Thalamic and hippocampal mechanisms in spatial navigation: A dissociation between brain mechanisms for learning how versus learning where to navigate

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1 Behavioural Brain Research 170 (2006) Research report Thalamic and hippocampal mechanisms in spatial navigation: A dissociation between brain mechanisms for learning how versus learning where to navigate Donald P. Cain a,b,, Francis Boon a, Michael E. Corcoran c a Department of Psychology, University of Western Ontario, London, Ont., Canada N6A 5C2 b Graduate Program in Neuroscience, University of Western Ontario, London, Ont., Canada N6A 5C2 c Department of Anatomy and Cell Biology, University of Saskatchewan, Saskatoon, Sask., Canada S7N 5E5 Received 23 September 2005; accepted 20 February 2006 Available online 29 March 2006 Abstract Various studies of hippocampus and medial thalamus (MT) suggest that these brain areas play a crucial, marginal, or no essential role in spatial navigation. These divergent views were examined in experiments using electrolytic Lesions of fimbria fornix (FF) or radiofrequency or neurotoxic Lesions of MT of rats subsequently trained to find a stable visible (experiment 1) or hidden platform (experiments 2 and 3) in a water maze (WM) pool. Rats with electrolytic Lesions of FF or radiofrequency Lesions of MT were impaired in swimming to a stable visible platform, particularly the MT Lesion Group, suggesting impairment of WM strategies acquisition. Additional Lesioned rats were then tested in a hidden platform version of the WM task. Some rats were given Morris s nonspatial pretraining prior to Lesioning to provide them with training in the required WM behavioral strategies. Nonspatially Pretrained rats with FF Lesions eventually were able to navigate to the hidden platform, but the accuracy of place responding was impaired. This impairment occurred without problems in the motoric control of swimming or the use of WM behavioral strategies, suggesting that these rats had a spatial mapping impairment. Radiofrequency MT Lesions blocked acquisition of WM behavioral strategies by Naive rats throughout 3 days of training, severely impairing performance on all aspects of the hidden platform task. Nonspatially Pretrained rats given the same MT Lesions readily learned the hidden platform location and were indistinguishable from controls throughout spatial training. Rats given neurotoxic Lesions of MT for removal of cells were only mildly impaired and improved considerably during training, suggesting an important role for fibers of passage in WM strategies learning. The results provide a clear dissociation between a role for MT in learning WM behavioral strategies and the hippocampal formation in spatial mapping and memory. This is the first identification of a brain area, MT, that is essential for learning behavioral strategies that by themselves do not constitute the solution to the task but are necessary for the successful use of an innate learning ability: place response learning using spatial mapping Elsevier B.V. All rights reserved. Keywords: Navigation; Learning; Memory; Hippocampus; Thalamus; Watermaze 1. Introduction The rat brain contains neural mechanisms for rapidly learning locations of objects in the environment, allowing efficient navigation. Early research suggested that hippocampal mechanisms are essential for this place learning [33,44], but intense debate centered around the mechanisms involved, particularly whether place learning requires NMDA receptor-dependant hippocampal Corresponding author. Tel.: x84628; fax: address: cain@uwo.ca (D.P. Cain). long term potentiation (NMDA-LTP) [32]. Recent work using refined testing techniques and dose-response designs to minimize confounding drug-induced sensorimotor disturbances indicates that robust place learning can occur during NMDA-LTP blockade, suggesting that NMDA-LTP might contribute to but is not essential for place learning [1,7,8,22,34,41,42]. This conclusion and the fact that extrahippocampal damage can impair water maze task (WM) acquisition [23,24,53,54] raises the question of whether the hippocampal formation (HF) is essential for this learning. Although Naive rats with HF damage were strongly impaired in the WM [33,44,45], spatially trained rats given HF damage were either impaired during post-surgical retraining [45] /$ see front matter 2006 Elsevier B.V. All rights reserved. doi: /j.bbr

2 242 D.P. Cain et al. / Behavioural Brain Research 170 (2006) or only weakly impaired on some but not all measures [35,55]. Minor alterations in WM training procedures allowed Naive rats with HF damage to acquire a place response [57,58,61]. Thus there is varying evidence that the HF plays an essential role, or only a marginal role, or no essential role in place learning. The HF and certain thalamic nuclei are interconnected, and the presence of head direction cells, which signal the directional heading of the animal [47], in the anterodorsal nucleus of the thalamus has focused attention on the role of the thalamus in the WM task. However, Lesions of the anterodorsal nucleus and adjacent anterior thalamic nuclei typically produce only mild to moderate WM impairments [54,55]. Although the impairment caused by anterodorsal nucleus damage is consistent with a role for the head direction system in navigation, both the electrophysiological [10] and behavioral evidence [33,35,44,54,55] suggest that the HF may be the more important component for navigation. On the other hand, Lesions of the medial thalamus that largely spare the anterodorsal nucleus head direction system produce severe impairment in a simple swim-to-visible platform task in a small glass acquarium [50]. This impairment was characterized by persistent swimming around the perimeter of the acquarium and failing to climb onto and use the visible platform as refuge. Large Lesions placed more caudally in the thalamus caused no impairment in this task. These findings suggest that important mechanisms for navigation, particularly for the acquisition of task-relevant behavioral strategies, may exist in the medial thalamus and that these mechanisms may be distinct from the head direction system of the anterior thalamic nuclei. Most of the above findings are limited by being obtained almost exclusively from Naive rats that had received no experience in the WM prior to the brain Lesions and the start of spatial training. This is important because, as with any task [26,56], the WM task requires acquisition of at least two components for its solution: (1) behavioral strategies for coping with and obtaining information in the task, and (2) information obtained through use of the behavioral strategies to solve the task [1,31,56]. Behavioral strategies that have been identified as essential for the WM task are suppression of thigmotaxic swimming to search for refuge away from the pool wall, and recognizing and using the hidden platform as refuge by climbing onto and remaining on it [1,31,56]. Naive rats given experimental treatments often have difficulty coping with the stressful conditions of the WM task [20] to acquire both components of the task, and often persist in swimming thigmotaxically, failing to make use of the hidden platform [5,56]. The difficulty in suppressing thigmotaxis and learning to search for refuge away from the pool wall is particularly problematic because it prevents rats from readily contacting the hidden platform and obtaining information about its location. This difficulty obscures a clear answer about whether a treatment impairs behavioral strategy learning, place learning, or both. Morris s [31] nonspatial pretraining technique (hereafter, pretraining) separates the strategies-learning and place-learning