Excitotoxic lesions of the rostral thalamic reticular nucleus do not affect the performance of spatial learning and memory tasks in the rat

Transcription

1 Behavioural Brain Research 120 (2001) Excitotoxic lesions of the rostral thalamic reticular nucleus do not affect the performance of spatial learning and memory tasks in the rat L.A.K. Wilton, A.L. Baird, J.L. Muir, J.P. Aggleton * School of Psychology, Cardiff Uni ersity, PO Box 901, CardiffCF10 3YG, UK Received 4 September 2000; received in revised form 10 November 2000; accepted 10 November 2000 Abstract Rats with cytotoxic lesions of the rostral pole of the thalamic reticular nucleus were compared with surgical control animals on a series of spatial learning and memory tests. While evidence was found for an initial, transient impairment on forced-choice alternation in a T-maze, this rapidly disappeared, and overall performance was unaffected. Subsequent experiments found no evidence that lesions of the rostral reticular nucleus affected the acquisition or performance of tests in the radial arm maze and the Morris water maze. Thus, it appears that the rostral pole of the thalamic reticular nucleus often does not play a necessary role in the performance of tests of spatial learning and memory, in spite of its interconnections with other regions that are required for normal spatial memory Elsevier Science B.V. All rights reserved. Keywords: Anterior thalamic nuclei; Attention; Rat; Spatial memory; Thalamic reticular nucleus 1. Introduction Pathology in the rostral thalamus has been linked to the development of anterograde amnesia in humans [1,16]. The rostral thalamus is, however, a complex region containing the confluence of many nuclei and tracts. As part of a series of studies comparing the contribution of different rostral thalamic nuclei to aspects of memory in rats, the present study examined the effects of rostral reticular nucleus lesions on tests of spatial memory. The thalamic reticular nucleus (TRN) surrounds much of the lateral margin of the thalamus, and it is through this nucleus that many fibres travelling either way between the thalamus and the cortex pass. Some of the fibres travelling through the TRN from both the thalamus and the cortex branch to form excitatory, glutamatergic synapses with TRN cells, which in turn send inhibitory GABAergic fibres back to the thalamus [7]. Thus, the TRN receives two excitatory * Corresponding author. Tel.: ; fax: address: aggleton@cardiff.ac.uk (J.P. Aggleton). inputs, those from the cortex and those from the thalamus [9], and sends GABAergic inhibitory projections back to thalamic nuclei. For this reason, this nucleus appears to be in a position to control the activity of the thalamo-cortical axis [9]. There is considerable evidence that the TRN is divided into several distinct sectors, each related to a particular functional group of thalamo-cortical pathways. It has been proposed, for example, that the more caudal TRN plays a role in selective attention via its interactions between sensory nuclei of the dorsal thalamus and respective cortical areas [19]. A different sector of the TRN, that has not been extensively investigated, is its rostral pole, which has dense reciprocal connections with the anterior thalamic nuclei [6,9]. In addition, this part of the TRN, like the anterior thalamic nuclei, receives inputs from the retrosplenial cortex [8]. Due to the dense connections that the rostral pole of the TRN has with the anterior thalamic nuclei, it is possible that this sector may play a role in learning and memory. The anterior thalamic nuclei have been shown to be especially important for the performance of spatial memory tasks by rats [2 4,15,18], and it is there /01/$ - see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S (00)

