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

Behavioural Brain Research 170 (2006) 241–256

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 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 b

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. © 2006 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.: +1 519 661 2111x84628; fax: +1 519 661 3961. E-mail address: [email protected] (D.P. Cain).

0166-4328/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2006.02.023

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]

242

D.P. Cain et al. / Behavioural Brain Research 170 (2006) 241–256

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 3. 2.1. Materials and methods 2.1.1. 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 12:12 h light:dark cycle for comparability with previous WM research. The rats had not been in water prior to the behavioral test

D.P. Cain et al. / Behavioural Brain Research 170 (2006) 241–256

243

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. 2.1.2. 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. 2.1.3. 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. 2.1.4. 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. 2.1.4.1. 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.

244

D.P. Cain et al. / Behavioural Brain Research 170 (2006) 241–256

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)).

2.2. 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.) mm3 (range: 5.1–7.4 mm3 ).

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

D.P. Cain et al. / Behavioural Brain Research 170 (2006) 241–256

245

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). 2.3. 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.

246

D.P. Cain et al. / Behavioural Brain Research 170 (2006) 241–256

3. 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]. 3.1. Material and methods 3.1.1. 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). 3.1.2. 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 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. 3.1.3. 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]. 3.1.4. Surgery Rats received FF Lesions, MT Lesions, or sham surgery 24–48 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. 3.1.5. 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.

3.1.6. 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. 3.1.6.1. 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.

3.2. 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 mm3 (range: 4.9–7.1 mm3 ). 3.2.1. 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]. 3.2.2. FF Lesion 3.2.2.1. 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

D.P. Cain et al. / Behavioural Brain Research 170 (2006) 241–256

247

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.

248

D.P. Cain et al. / Behavioural Brain Research 170 (2006) 241–256

(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). 3.2.2.2. 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 3. 3.3. MT Lesion 3.3.1. 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.

D.P. Cain et al. / Behavioural Brain Research 170 (2006) 241–256

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). 3.3.2. 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 (6.8–18.1 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 79.7–99.5%, 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

249

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 mm3 ). 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. 3.4. 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

250

D.P. Cain et al. / Behavioural Brain Research 170 (2006) 241–256

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]. A novel 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]. 4.1. Material and methods 4.1.1. 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). 4.1.2. Apparatus The same pool, testing room, and tracking equipment used in experiments 1 and 2 were used. 4.1.3. Pretraining Pretraining for the Pretrained Groups was carried out prior to surgery as described above for experiment 2. 4.1.4. 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. 4.1.5. Spatial training and analysis Spatial training, including reversal training, and behavioral analysis were as described above for experiment 2. 4.1.5.1. 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% H2 O2 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

D.P. Cain et al. / Behavioural Brain Research 170 (2006) 241–256 on any measure. Therefore they were collapsed into a single NMDA Control Group.

4.2. 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

251

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 mm3 (range: 4.6–8.1 mm3 ). 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)).

252

D.P. Cain et al. / Behavioural Brain Research 170 (2006) 241–256

anteromedial, gelatinosus, and rhomboid nuclei, the following areas were spared in this rat: anterior portions of the paraventricular, central medial, and dorsomedial nuclei, and the whole paratenial nucleus. Cell loss was not evident in the laterodorsal nuclei in any rat, and there was no evidence of cell loss in any control rat. 4.2.1. Pretraining Mean search time for the Pretrained rats to find the hidden platform decreased from 63.8 ± 13.6 s (mean ± S.E.M.) on the first pretraining Trial to 19.6 ± 7.4 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. 4.2.2. NMDA Lesion 4.2.2.1. Spatial training. As shown in Fig. 8, the Naive NMDA Lesion Group was mildly impaired and Pretrained NMDA Lesion rats were unimpaired relative to the NMDA Control Group. These impressions were confirmed by analysis that revealed significant differences in search time between the Naive NMDA Lesion Group and the Pretrained NMDA Lesion and NMDA Control Groups on day 2 only (day 2, Group: F(2,9) = 11.0, p < .0001; Trial: F(9,189) = 7.3, p < .0001; Naive NMDA Lesion versus Pretrained NMDA Lesion and NMDA Control, p < .05, Fig. 8A). Analyses of time in the periphery and percent time in the periphery revealed small but significant impairments in the Naive NMDA Lesion Group (time in periphery, days 1,2, Group: Fs(2,9) = 5.4, 7.3, ps = .013, .004; Trial: Fs(9,189) = 9.4, 18.3, ps = .0001; Naive NMDA Lesion versus Pretrained NMDA Lesion and NMDA Control, p < .05 both days, Fig. 8B; percent time in the periphery,