components, providing two important advantages over the conventional WM task as used with Naive rats. First, exposure during pretraining to the handling and pool involved in the WM task reduces the strong stress response triggered by testing in a WM [4]. Reduction of the stress response prior to HF damage may be especially important in WM studies because stress impairs performance in this task [14,20] and the HF normally modulates the stress response through a negative feedback mechanism, which is impaired by HF damage [27]. A second advantage is that during pretraining curtains surround the pool and the platform is moved after each Trial, preventing the learning of cue configurations and associations with pool geometry or a platform position prior to spatial training. Thus pretraining separates the behavioral strategies-learning component from the spatial learning component. This allows evaluation of initial spatial learning ability under experimental treatment in animals that already know the required behavioral strategies, providing a targeted test of an animal s place learning ability under the treatment, yielding clearer answers to questions about brain mechanisms of place learning. No WM study has used Pretrained controls and a detailed behavioral analysis with HF or thalamic animals, providing the impetus for this study. We first Pretrained Naive intact rats, then made FF or medial thalamic (MT) Lesions [50], then gave conventional WM training with a stable hidden platform. If Pretrained Lesioned rats can acquire a place response the Lesioned area must not be essential for place learning, suggesting that in cases where Naive rats given the same Lesion were impaired in the task the impairment may be in acquiring and using WM behavioral strategies. The latter possibility is evaluated by a detailed behavioral analysis that involves quantitative measurement of behavioral strategy use [8]. Conversely, if Pretrained Lesioned rats employ the necessary WM behavioral strategies during spatial training but remain impaired in place responding, this would imply that the Lesioned structure is necessary for place learning. Results revealed a clear dissociation between a role for the MT in learning WM behavioral strategies, and HF in spatial mapping and memory. 2. Experiment 1 visible platform task Naive rats with HF or MT Lesions swim predominantly in the periphery of a pool or acquarium and are impaired in swimming to a visible platform (Fig. 1 in [33])([50], D.P. Cain, unpublished data). This raises the possibility that these rats harbor an impairment in generating navigation behavior even when the goal is clearly visible. Therefore we tested Naive rats with FF or MT Lesions in a simple swim-to-visible platform task to evaluate swimming and navigation behavior in order to: (1) replicate and better document the findings of impaired swimming to a visible platform [33,50], (2) extend testing to determine the persistence of the impairments over multiple testing sessions on different days, and (3) document the normal swim trajectory to a visible refuge in Naive intact rats to aid the selection of optimal measures of navigation ability in experiments 2 and Materials and methods 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 12:12 h light:dark cycle for comparability with previous WM research. The rats had not been in water prior to the behavioral test

3 D.P. Cain et al. / Behavioural Brain Research 170 (2006) Fig. 1. 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 are overlain on an outline of the pool as seen from above. Paths from the FF Lesion and control Groups on days 1 and 2 are shown in (A); paths from the MT Lesion and control Groups on days 1, 2, and 3 are shown in (B). The positions of the release points and the visible platform are shown in (C). Note that the start point was moved closer to the visible platform for the MT Lesion Group on day 3. There was a 9 cm gap between the pool wall and the closest edge of the visible platform. The MT Lesion rats failed to generate any direct swims to the visible platform on days 1 and 2. procedures described below and were Naive to all experimental procedures prior to surgery and random allocation to Groups. Controls received sham surgery. Rats recovered for 14 days before testing. Procedures in all experiments were in accordance with guidelines of the Canadian Council on Animal Care and approved by the University of Western Ontario Surgery Lesioned rats received either bilateral cathodal Lesions that severed the axons of the FF (FF Lesion Group, n = 7) or radiofrequency Lesions of MT that were intended to damage the medial thalamus without damaging the HF or head direction system in the anterior thalamus (MT Lesion Group, n = 8). Electrolytic FF Lesions were used because: (1) they deprive HF of a major cholinergic input and impair normal hippocampal electrographic and place cell activity [28,43,49]; (2) they produce strong behavioral impairments in the WM task that are comparable to impairments produced by removal of hippocampal cells [13,58,61]; and (3) they do not produce thinning of overlying eocortex and the risk of Lesion confound that typically accompanies the direct removal of hippocampal tissue [12]. Radiofrequency Lesions of MT were used because they produce reliable and circumscribed damage to heterogeneous tissue such as thalamus [50]. Rats received atropine pretreatment and sodium pentobarbital anesthesia (60 mg/kg i.p.) and were placed in a stereotaxic apparatus. Body temperature was maintained at normothermia using a heating pad. Surgical procedures and introduction of electrodes were carried out under aseptic conditions. FF Lesions were produced by passing cathodal current at 1.5 ma for 40 s through electrodes insulated except at the tips at the following coordinates with respect to bregma and dura surface: 1.3 mm posterior, 0.0 mm lateral, 3.6 mm ventral. MT Lesions were produced using a Grass radiofrequency Lesion maker to heat tissue to 65 C for 60 s at one site in each hemisphere. The following coordinates with respect to bregma and dura surface were used for MT Lesions: 1.8 mm posterior, 0.7 mm lateral, 4.5 mm ventral. Sham controls received the same treatment as Lesioned rats up to and including drilling holes in the skull but electrodes were not inserted (FF control Group, n = 7; MT control Group, n = 8). Behavioral testing began 14 days after surgery Apparatus The WM was a white circular pool (1.5 m dia) 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 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 swimming into the platform while swimming thigmotaxically (see Fig. 1C). 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 [9]. 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 Training Visible platform training took place on days 1 and 2 (10 Trials per day; 5 min intertrial interval). A rat was released into the water immediately adjacent to and facing the pool wall opposite the visible platform, which remained in a fixed position throughout training (see Fig. 1C). 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. Due to the severe impairment of the MT Lesion rats, which did not produce any direct swims to the visible platform on days 1 and 2 (see Section 2.2), 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 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 m thickness. Every 5th 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 a brain atlas [38]. Volumetric measures of damage were calculated by multiplying the area of damage by the distance between sections and summing damage volume for each rat. Behavioral data were analyzed using 1-way and repeated measures ANOVA and product moment correlations. Only significant results are reported.