2 178 L.A.K. Wilton et al. / Beha ioural Brain Research 120 (2001) fore noteworthy that M Harzi et al. [10] and Collery et al. [5] reported that ibotenate lesions of the rostral pole of the TRN can also impair spatial memory performance in the radial arm maze and T-maze, respectively. These findings are not, however, conclusive as in both studies, some of the reticular nucleus lesions also involved the adjacent anterior ventral nucleus. This is potentially important as even subtotal lesions of the anterior thalamic nuclei can disrupt spatial memory tests [2,4]. Thus, in the present study, we re-examined the role of this area in spatial learning and memory, taking special care to restrict our lesions to the region of the rostral TRN. The lesions were made by injecting N-methyl-D-aspartic acid (NMDA) in order to limit damage to fibres of passage. The study used tasks known to be sensitive to lesions of the anterior thalamic nuclei. The spatial forcedchoice task (delayed non-matching to place) in the T-maze has been shown to be affected by both complete [3,18] and selective [2,4,20] lesions within the anterior thalamic nuclei. Likewise, the standard reference memory version of the Morris water maze [11] has repeatedly been shown to be sensitive to anterior thalamic lesions [14,17]. The final behavioural task used a radial arm maze to test spatial working memory, and was similar to that used by M Harzi et al. [10]. Again, this task is known to be sensitive to both complete anterior thalamic lesions and to selective lesions within the group of nuclei [2,4]. 2. Methods 2.1. Subjects The study involved 36 naïve, male rats of the Lister Hooded strain (Harlan-Olac, Bicester, UK). Throughout the period of the experiment, the rats were housed in pairs under diurnal conditions (14 h light/10 h dark). At the time of surgery, they were aged around 4 months and weighed between 280 and 360 g. All animals were given a minimum of 2 weeks to recover from surgery before the first test began. Prior to training on the T-maze alternation task, the animals were food-deprived and maintained at 85% of their normal body weight by restricted access to food. For the water-maze task, the animals were placed on ad-lib food. Those animals that were tested on the radial maze task were again food-deprived and maintained at 85% of their normal weight for the duration of testing. Throughout the study, all animals had unrestricted access to water. All experiments were conducted in accordance with the United Kingdom Animals (Scientific Procedures) Act (1986) Surgical and histological procedures A total of 36 rats were randomly divided into two surgical groups. These groups were surgical controls (SHAM, n=15), and cytotoxic lesions of the rostral reticular thalamic nuclei (TRNX, n=21). Each of the 36 animals was deeply anaesthetized by intraperitoneal injection of pentobarbitone sodium (Sagatal) at a dose of 60 mg/kg. The animal was then placed in a stereotaxic headholder (David Kopf Instruments, Tujunga, CA), with the incisor bar set at +5.0 to the horizontal plane, and the scalp retracted to expose the skull. A craniotomy was made above the sagital sinus and the dura cut to expose the cortex above the target region. For the animals receiving lesions of the reticular thalamic nucleus, injections of 0.12 M N-methyl-D-aspartic acid (NMDA) (Sigma Chemicals, Poole) dissolved in phosphate buffer (ph 7.2) were made through a 1 l Hamilton syringe into two sites in each hemisphere. The stereotaxic co-ordinates relative to bregma were AP-0.2, Lat 2.4, with 0.2 l of NMDA injected, and AP-0.4, Lat 2.8, with 0.1 l of NMDA injected. The height of all of the injection sites was 6.2 mm below the top of cortex at the injection site. Each injection was made gradually over a 3 min period, and the needle was left in situ for a further 4 min before being withdrawn. The surgical control animals (SHAM) received a craniotomy that corresponded with that required for the TRN surgeries. The dura was then cut to expose the dorsal cortex and a Hamilton syringe lowered to the TRN co-ordinates, but no injection was made. On completion of all of the surgeries, the skin was sutured and a topical antibiotic (Aureomycin) powder applied. The rats also received 5 ml of glucose/saline as fluid replacement. Following completion of the experiment, subjects were anaesthetized with Euthatal and transcardially perfused with saline followed by 10% formol-saline. The brain was removed and post-fixed in formol-saline overnight, before being transferred into a 25% sucrose solution and again left overnight. Coronal sections were cut at 60 m on a freezing microtome and stained with cresyl-violet, a Nissl stain. Five animals (three TRNX, two SHAM) were not perfused directly following the completion of behavioural testing but were returned to surgery for injection of the retrograde tracer, fluorogold, into the anterior thalamic nuclei. If there were intact projections from the TRN to the anterior thalamus, injections of fluorogold into the anterior thalamic nuclei would result in fluorescence staining in the TRN. By this means, the extent to which the lesions overlapped with the area of the TRN supplying the anterior thalamic nuclei can be verified. Surgical procedures were as before, and the animals received two injections of 0.14 l of 4% fluorogold (Fluorochrome Inc., Denver, CO) solution

