of muscarinic, NMDA and benzodiazepine receptor activity in the water maze task: implications for a rat model of Alzheimer dementia. Behav Brain Res 2000;111:125–37. [13] Cain DP, Saucier D, Boon F. Testing hypotheses of spatial learning: the role of NMDA receptors and NMDA-mediated long term potentiation. Behav Brain Res 1997;84:179–93. [14] Cain DP, Saucier D, Hall J, Hargreaves EL, Boon F. Detailed behavioral analysis of water maze acquisition under APV or CNQX: contribution of sensorimotor disturbances to drug-induced acquisition deficits. Behav Neurosci 1996;110:86–102.

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