Fs(2,21) = 3.9, 6.4, ps = .04, .007; Naive NMDA Lesion versus Pretrained MT Lesion and NMDA Control, p < .05 both days, Fig. 8C). Group mean percent direct and circle swims are shown in Fig. 8D. Naive NMDA Lesion rats were impaired on day 2 only (F(2,21) = 7.5, p < .003; Naive NMDA Lesion versus Pretrained NMDA Lesion and NMDA Control, p < .05, Fig. 8C). All Groups displayed normal swimming behavior, used the hidden platform as a refuge, and had similar swim speeds (data not shown). 4.2.2.2. 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. 8A, and indicate that all Groups displayed an increase in search time on the first Trial of day 3 relative to the last Trial on day 2 (ts = 3.3–3.5, ps = .01–.02) suggesting that rats were searching for the hidden platform in its former position. During reversal training the Naive NMDA Lesion Group was impaired on time in the periphery and percent time in the periphery (time in periphery, Group: F(2,9) = 5.8, p < .001; percent time in the periphery, F(2,21) = 5.5, p < .01; Naive NMDA Lesion versus Pretrained NMDA Lesion and NMDA Control, p < .05, Fig. 8B and C). The Naive NMDA Lesion Group was impaired in percent direct and circle swims relative to the NMDA Control Group (F(2,21) = 3.6, p < .05) but did not differ from the Pretrained NMDA Lesion gtroup (Fig. 8D). To evaluate whether the Naive NMDA Lesion Group improved in strategies use and spatial memory across the three training days, one-way repeated measures ANOVAs were calculated on percent of time in the periphery and percent direct and circle swims. The Naive NMDA Lesion Group improved on both measures across training days (percent time in periph-

Fig. 8. Neurotoxic MT Lesion and control rats in the WM task, experiment 3. 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 Naive NMDA Lesion Group was mildly impaired on some measures on some days but performed as well as controls on other measures and days, and improved across days in WM strategies (percent time in the periphery) and spatial memory (percent direct and circle swims). All 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). * : 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.