4 244 D.P. Cain et al. / Behavioural Brain Research 170 (2006) Fig. 2. Photomicrographs showing FF (A) and MT damage (B, C). Damage in (A) shows the severing of the fimbria fornix in FF Lesioned rats in experiments 1 and 2. The sections in (A) are approximately at the level of plates 22 (top, 0.92 mm relative to Bregma) and 23 (bottom, 1.30 mm) of Paxinos and Watson (1986). Damage in (B) shows damage in rats given MT Lesions in experiments 1 and 2. The sections in (B) are approximately at the level of plates 23, 25, 27, and 30 from top to bottom ( 1.30, 1.80, 2.30, and 3.14 mm relative to Bregma, respectively). Sections in (C) show the brain of rat MT7, which had the smallest Lesion in the Naive MT Leison Group in experiment 2. Damage in (C) does not extend as far rostrally as in (B), and the anterior thalamic nuclei are intact in this rat (top panel of (C)) Results The dorsal fornix and the fimbria were completely severed in all rats given FF Lesions (Fig. 2A). Minor damage to adjacent septal nuclei and the hippocampal commissure occurred in some brains. Damage to the overlying medial neocortex caused by the Lesioning electrode was small and well localized. Previous research has shown that similar damage to supracallosal tissue does not produce additional impairment on the WM [45] and that limited neocortical damage does not impair performance in the WM in Pretrained rats [19]. As shown in Fig. 2B, damage to the MT included part or all of the following: the anteromedial, mediodorsal, paraventricular, paratenial, paracentral, central medial, and rhomboid nuclei. Partial damage also occurred to the anterodorsal, anteroventral, and ventrolateral nuclei on their medial borders, and to the reuniens and gelatinosus nuclei on their dorsal or dorsomedial borders, respectively; when present the damage typically involved approximately 5 15% of the volume of these structures. The medial habenular nucleus and stria medullaris were damaged in most cases but the mammilothalamic tract was damaged, in part, in fewer than half of Lesioned rats. The laterodorsal nuclei were not damaged in any rat. The mean volume of the MT Lesions was 6.1 ± 0.59 (mean ± S.E.M.) mm 3 (range: mm 3 ). All rats displayed normal swimming behavior, with typical forelimb inhibition and alternate thrusting of the hindlimbs, and used the platform as refuge when they contacted it. As shown in Fig. 1A and B, which displays every swim path for all rats in each Group, both FF and MT Lesioned rats swam more in the periphery than controls, generating less efficient swims than controls. Remarkably, there were no direct swims to the visible platform by any rat in the MT Lesion Group on days 1 and 2. Control rats displayed a mix of both periphery and efficient swims on day 1, and more efficient swims on day 2 (Fig. 1A and B), an outcome similar to our previous findings [4,6]. 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 [8], where the hidden platform was never placed during spatial training in experiments 2 and 3. A heading error was scored if a rat swam away from the visible platform (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. The two control Groups did not differ on any measure on either day

5 D.P. Cain et al. / Behavioural Brain Research 170 (2006) p <.007; MT Lesion and FF Lesion versus Pooled Controls, p <.05 all measures). An attempt was made to relate behavioral impairment to gross MT Lesion size but this yielded no useful relationships (all r < 0.15, all p > 0.05). This was likely due to the fact that every MT Lesion rat was severely and similarly impaired in the task, and the range of Lesion volumes was restricted. For example, no rat with an MT Lesion displayed even a single direct swim to the visible platform on either day 1 or day 2 (Fig. 2B). On day 3 when the start point was moved closer to and facing the visible platform, the MT Lesion Group remained severely impaired on all measures (ANOVA, mean swim time, F(1,20) = 11.1, p <.003; time in periphery, F(1,20) = 11.2, p <.003; heading errors, F(1,20) = 19.5, p <.0001; see Fig. 3A C). This was caused by frequent swims directly to the wall upon release, followed by swimming in the periphery to the visible platform (Fig. 1B) Discussion 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 two rats were started at and facing the wall. On day three rats were started away from the wall, closer to and facing the visible platform (see Fig. 1). Heading errors occurred among the MT Lesion Group on day 3 because they frequently swam directly toward the wall immediately upon release. Plots and points that differ statistically from Pooled Controls are indicated by an *. For data symbols that do not have error bars, the bars are contained entirely within the symbols. Error bars indicate ±S.E.M. in all figures. (p >.05) and were collapsed for statistical analysis. As shown in Fig. 3A C, analysis indicated longer swim times, greater time in the periphery, and more heading errors by both FF and MT Lesioned Groups than controls (repeated measures ANOVA, swim time per Trial, Group: F(2,1) = 15.4, p <.0001, Trials: F(1,1) = 55.8, p <.0001; time in periphery per Trial, Group: F(2,1) = 22.7, p <.0001, Trials: F(1,1) = 62.6, p <.0001; heading errors, Group: F(2,1) = 10.1, p <.001, Trials: F(1,1) = 8.6, The visible platform task used here involved repeated Trials with fixed start and goal locations and appears to be the simplest WM task used to date. Despite the utter simplicity of the task, and with no impairment in swimming behavior as such, both Lesioned Groups were impaired on all measures. This confirms and extends the finding that hippocampal rats were impaired in swimming to a visible platform (Fig. 1 in [33]) and adds the novel finding that Naive MT Lesioned rats were severely impaired in swimming to a visible platform even when starting close to and facing the visible platform. The significant Trials effects indicate that rats improved in performance from day 1 to day 2. However, the MT Lesion Group nevertheless was severely impaired throughout testing on days 1 and 2 and failed to generate a single direct swim on either day. Although they generated some direct swims on day 3 when the start point was moved closer to and facing the visible platform, they remained severely impaired relative to controls, suggesting that the impairment was a lasting one. 