D.P. Cain et al. / Behavioural Brain Research 170 (2006) 241–256

ery, F(2,10) = 19.5,l p < .001; percent direct and circle swims, F(2,10) = 4.3, p < .05). Correlations between total volume of cell loss and behavioral impairments again yielded no useful relationships (all rs < 0.21, all ps > 0.05), which was likely due to the overall good performance of the Groups and the consistency of the Lesions. The most instructive neuroanatomical outcome appears to be from rat NMDA10 of the Naive NMDA Lesion Group (see Fig. 7B). As indicated above, this rat had the least cell loss in its Group, with almost no damage to anterior thalamus. Yet this rat had mean search times on each of days 1–3 (20.2, 12.3, 8.4 s, respectively) that were comparable to the mean search times of the rest of the rats in its Group (18.2, 12.9, 9.3 s, respectively), all of which had cell loss in the anterior thalamus. This suggests that the relevant area of damage for the mild impairments found in the Naive NMDA Lesion Group was likely damage to cells in MT but not damage to cells in anterior thalamus. In sum, during spatial training Naive NMDA Lesion rats were mildly impaired but there was evidence that these rats acquired sufficient command of WM strategies to learn a considerable amount about the hidden platform location during both spatial and reversal training. Pretrained NMDA Lesion rats were unimpaired relative to controls. Results of immunostaining for cell loss suggested that MT, but not anterior thalamus, was important for acquiring WM strategies. 4.3. Discussion In contrast to results from experiment 2 obtained with radiofrequency Lesions, neurotoxic Lesions of MT produced only mild impairment in the Naive NMDA Lesion Group on some measures and days of testing. Despite this mild impairment the Naive NMDA Lesion rats acquired sufficient command of WM strategies to learn a considerable amount about the hidden platform location during both spatial and reversal training, with significant improvements in both strategies use and spatial memory across days. The fact that impairments were found in both strategies and spatial memory measures in the Naive NMDA Lesion Group together with the fact that no impairments were found in the Pretrained NMDA Lesion Group suggests that the impairment in the Naive Group was in WM strategies acquisition rather than in spatial memory. Examination of brain sections from individual rats immunostained with NeuN suggested that the impairment seen in the Naive NMDA Lesion Group was due to cell loss in MT and that damage to anterior thalamus was not necessary for the impairment to occur. Taken together the results suggest that cells in MT make a small but significant contribution to acquisition of WM strategies. 5. General discussion Experiment 1 showed that radiofrequency Lesions of MT severely impaired Naive rats in navigating efficiently from a fixed start point to a fixed visible platform but did not impair swimming behavior as such. Experiment 2 showed that the same Lesions severely impaired Naive rats in acquiring WM behavioral strategies throughout 3 days of training, but that Pretrained

253

rats given the same Lesions made effective use of the WM strategies they acquired prior to the Lesion and learned the location of a hidden platform as rapidly as controls. Experiment 3 showed that removal of MT cells by direct injection of NMDA produced only a mild impairment in Naive rats that did not prevent them from acquiring sufficient WM strategies to learn a considerable amount about the hidden platform location during both spatial and reversal training. Histological analysis of brains from the three experiments indicated that the Lesions were comparable in location and overall volume. The simplest interpretation of these results is that neural structures in MT, but not in anterior thalamus, are crucial for the acquisition of behavioral strategies needed for effective navigation in a WM [31,56] but are not required for place learning provided the rat has command of water maze strategies. Radiofrequency Lesion damage to MT specifically impaired the learning of WM strategies without causing thigmotaxis or impairing the rats’ ability to generate the movements required in the task. This was shown by the fact that the Pretrained MT Lesion Group did not swim excessively in the periphery and was not motorically impaired. Thus once WM strategies were acquired MT was no longer needed for their successful use in the task. This is the first identification of a brain area, MT, that is essential for learning behavioral strategies [26,56] 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. 5.1. Detailed behavioral analysis and inferences about learning Naive rats with FF or MT Lesions were impaired in experiment 2. As these Groups swam extensively in the periphery and often failed to contact the hidden platform as a consequence, their poor performance may be more attributable to a failure to obtain adequate information about the platform location than to a failure to remember its location. Pretrained FF Lesion rats used appropriate behavioral strategies and learned something about the platform location but nevertheless failed to match the performance of the Pooled Control or Pretrained MT Lesion Groups on days 1 and 2, indicating a place memory impairment. This inference differs from some previous conclusions about impairments in hippocampal rats, in that we infer that HF contributes an important spatial processing/place memory function as opposed to a navigational motor control function. According to the latter view, hippocampal rats are impaired in place responding due to a problem in some process of motoric control, or ‘getting there’ [57,59], and are not impaired in learning the location of a platform in relation to distal cues. In those studies rats were initially trained to swim to a stable visible platform from various starting points along the wall. For testing, the visible platform was removed and replaced by a hidden platform or no platform. Hippocampal rats swam to the vicinity of the platform location but their swim latencies were longer, they made more errors, and they gradually developed an overlapping-loop search strategy that was interpreted as a motor control problem and thought to be the main cause of the spatial impairment [57,59].

254

D.P. Cain et al. / Behavioural Brain Research 170 (2006) 241–256

In contrast, our data from experiment 2 show that both Pretrained Lesion Groups used appropriate behavioral strategies and did not develop a looping search strategy. The swim paths in Fig. 4 show that the median rat in the Pretrained FF Lesion Group was impaired because it narrowly missed finding the hidden platform on some Trials. During the last half of training on day 1, when it missed the hidden platform despite swimming in the correct general direction, the rat always made a tight turn, returned to the vicinity of the platform, and found the refuge. Group data in Fig. 5A and D for the Pretrained FF Lesion Group reflect similar events. The same figures show that the Pooled Control and Pretrained MT Lesion rats made many accurate place responses. Pretrained FF Lesion rats had command of swimming and navigation behavior (otherwise they could not have swum or performed tight turns) and appropriate behavioral strategies (otherwise they would have swum in the periphery or in loops and failed to use the hidden platform as refuge). Their navigation behavior was not random but it was not as accurate as that of controls. The impaired performance of the Pretrained FF Lesion Group seems most easily understood as impaired spatial place memory. Importantly, Fig. 4 and review of videotapes revealed no evidence of motor control impairments in this crucial Group, including no abnormal overlapping-loop search strategies as reported for hippocampal rats trained with a visible platform and tested in probe Trials with a hidden or no platform [57,59]. In contrast to this earlier work, we used a hidden platform throughout. Why would different WM training regimens with a visible or no platform versus a consistently present hidden platform produce such different behavioral outcomes, with different implications for hippocampal function? Data from the current and previous studies [3,5,6,8, 18,19,34,37,41,42] indicate that an animal’s specific initial experience in the WM can markedly affect its behavior thereafter, particularly the behavioral strategies employed in this task. In experiment 2 rats with the same Lesion behaved very differently in the same spatial training situation depending on whether they were Naive or Pretrained prior to the Lesion. Rats given training with a hidden platform, as in experiment 2, learn that there is always an unseen refuge in the pool and likely come to expect that the refuge will always be present, but hidden [30,31]. With initial training using a visible platform followed by probe tests with a hidden or no platform [57,59] the behavioral strategies adopted are likely to be different. The unpredictable presence or absence of an unseen refuge in repeated probe Trials might be expected to lead to gradual adoption of a looping search strategy to maximize the opportunity of finding refuge in the area of the pool that formerly contained the clearly visible platform. This idea is supported by evidence and discussion [57–59] that the adoption of a looping search strategy occurs gradually during the testing, that it is reinforced by this particular testing regimen, and that it contributes to the spatial reversal impairment that occurs with hippocampal rats tested in this manner. For these reasons we did not employ a visible platform during training, or probe Trials with a hidden or no platform after training. Whatever the reason for the adoption of a looping search strategy, our use of a hidden platform and direct and circle swims as a measure of spatial memory instead of no-platform probe Trials