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) there was no damage to any main visual area of the brain, (4) Lesions of the FF or MT do not impair visual processing [16,53], (5) Pretrained MT Lesioned rats used visual cues effectively in the WM (see Section 3). An alternate explanation may be that the MT is important for acquiring WM behavioral strategies, a possibility that is pursued in experiment 2. The swim paths reveal that swim trajectories are frequently curved in both Lesioned and control rats even if the refuge is clearly visible. The data also show that Naive Lesioned rats gradually improve in task-relevant navigation behaviors from day 1 to day 2, even for this simplest version of the WM. These facts were taken into consideration in experiment 2 by including Pretrained Groups and a detailed behavioral analysis that scored direct or efficient curved swim trajectories to the platform.

6 246 D.P. Cain et al. / Behavioural Brain Research 170 (2006) Experiment 2 effect of radiofrequency Lesions of FF or MT on spatial learning Experiment 2 examined the effect of Lesions of FF or MT on WM acquisition with a hidden platform. Some Groups consisted of rats given pretraining prior to the Lesion to separate the strategies-learning and spatial-learning components of the task [1,31] Material and methods Subjects and experimental Groups Similar subjects as in experiment 1 were used. All rats were Naive to experimentation and no rats from experiment 1 were used. General housing and testing conditions were as in experiment 1. Rats were randomly allocated to Groups that were given pretraining or no pretraining, and either FF Lesions, MT Lesions, or sham Lesion treatment. Groups were: Naive FF Lesion (n = 10), Pretrained FF Lesion (n = 14), Naive MT Lesion (n = 8), Pretrained MT Lesion (n = 8), Naive sham Lesion (n = 7), Pretrained sham Lesion (n = 7) 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 1 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 [9]. Data were again recorded and analyzed using the VCR and Poly-Track system Pretraining Pretraining was carried out prior to surgery, with 3 Trials per day on each of 4 days for a total of 12 pretraining Trials, with a 5 min intertrial interval [18,31]. 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 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 the television monitor, and search times were recorded. The acquisition of WM behavioral strategies during pretraining has been documented [18,31,39] Surgery Rats received FF Lesions, MT Lesions, or sham surgery h after the completion of pretraining, if given. The surgical procedures were as described in experiment 1. Sham rats received anesthesia and had holes drilled in the skull for each of the Lesion locations but no electrode was lowered. Spatial training began 14 days after surgery Spatial training For spatial training the curtains were removed from around the pool, allowing 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 southeast quadrant (5 min intertrial interval). Identical spatial training was repeated on day 2. Reversal spatial training was given on day 3 and was identical except that the hidden platform was in the center of the northwest quadrant. 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 2-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 Behavioral analysis Digitized swim paths of spatial training Trials were objectively analyzed for: (1) swim time from release to contact with the hidden platform, (2) time swum in the pool periphery, (3) direct and circle swims, and (4) swim speed. A direct swim remained within an 18 cm-wide virtual alley from the start to the platform [23]. A circle swim approximated an arc of a circle from the start to the platform without exceeding 360 of circling or crossing over itself [6]. 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 [23,58] and because experiment 1 showed that control rats frequently navigate efficiently to a goal using direct and circle swims. Swim speed was obtained by dividing the total distance swum in a training session by the total search time Histological and data analysis. Histological analysis was performed as in experiment 1. Data were analyzed using 1-way and repeated measures ANOVA with Newman Keuls post hoc tests, and product-moment correlations. Only significant results are reported. Data from days 1 and 2 were analyzed separately because, as expected from previous research with brain Lesions [19] there was heterogeneity of variance in some measures between days, and the brain mechanisms underlying initial acquisition of a place response and subsequent reversal place learning are reported to be different [57], 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 they were collapsed into a single Pooled Control Group Results Histological results were comparable to those reported in experiment 1, and involved damage to the same structures reported earlier for the FF and MT Lesion Groups, respectively (see Fig. 2A and B). MT Lesion placement and volumes were comparable to those in experiment 1. The laterodorsal nuclei were not damaged in any rat. Mean MT Lesion volume was 5.9 ± 0.68 mm 3 (range: mm 3 ) Pretraining Mean search time for the Pretrained rats to find the hidden platform decreased from 49.7 ± 11.9 s (mean ± S.E.M.) on the first pretraining Trial to 24.2 ± 9.4 