had the advantage of using a conventional WM training regimen with direct comparability to most other WM work, under conditions consistent with the environment that rats evolved and normally live in that contains stable refuges that do not appear and disappear from moment to moment, thus avoiding the reinforcement of abnormal looping search strategies. Further, the use of the direct and circle swim measure of spatial memory allowed scoring navigation accuracy on every training Trial based on swim trajectories that were shown in experiment 1 to be normally used by rats swimming to a refuge. Therefore we conclude that the navigation impairment displayed by hippocampal rats resulted from damage to a spatial mapping mechanism, not from a problem in the motoric control of navigation behavior. 5.2. Brain mechanisms of spatial navigation Two main hypotheses of spatial navigation have been advanced: spatial mapping, which uses mainly visual information to create a cognitive map of space that allows efficient navigation [36], and path integration, which tracks self-generated movement through space [2]. In principle a rat could use either or both mechanisms but this would depend on the availability of cues, level of ambient light, training regimen, etc. Hippocampal rats tested in darkness cannot return directly to the start of a foraging foray and instead retrace the less efficient outgoing path, indicating that the HF is crucial for path integration in rats [60]. As used here, the WM allowed testing of spatial mapping ability [30], with multiple start points along the pool wall preventing the use of path integration. Our results show that Pretrained FF Lesion rats navigated to the vicinity of a hidden refuge but often were impaired in finding its exact location, and that the impairment was not the result of failure to employ appropriate behavioral strategies. Thus HF is involved in both path integration and spatial mapping, but while it appears to be essential for path integration [60] our data suggest that it is only important for maximal spatial mapping accuracy early in spatial training, and that rats with FF Lesions eventually learn to make accurate place responses whether they are Naive or Pretrained. The impairment in rats with FF Lesions is consistent with the finding that place cells in rats with these Lesions failed to follow distal cues as effectively as those of normal rats, and had lower reliability of place fields [43]. The fact that rats with FF Lesions eventually learn to make acurate place responses is consistent with evidence implicating a variety of extrahippocampal brain circuits in spatial mapping and navigation behavior [17,23,44], including extrahippocampal areas that contain place cells, such as entorhinal cortex and striatum [40,62], or head direction cells, such as thalamus and cortex [10,11,29,46,47]. Conceivably some of these areas might communicate with the HF to control navigation behavior even if the FF is severed. Comparison of data from experiments 2 and 3 indicates a striking difference in the behavioral impairment of Naive animals given the two kinds of thalamic Lesion, with the Naive MT Lesion Group remaining severely impaired throughout 3 days of training and the Naive NMDA Lesion Group showing only mild impairment on certain measures and days. Histo-

D.P. Cain et al. / Behavioural Brain Research 170 (2006) 241–256

logical analysis indicated approximately equivalent placement and overall volumes of the two kinds of Lesion. The fact that NMDA Lesions spare fibers of passage [25] whereas the heat associated with radiofrequency Lesions damages all tissue including fibers implies that fibers of passage in MT may be important for acquisition of WM strategies. Given that extensive neurotoxin-induced cell loss in MT could be expected to result in degeneration of axons arising from neurons whose cell bodies lie within the Lesioned area, the severe behavioral impairment caused by the radiofrequency Lesion damage in the Naive MT Lesion Group implies that fibers originating and terminating outside MT may be important for learning WM strategies. Leaving aside the damage to portions of the anterior thalamic nuclei in the current experiments, which was not clearly associated with behavioral impairment, the MT damage reported here largely overlaps the midline and intralaminar areas of thalamus, which have been clustered into four Groups differentiated on neuroanatomical and functional grounds [48]. Results from experiment 3 pointing to a modest contribution of MT cells to acquisition of WM strategies are consistent with many of the functions associated with these Groups, such as sensory processing and cognitive functions. However, as damage occurred in at least three of these four neuroanatomical Groups in most Lesioned rats it is not yet possible to usefully interpret the impairment in the Naive NMDA Lesion Group in terms of a particular neuroanatomical Group within the midline and intralaminar thalamus [48]. To date there has been no research directed at the role of individual nuclei or fiber pathways in MT in studies of spatial navigation in which behavior was studied in sufficient detail to allow distinctions between impairments in WM strategies acquisition versus place memory. Although the mammilothalamic tract is associated with memory function in both animal [52] and human clinical research, this fiber pathway was undamaged in most of the rats that were severely impaired in the Naive MT Lesion Group (experiment 2, Fig. 2). A study of subregions within the midline and intralaminar thalamus found that small electrolytic Lesions that damaged the central lateral nucleus together with at least 50% of the anteroventral and anterodorsal thalamic nuclei were without effect in Naive rats trained in the WM [21], which is consistent with our conclusion that damage to anterior thalamus is not essential for the impairments seen in our Naive MT Lesion rats. In the same study more posterior electrolytic Lesions that damaged portions of both the midline and intralaminar areas produced a modest WM impairment in Naive rats, but no assessment of WM strategies versus spatial memory impairments was made. Specific neurotoxic Lesions of nucleus reuniens failed to result in any WM impairment [15]. Additional research using small neurotoxic Lesions aimed at individual nuclei or neuroanatomical Groups, or small radiofrequency Lesions of specific fiber pathways, together with a detailed behavioral analysis, is needed to identify specific structures important for acquisition of WM strategies. Further studies exploring the role of any structures so identified in the acquisition of other behavioral tasks would be useful. Our data suggest that these approaches should be directed to structures contained within a small area of MT centered on the midline and intralaminar areas.