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 [18,31,39] FF Lesion Spatial training. As shown in the representative swim paths in Fig. 4 and Group data in Fig. 5, the Naive FF Lesion and Pretrained FF Lesion Groups were impaired on the spatial learning measures relative to Pooled Controls throughout acquisition on days 1 and 2, and showed no memory for the hidden platform position on the first Trial on day 3. The Pretrained FF Lesion Group learned something about the platform position during acquisition as indicated by better performance than the Naive FF Lesion Group. Only the Naive FF Lesion Group was impaired in WM behavioral strategies as indicated by periphery swimming. By the end of training on day 3 the Groups did not differ on any measure. These impressions were confirmed by analyses indicating impairments in both search time and direct and circle swims in both Lesioned Groups relative to Pooled Controls

7 D.P. Cain et al. / Behavioural Brain Research 170 (2006) Fig. 4. Swim paths for all 10 Trials on day 1 of spatial training with a hidden platform (experiment 2) for the rat in each Group with the summed total swim time closest to the mean summed total swim time of its Group. During the last 5 Trials of day 1 the Pooled Control and Pretrained MT Lesion rats navigated to the hidden platform effectively. In contrast, during the last 5 Trials the Pretrained FF Lesion rat typically swam toward the hidden platform, narrowly missed finding it, performed a tight turn, and swam back to find the hidden platform. This was typical of this Group s navigation ability, which was not as accurate as that of the Pooled Controls. The Naive Lesioned rats swam predominantly in the periphery of the pool and displayed poor navigation strategies. Fig. 5. Electrolytic FF Lesion and control rats in the WM task, experiment 2. Mean hidden platform search time (A); mean time swum in the pool periphery (B); mean percent of time spent in the pool periphery (C); percent of swims that were either direct or circle (D). Both Lesioned Groups had longer search times than Pooled Controls on days 1 and 2 but did not differ from Pooled Controls on day 3. In (A), L: last Trial on day 2 of acquisition; F: first Trial on day 3, reversal. Only the Pooled Control Group exhibited a significant increase from L to F. Only the Naive FF Lesion Group had a longer mean time and percent time in the periphery on days 1 and 2, indicating that the Naive FF Lesion Group failed to employ appropriate navigation strategies whereas the Pretrained FF Lesion Group employed appropriate strategies. The Naive FF Lesion Group was impaired in the percent direct and circle swim measure on days 1 and 2 (B). The Pretrained FF Lesion Group was impaired relative to Pooled Controls on days 1 and 2 but had more direct and circle swims on day 1 than the Naive FF Lesion Group. By the end of day 3 the Groups did not differ on any measure. * : Significantly different from Pooled Controls; : significantly different from Naive FF Lesion Group. Plots within a day that differ significantly from other plots are indicated by symbols associated with the first and last symbol of the plot. For datapoint symbols that do not have error bars, the bars are contained entirely within the symbols.

8 248 D.P. Cain et al. / Behavioural Brain Research 170 (2006) (search time, day 1, Group: F(2,9) = 19.7, p <.0001; Trial: F(9,315) = 7.5, p <.0001; day 2, Group: F(2,9) = 14.8, p <.0001; Naive FF Lesion and Pretrained FF Lesion > Pooled Control, p <.05 both days, Fig. 5A; percent direct and circle swims, day 1, F(2,35) = 12.2, p <.0001; Naive FF Lesion and Pretrained FF Lesion < Pooled Control, p <.05; day 2, F(2,35) = 10.9, p <.0001; Naive FF Lesion and Pretrained FF Lesion < Pooled Control, p <.05, Fig. 5D). The Pretrained FF Lesion Group performed better than the Naive FF Lesion Group in most comparisons (search time, days 1 and 2, Pretrained FF Lesion < Naive FF Lesion, p <.05, Fig. 5A; percent direct and circle swims, day 1, Pretrained FF Lesion > Naive FF Lesion, p <.05, Fig. 5D). Only the Naive FF Lesion Group had a WM strategy impairment (time in periphery, day 1, Group: F(2,9) = 24.3, p <.0001; Trial: F(9,315) = 9.9, p <.0001; day 2, Group: F(2,9) = 10.9, p <.0001; percent time in periphery, day 1, F(2,35) = 24.5, p <.0001; day 2, F(2,35) = 14.7, p <.0001; Naive FF Lesion > Pretrained FF Lesion and Pooled Control, p <.05 both measures, both days). All Groups displayed normal swimming behavior, used the hidden platform as a refuge, and had similar swim speeds (p >.05, data not shown) Reversal training. If Groups learned and remembered the hidden platform location during acquisition, they should search in the old platform location, with long search times at the start of reversal training. To evaluate this possibility the last Trial on day 2 and the first reversal Trial of day 3 were plotted separately in Fig. 5A and Group mean times were compared. Pooled Controls were the only Group to exhibit an increase in search time from day 2 to day 3 on these Trials (Pooled Control, t = 3.5, P <.001). All Groups readily acquired the reversal on day 3 and did not differ on any measure (p >.05; Fig. 5). In sum, the Naive FF Lesion Group had both behavioral strategies and place response impairments, while the Pretrained FF Lesion Group had a place response impairment without a behavioral strategies impairment. Although the Pretrained FF Lesion Group performed better than the Naive FF Lesion Group, it failed to match the performance of the Pooled Control Group on days 1 and 2, and displayed no evidence of memory for the platform position on the first Trial on day 3. All Groups acquired the reversal by the end of training on day MT Lesion Spatial training As shown in the representative swim paths in Fig. 4 and Group data in Fig. 6, the Naive MT Lesion rats were severely impaired on all measures on all days. This included extreme periphery swimming, the Group mean approaching 95% of the time in the periphery on day 1 (Fig. 6C). In contrast, Pretrained MT Lesion rats readily swam away from the wall and learned the location of the hidden platform quickly. These impressions were confirmed by analysis that revealed highly significant differences in search time between the Naive MT Lesion Group and both the Pretrained MT Lesion and Pooled Control Groups, which did not differ (day 1, Group: F(2,9) = 185.2, p <.0001; Trial: F(9,243) = 7.5, p <.0001; Interaction: F(18,243) = 3.7, p <.0001; day 2, Group: F(2,9) = 66.3, p <.0001; Trial: F(9,243) = 3.3, p <.001; Interaction: F(18, 243) = 2.7, p <.001; Naive MT Lesion versus Pretrained MT Lesion and Pooled Control, p <.05 both days, Fig. 6A). Analyses of time in the periphery and percent time in the periphery revealed similar impairments in the Naive MT Lesion Group but no difference between the Pretrained MT Fig. 6. Radiofrequency MT Lesion and control rats in the WM task, experiment 2. Mean hidden platform search time (A); mean time swum in the pool periphery (B); mean percent of time spent in the pool periphery (C); percent of swims that were either direct or circle (D). The percent direct and circle swims measure was 0.0% for the Naive MT Lesion Group on day 1. The Naive MT Lesion Group was impaired on all measures on all days relative to both the Pretrained MT Lesioned Group and the Pooled Controls, which did not differ on any measure on any day. The Pretrained MT Lesion and Pooled Control Groups displayed an increase in search time from the last acquisition Trial on day 2 (L) to the first reversal Trial on day 3 (F) but the Naive MT Lesion Group did not. * : Significantly different from Pooled Controls. Plots within a day that differ significantly from other plots are indicated by symbols associated with the first and last symbol of the plot. For datapoint symbols that do not have error bars, the bars are contained entirely within the symbols.

9 D.P. Cain et al. / Behavioural Brain Research 170 (2006) Lesion Group and controls (time in periphery, day 1, Group: F(2,9) = 182.6, p <.0001; Trial: F(9,243) = 7.6, p <.0001; Interaction: F(18,243) = 3.6, p <.0001; day 2, Group: F(2,9) = 65.6, p <.0001; Trial: F(9,243) = 3.8, p <.0001; Interaction: F(18, 243) = 2.5, p <.001, Naive MT Lesion versus Pretrained MT Lesion and Pooled Control, p <.05 both days, Fig. 6B; percent time in the periphery, day 1, F(2,27) = 39.8, p <.0001; day 2, F(2,27) = 53.4, p <.0001; Naive MT Lesion versus Pretrained MT Lesion and Pooled Control, p <.05 both days, Fig. 6C). Group mean percent direct and circle swims are shown in Fig. 6D. As would be expected, Naive MT Lesion rats were severely impaired on both days 1 and 2 whereas both the Pretrained MT Lesion and Pooled Control Groups produced efficient swims and did not differ on either day (day 1, F(2,27) = 46.5, p <.0001; day 2, F(2,27) = 41.0, p <.0005; Naive MT Lesion versus Pretrained MT Lesion and Pooled Control, p <.05 both days). All Groups displayed normal swimming behavior, used the hidden platform as a refuge, and had similar swim speeds (data not shown) Reversal training Search time data from the last Trial on day 2 and the first reversal Trial of day 3 are plotted separately in Fig. 6A, and indicate that both the Pretrained MT Lesion and Pooled Control Groups displayed an increase in search time on the first Trial of day 3 relative to the last Trial on day 2 (t = 2.6, p <.02), whereas the Naive MT Lesion Group did not (p >.05). During reversal training the Naive MT Lesion Group was impaired on all measures whereas the Pretrained MT Lesion Group was not impaired (search time, Group: F(2,9) = 9.3, p <.001; Trial: F(9,243) = 10.4, p <.001; time in periphery, Group: F(2,9) = 8.9, p <.001; percent time in the periphery, F(2,27) = 10.8, p <.0001; percent direct and circle swims, F(2,27) = 6.0, p <.007; Naive MT Lesion versus Pretrained MT Lesion and Pooled Control, p <.05 all measures, Fig. 6). An attempt was made to relate behavioral impairments to gross MT Lesion size but this yielded no useful relationships (all rs < 0.12, all ps > 0.05). As in experiment 1, this was likely due to the facts that every Naive MT Lesion rat was severely impaired in the task and the range of Lesion volumes was restricted. For example, day 1 individual animal mean search times ranged from 40.8 to 60 s per Trial, all extremely poor scores and well above the range of individual animal mean search times of the the Pooled Control Group ( s). Further, day 1 median and modal search times for individual Naive MT Lesion rats were all 60 s with no range in either measure, indicating that on most Trials these animals did not find the hidden platform. Similarly, the range of individual means for Naive MT Lesion rats for percent time swum in the periphery was %, and the percent of direct + circle swims was 0.0% for every rat. No Naive MT Lesion rat performed a direct or circle swim to the hidden platform on day 1 and there was no meaningful improvement in this Group s performance throughout the 3 days of spatial training, an outcome consistent with the failure of rats in the MT Lesion Group to swims directly to the visible platform on day 1 in experiment 1. With so such restricted ranges in the data of the Naive MT Lesion rats it would not be expected that a correlational analysis would yield significant outcomes. To evaluate the possible role of damage to the anterior thalamic nuclei in the impairments we further examined the histology and behavioral performance of the rat with the smallest thalamic Lesion (rat MT7, Lesion volume 4.9 mm 3 ). Illustrations of this rat s Lesion appear in Fig. 2C and indicate notably less rostral damage than found in the other rats in the Naive MT Lesion Group, with