255

Acknowledgements Supported by grants from the Natural Science and Engineering Research Council of Canada to DPC and to MEC. We thank S. MacDougall-Shackleton for helpful comments and Ken Wolfe for technical assistance. References [1] 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. [2] Barlow JS. Inertial navigation as a basis for animal navigation. J Theor Biol 1964;6:76–117. [3] Beiko J, Candusso L, Cain DP. The effect of nonspatial water maze pretraining in rats subjected to serotonin depletion and muscarinic receptor antagonism. Behav Brain Res 1997;88:201–11. [4] 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. [5] Cain DP. Testing the NMDA, long-term potentiation, and cholinergic hypotheses of spatial learning. Neurosci Biobehav Rev 1998;22:181–93. [6] 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. [7] 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. [8] 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. [9] Cain DP, Saucier D, Hargreaves EL, Wilson E, deSouza JF. Polypropylene pellets as an inexpensive reusable substitute for milk in the Morris milk maze. J Neurosci Meth 1993;49:193–7. [10] Calton JL, Stackman RW, Goodridge JP, Archey WB, Dudchenko PA, Taube JS. Hippocampal place cell instability after Lesions of the head direction cell network. J Neurosci 2003;23:9719–31. [11] Chen LL, Lin LH, Green EJ, Barnes CA, McNaughton BL. Head direction cells in the rat posterior cortex. I. Anatomical distribution and behavioral modulation. Exp Brain Res 1994;101:8–23. [12] Clark RE, Broadbent NJ, Squire LR. Hippocampus and remote spatial memory in rats. Hippocampus 2005;15:260–72. [13] de Bruin JPC, Moita MP, de Brabander HM, Jooster R. Place response learning of rats in a Morris water maze: differential effects of fimbria–fornix and medial prefrontal cortex Lesions. Neurobiol Learn Mem 2001;75:164–78. [14] de Quervain DJ, Roozendaal B, McGaugh JL. Stress and glucocorticoids impair long-term spatial memory. Nature 1998;394:787–90. [15] Dolleman-van der Weel MJ, Morris RGM, Witter MP. Ibotenate Lesions of the nucleus reuniens thalami fail to impair spatial learning and memory. Soc Neurosci Abstr 1994;20:1212. [16] Gaffan EA, Bannerman DM, Warburton EC, Aggleton JP. Rats’ processing of visual scenes: Effects of Lesions to fornix, anterior thalamus, mamillary nuclei or the retrohippocampal region. Behav Brain Res 2001;121:103–17. [17] Harker KT, Whishaw IQ. Impaired spatial performance in rats with retrosplenial Lesions: Importance of the spatial problem and the rat strain in identifying Lesion effects in a swimming pool. J Neurosci 2002;22:1155–64. [18] Hoh TE, Cain DP. Complex behavioral strategy and reversal learning in the water maze without NMDA receptor-dependent long-term potentiation. J Neurosci 1999;19(RC2):1–5. [19] Hoh TE, Kolb B, Eppel A, Vanderwolf CH, Cain DP. Role of the neocortex in the water maze task in the rat: a detailed behavioral and Golgi-Cox analysis. Behav Brain Res 2003;138:81–94.