no damage to the anterior thalamic nuclei. Despite having the smallest Lesion with no damage to anterior thalamic nuclei, rat MT7 exhibited WM performance comparable to that of the worst-performing rat in the Naive MT Lesion Group (rat MT7: day 1 mean search time, 60 s; percent direct and circle swims, 0.0%; percent of time swum in periphery, 98.9%; worst rat, MT1: day 1 mean search time, 60 s; percent direct and circle swims, 0.0%; percent of time swum in periphery, 99.5%). The converse analysis of the rat with the best behavioral performance in the Naive MT Lesion Group indicated slightly better, but still extremely poor performance (rat MT6: day 1 mean search time, 40.8 s; percent direct and circle swims, 0.0%; percent of time swum in periphery, 79.7%). This rat was typical of its Group in having partial damage to the medial aspects of the anterior thalamic nuclei. Thus analysis of rats MT1, MT6, and MT7 failed to indicate an obvious association between anterior thalamic damage and behavioral impairment and instead suggested that damage to MT may be responsible the behavioral impairments. In sum, in Naive rats FF and MT Lesions impaired both behavioral strategies and place responding, whereas Pretrained FF Lesion rats used appropriate behavioral strategies but were impaired in place responding. Pretrained MT Lesion rats performed as well as controls on all measures. Histological analysis of individual rats suggested that MT, but not the anterior thalamic nuclei, may be important for acquisition of WM strategies Discussion These data provide the most extreme dissociation reported to date between the performance of rats that do versus do not know the required WM behavioral strategies prior to spatial training. Naive MT Lesion rats were unable to acquire the task and remained severely impaired throughout the 3 days of training. In contrast, the Pretrained MT Lesion rats performed well and were indistinguishable from controls. The most important outcome of experiment 2 is the identification of a brain area, MT, that is essential for acquiring WM behavioral strategies. Although the specific thalamic circuitry within the MT Lesioned area that is important for WM strategies acquisition is not known, the anterior thalamic nuclei, which were partly damaged in most Naive MT Lesion rats, may be involved because of the prominent population of head direction cells they contain [46,47]. The fact that MT Lesions impaired Naive but not Pretrained rats is consistent with the idea that head direction information may be important for initial orientation and acquisition of strategies for navigating in novel environments. However, examination of two rats in the Naive MT Lesion Group suggests that the anterior thalamic area may not be crucial for acquisition

10 250 D.P. Cain et al. / Behavioural Brain Research 170 (2006) of WM strategies. Rat MT7, which had the smallest MT Lesion, with no damage to the anterior thalamic nuclei (see Fig. 2C), had an impairment comparable to the worst-performing rat in its Group. And rat MT6, whose anterior thalamic nuclei were damaged, performed slightly better than rat MT7. Although based on limited information, the available evidence points to damage to MT, but not damage to the anterior thalamic area, as being important for producing severe WM strategies impairments in Naive rats. The fact that Pretrained FF Lesion rats used appropriate behavioral strategies but were impaired on days 1 and 2 and had no memory for the platform location on the first reversal Trial on day 3 is consistent with the widely held view that the HF is important for acquiring a place response [33,36,44]. Anovel contribution of experiment 2 was to show that, in rats confirmed to be using appropriate behavioral strategies, place responding was significantly more accurate in rats with an intact HF than in rats with an FF Lesion. This suggests that a role of HF is to maximize the accuracy of spatial memory for the location of places or objects in the environment based on distal visual cues [36]. The fact that the Naive FF Lesion rats gradually acquired both components of the task and were no longer impaired by the end of training on day 3 suggests that the HF may not be crucial for spatial navigation in a WM if sufficient training is given. The usefulness of this suggestion depends on the adequacy of FF Lesions for evaluating HF contributions to spatial navigation. This issue was studied directly by Whishaw and Jarrard [58], who concluded that FF Lesions and ibotenic acid Lesions of hippocampal cells produce very similar behavioral impairments, with FF rats slightly more impaired than ibotenic Lesioned rats. Therefore it appears that FF Lesions are adequate for evaluating the HF contribution to spatial navigation and that the HF may not be crucial for spatial navigation in a WM if sufficient training is given. In contrast to earlier research with neurotoxic Lesions of thalamus that damaged cells of the anterior thalamic nuclei but not fibers of passage [51,55], we used radiofrequency Lesions that damaged both cells and fibers in MT. The fact that the neurotoxic Lesions used previously [51,55] were centered on a different region of thalamus than our Lesions and produced a much more modest behavioral impairment than we found in our radiofrequency Lesioned Naive MT Lesion Group suggests that the severe impairment we found may have resulted from damage to either different thalamic nuclei than were Lesioned previously [51,55] or from damage to fibers of passage, or both. Analysis of data from the Naive MT Lesion Group suggests that the severe WM strategy impairments we found were associated with damage to MT and not the anterior thalamic nuclei. To clarify the MT structures involved in WM strategies acquisition, experiment 3 was designed to study the effect of damage to MT by a neurotoxic treatment that damages cells. 4. Experiment 3 spatial learning task with NMDA Lesions of thalamus Experiment 3 examined the effect of NMDA Lesions [25] of MT on acquisition of the hidden platform version of the WM. Both Pretrained and Naive Groups were again used to allow separation of strategies-learning and spatial-learning components of the task [1,31] Material and methods Subjects and experimental Groups Similar subjects as in experiments 1 and 2 were used. All rats were Naive to experimentation and no rats from the earlier experiments were used. General housing and testing conditions were as in experiments 1 and 2. Rats were randomly allocated to Groups that were given pretraining or no pretraining, and either MT Lesions or sham Lesion treatment. Groups were: Naive NMDA Lesion (n = 7), Pretrained NMDA Lesion (n = 8), Naive NMDA sham Lesion (n = 5), Pretrained NMDA sham Lesion (n = 5) Apparatus The same pool, testing room, and tracking equipment used in experiments 1 and 2 were used Pretraining Pretraining for the Pretrained Groups was carried out prior to surgery as described above for experiment Surgery NMDA Lesions were made using general surgical techniques similar to those used in experiments 1 and 2. A sterile stainless steel 30-ga injection cannula was used to stereotaxically inject NMDA into two target sites in the thalamus. With bregma and lambda on the same plane [38], coordinates with respect to bregma and dura surface were: 2.3 mm posterior, 0.0 mm lateral, 5.7 mm ventral; and 3.3 mm posterior, 0.0 mm lateral, 5.7 mm ventral. NMDA (Sigma, St. Louis, MO) was mixed in fresh PBS at a concentration of 0.12 M immediately before use. Injections were made over a 5 min period using a Sage syringe pump connected to the injection cannula with PE10 tubing with no dead space. The volume of each injection was 0.2 microliters and the cannula was left in place for 5 min after the completion of each injection. Sham controls received the same treatment as Lesioned rats up to and including drilling holes in the skull but the injection cannula was not inserted. Behavioral testing began 14 days after surgery Spatial training and analysis Spatial training, including reversal training, and behavioral analysis were as described above for experiment Immunohistochemical and data analysis. Within 48 h after completion of behavioral testing all rats were transcardially perfused under deep anesthesia with 0.9% saline followed by 4% paraformaldehyde in 0.1 M PBS at ph7.4. Brains were removed from the skull and post-fixed in the same fixative for 2 h and then placed in a 25% sucrose solution until they sank. Coronal sections 40 m thick were cut through the regions of the Lesions on a cryostat and sections were stored in PBS. Immunostaining with NeuN (Neuronal Nucleus) antibody (Chemicon) was used to verify the neurotoxic Lesion [25]. We used NeuN immunohistochemistry because, in our experience, neurotoxin-induced damage is not always evident in tissue stained with conventional Nissl stains (Sheerin and Corcoran, unpublished observations). Briefly, free-floating sections were prepared using a conventional avidin biotin immunoperoxidase technique by pretreating sections at room temperature in 0.2% H 2 O 2 in PBS for 30 min followed by rinsing in PBS. Sections were blocked in PBS-T/5% Normal Horse Serum (NHS) for 1 h and then incubated with NeuN (1:2000 in PBS-T/5% NHS) for 72 h at 4 C. Sections were rinsed and incubated with biotinylated horse anti-mouse (1:250) for 2 h and in 1:400 extravidin in PBS for an additional 2 h. The reaction product was developed using the conventional diaminobenzidine method and sections were mounted on poly-l-lysine coated slides and coverslipped with permount for later analysis. Determination of Lesion extent and volume were made using equipment and techniques described for experiments 1 and 2. Data analysis was as in experiment 2. Preliminary analysis indicated that the Naive and Pretrained NMDA sham Lesion subgroups did not differ

11 D.P. Cain et al. / Behavioural Brain Research 170 (2006) on any measure. Therefore they were collapsed into a single NMDA Control Group Results One rat in the Pretrained NMDA Lesion Group had evidence of less cell loss than the other NMDA Lesioned rats and was dropped from the experiment, leaving seven rats in the Pretrained NMDA Lesion Group. Because the behavioral impairments in the Naive NMDA Lesioned Group were less severe than in the Naive MT Lesioned Group in experiment 2, results of NeuN immunostaining are presented for both the largest and smallest Lesions in the Naive NMDA Lesion Group (see Fig. 7). NMDA Lesioned rats had evidence of loss of cells in areas comparable to areas damaged by radiofrequency Lesions in experiments 1 and 2 (see Fig. 2). The mean volume of the area of cell loss was 6.4 ± 0.97 mm 3 (range: mm 3 ). Rat NMDA10 had the smallest Lesion in the Naive NMDA Lesion Group (see Fig. 7B) and will be discussed further below. Although this rat showed evidence of cell loss in many areas of MT that were damaged in experiments 1 and 2, including parts of the mediodorsal, paraventricular, paracentral, central medial, inter- Fig. 7. Photomicrographs showing neurotoxic NMDA-induced cell damage in MT in experiment 3. Because impairment in the Naive NMDA Lesion Group was much less than in the radiofrequency-lesioned MT Groups in experiment 2, the largest (A) as well as the smallest (B) Lesions are shown. The sections in (A) are approximately at the level of plates 25, 26, 27, and 38 from top to bottom ( 1.80, 2.12, 2.56, and 3.6 mm relative to Bregma, respectively). Sections in (B) show the brain of rat NMDA10, which had the smallest Lesion in the Naive NMDA Leison Group, experiment 3. Damage in (B) does not extend as far rostrally as in (A), indicating no damage in the anterior thalamic nuclei in this rat (top panel of (B)).