256

D.P. Cain et al. / Behavioural Brain Research 170 (2006) 241–256

[20] Holscher C. Stress impairs performance in spatial water maze learning tasks. Behav Brain Res 1999;100:225–35. [21] Leljeli M, Strazielle C, Caston J, Lallonde R. Effects of centrolateral or medial thalamic Lesions on motor coordination and spatial orientation in rats. Neurosci Res 2000;38:155–64. [22] Keith JR, Galizio M. Acquisition in the Morris swim task is impaired by a benzodiazepine but not an NMDA antagonist: a new procedure for distinguishing acquisition and performance effects. Psychobiology 1997;25:217–28. [23] Kolb B, Buhrmann K, McDonald R, Sutherland RJ. Dissociation of the medial prefrontal, posterior parietal, and posterior temporal cortex for spatial navigation and recognition memory in the rat. Cereb Cortex 1994;4:664–80. [24] Kolb B, Pittman K, Sutherland RJ, Whishaw IQ. Dissociation of the contributions of the prefrontal cortex and dorsomedial thalamic nucleus to spatially guided behavior in the rat. Behav Brain Res 1982;6:365–78. [25] Koo JW, Han JS, Kim JJ. Selective neurotoxic Lesions of bilateral and central nuclei of the amygdala produce differential effects on fear conditioning. J Neurosci 2004;24:7654–62. [26] Krechevsky I. A study of the continuity of the problem solving process. Psychol Rev 1938;45:107–33. [27] McEwen BS, Sapolsky RM. Stress and cognitive function. Curr Opin Neurobiol 1995;5:205–16. [28] Miller VM, Best PJ. Spatial correlates of hippopocampal unit activity are altered by Lesions of the fornix and entorhinal cortex. Brain Res 1980;194:311–23. [29] Mizumori SJY, Williams JD. Directionally selective mnemonic properties of neurons in the lateral dorsal nucleus of the thalamus of rats. J Neurosci 1993;13:4015–28. [30] Morris RGM. Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Meth 1984;11:47–60. [31] Morris RGM. Synaptic plasticity and learning: Selective impairment of learning in rats and blockade of long-term potentiation in vivo by the Nmethyl-d-aspartate receptor antagonist AP5. J Neurosci 1989;9:3040–57. [32] Morris RGM, Anderson E, Lynch GS, Baudry M. Selective impairment of learning and blockade of long-term potentiation by an N-methyl-daspartate receptor antagonist, AP5. Nature 1986;319:774–6. [33] Morris RGM, Garrud P, Rawlins JNP, O’Keefe J. Place navigation impaired in rats with hippocampal Lesions. Nature 1982;297:681–3. [34] Morris RGM, Inglis J, Upstairs/Downstairs revisited: The pretraining rescue of spatial learning during blockade of NMDA receptors is hippocampally mediated. Soc Neurosci Abstr, 2003;29:717.7. [35] Morris RGM, Schenk F, Tweedie F, Jarrard LE. Ibotenate Lesions of hippocampus and/or subiculum: dissociating components of allosteric spatial learning. Eur J Neurosci 1990;2:1016–28. [36] O’Keefe J, Nadel L. The hippocampus as a cognitive map. London: Oxford; 1978. [37] Otnaess MK, Brun VH, Moser MB, Moser EI. Pretraining prevents spatial learning impairment after saturation of hippocampal long-term potentiation. J Neurosci 1999;19(RC29):1–5. [38] Paxinos G, Watson C. The rat brain in stereotaxic coordinates. New York: Academic; 1986. [39] Perrot-Sinal TS, Kosteniuk MA, Ossenkopp KP, Kavaliers M. Sex differences in the Morris water maze and the effects of initial nonstationary hidden platform training. Behav Neurosci 1996;110:1309–20. [40] Quirk GJ, Muller RU, Kubie JL, Ranck Jr JB. The positional firing properties of medial entorhinal neurons: description and comparison with hippocampal place cells. J Neurosci 1992;12:1945–63. [41] Saucier D, Cain DP. Spatial learning without NMDA receptor-dependent long-term potentiation. Nature 1995;378:186–1189. [42] Saucier D, Hargreaves EL, Boon F, Vanderwolf CH, Cain DP. Detailed behavioral analysis of water maze acquisition under systemic NMDA or muscarinic antagonism: nonspatial pretraining eliminates spatial learning deficits. Behav Nerurosci 1996;110:103–16.

[43] Shapiro ML, Simon DK, Olton DS, Gage FH, Nilsson O, Bjorklund A. Intrahippocampal grafts of fetal basal forebrain tissue alter place fields in the hippocampus of rats with fimbria–fornix Lesions. Neuroscience 1989;32:1–18. [44] Sutherland RJ, Kolb B, Whishaw IQ. Spatial mapping: definitive disruption by hippocampal or medial frontal cortical damage in the rat. Neurosci Lett 1982;31:271–6. [45] Sutherland RJ, Rodriguez AJ. The role of the fornix/fimbria and some related structures in place learning and memory. Behav Brain Res 1989;32:265–77. [46] Taube JS, Muller RU, Ranck Jr JB. Head direction cells recorded from the postsubiculum in freely moving rats. I. Description and quantitative analysis. J Neurosci 1990;10:420–35. [47] Taube JS. Head direction cells recorded in the anterior thalamic nuclei of freely moving rats. J Neurosci 1995;15:70–86. [48] van der Werf YD, Witter M, Groenewegen HJ. The intralaminar and midline nuclei of the thalamus. Anatomical and functional evidence for participation in processes of arousal and awareness. Brain Res Rev 2002;39:107–40. [49] Vanderwolf CH. Cerebral activity and behavior: control by central cholinergic and serotonergic systems. Int Rev Neurobiol 1988;30:225–340. [50] Vanderwolf CH, Penava D. Potentiation of the effects of antimuscarinic drugs on behavior by serotonin depletion: specificity and relation to learning and memory. In: Levin ED, Decker MN, Butcher LL, editors. Neurotransmitter interactions and cognitive function. Boston: Birkhauser; 1992. p. 257–76. [51] van Groen T, Kadish I, Wyss JM. Role of the anterodorsal and anteroventral nuclei of the thalamus in spatial memory in the rat. Behav Brain Res 2002;132:19–28. [52] Vann SD, Aggleton JP. Evidence of a spatial encoding deficit in rats with Lesions of the mammillary bodies or mammillothalamic tract. J Neurosci 2003;23:3506–14. [53] Warburton EC, Aggleton JP. Differential deficits in the Morris water maze following cytotoxic Lesions of the anterior thalamus and fornix transection. Behav Brain Res 1999;98:27–38. [54] Warburton EC, Baird A, Morgan A, Muir JL, Aggleton JP. The conjoint importance of the hippocampus and anterior thalamic nuclei for allocentric spatial learning: Evidence from a disconnection study in the rat. J Neurosci 2001;21:7323–30. [55] Warburton EC, Morgan A, Baird AL, Muir JL, Aggleton JP. Does pretraining spare the spatial deficit associated with anterior thalamic damage in rats? Behav Neurosci 1999;113:956–67. [56] Whishaw IQ. Dissociating performance and learning deficits on spatial navigation tasks in rats subjected to cholinergic muscarinic blockade. Brain Res Bull 1989;23:347–58. [57] Whishaw IQ, Cassel JC, Jarrard LE. Rats with fimbria–fornix Lesions display a place response in a swimming pool: a dissociation between getting there and knowing where. J Neurosci 1995;15:5779–88. [58] Whishaw IQ, Jarrard LE. Similarities versus differences in place learning and circadian activity in rats after fimbria–fornix section or ibotenate removal of hippocampal cells. Hippocampus 1995;5:595–604. [59] Whishaw IQ, Jarrard LE. Evidence for extrahippocampal involvement in place learning and hippocampal involvement in path integration. Hippocampus 1996;6:513–24. [60] Whishaw IQ, Maaswinkle H. Rats with fimbria–fornix Lesions are impaired in path integration: A role for the hippocampus in sense of direction. J Neurosci 1998;18:3050–8. [61] Whishaw IQ, Tomie J. Perseveration on place reversals in spatial swimming pool tasks: Further evidence for place learning in hippocampal rats. Hippocampus 1997;7:361–70. [62] Wiener SI. Spatial and behavioral correlates of striatal neurons in rats performing a self-initiated navigation task. J Neurosci 1993;13: 3802–17.