3 L.A.K. Wilton et al. / Beha ioural Brain Research 120 (2001) per hemisphere. The stereotaxic co-ordinates were, from Ear Bar Zero, AP +4.9, Lat 1.0, and Depth +4.6, and AP +4.9, Lat 1.6, and Depth Following injection of the fluorogold, the animals were returned to their home cage for 5 days, before perfusion as described above. Coronal sections were cut at 60 m on a freezing microtome and alternate sections stained with cresyl-violet, or cover-slipped without staining for fluorescence microscopy T-maze tasks Apparatus The floors of the T-maze were 10 cm wide and made of wood painted white. The stem of the T-maze was 70 cm long with a guillotine door located 25 cm from the beginning. Closure of this door created a start box. The cross piece of the T-maze was 140 cm long, and at each end, there was a food well 2 cm in diameter and 0.75 cm deep. The walls of the maze were 17 cm high and made of clear Perspex. The maze was supported on two stands 94 cm high. Lighting was provided by a fluorescent light suspended 164 cm above the apparatus Spatial forced alternation Procedure Pretraining. After post-operative recovery, the animals were habituated to the T-maze. Each animal received three 5 min sessions during which reward pellets were scattered throughout the maze, and the animals were allowed to explore freely. By the end of habituation, the animals would reliably run down the stem of the maze to find food pellets in both arms Acquisition. Following habituation the animals received 12 sessions of acquisition training. During each session, six trials were presented with an inter-trial interval (ITI) of, approximately, 4 min. Each trial consisted of two stages. In the first stage ( sample run ), three food pellets (45 mg Campden Instruments, Loughborough) were placed in each food well, and a metal barrier was used to restrict access to one of the arms. As a consequence, the animal was forced to enter a pre-selected arm on each sample run and then allowed to eat the food there. The animal was then picked up and confined in the start box for a delay of 15 s, during which the metal barrier was removed. During the test run, the door to the start box was opened and the animal allowed a free choice between the two arms of the T-maze. The criteria for selecting an arm consisted of the rat placing a back foot in one of the arms. No retracing by the rat was permitted. If the rat had alternated (i.e. had entered the arm not previously visited on the sample run ), it was allowed to eat the food reward before being returned to its cage. If the other arm was chosen (i.e. the same arm as visited on the sample run ), the rat was confined to that arm for approximately 10 s and then returned to its cage Mass trials spatial forced alternation During this phase of testing, the animals received 12 trials per session. Training was similar to acquisition, except that now the intertrial interval between the test run of one trial, and the sample run of the following trial was approximately 15 s. All 12 trials were run consecutively for each rat. The animals received three sessions of such testing, i.e. with 15 s between sample and test runs, and 15 s between test run and next sample run. Following this, they received another three sessions of mass trials testing, but with two different delays interpolated between the sample and test phases of each trial. For these sessions, the rat was confined to the start box after the sample run for either no delay ( 0 s) or for a delay of 30 s, after which the door rose. During each 12-trial session, there were equal numbers of the two delays intermixed in a pseudo-random fashion Continuous alternation For this final task in the T-maze, the rats received two sessions of 12 trials per session, with each trial consisting of a single run in the maze. The initial sample run was a free choice of both arms, both of which were baited. Following choice of one of the arms, the rat was returned to the start box, and allowed a second test run after an inter-trial interval of approximately 15 s. The rat was always rewarded for alternating (i.e. entering the opposite arm to that entered on the previous trial) irrespective of whether the previous arm choice was correct or incorrect. Thus, if an animal made an error (e.g. turned right on successive trials), the rat would still rewarded for alternating on its next run (turn left on next trial) Water maze Apparatus The water maze was a circular pool (painted white, 2.0 m in diameter, 0.6 m high) constructed from fibreglass. The pool contained water that was maintained at a temperature of 26 1 C. The water was made opaque by the addition of three pints of semi-skimmed milk. The pool was situated in the centre of a room that contained prominent visual cues, such as posters on the wall, curtains, a screen, and a door. During testing in the water maze, a platform, 10 cm in diameter, was located 1 cm below the water in one of

4 180 L.A.K. Wilton et al. / Beha ioural Brain Research 120 (2001) four locations in the pool, approximately, 50 cm from the side walls. A video-camera was mounted to the ceiling above the pool and was connected to a videorecorder and tracking device (HVS Image analysing Ltd., Kingston, UK), which permitted on- and offline automated tracking of the path taken by the rat. The resulting x and y co-ordinates were digitized by a RISC PC computer, and stored on disc Procedure The animals received four trials per session. For each trial, the animal was placed in the pool facing the wall at one of eight pseudo-randomly assigned start positions. The rats were divided into four groups, each with approximately equal numbers of animals from Groups SHAM and TRNX, and were trained to locate the hidden escape platform, which remained in a fixed location throughout testing. Each of the four groups was trained to a different platform location. Trials lasted a maximum of 120 s, with a 30 s ITI. The latency and path length to find the submerged platform were recorded. On the rare occasions when the animals did not locate the platform within 120 s, the experimenter guided them onto the platform. The animals were tested in this way for 6 days, after which they received a probe trial. For the probe trial, the platform was removed from the pool and the animal released from the quadrant opposite to where the platform would have been located. The length of the trial was 60 s, after which the rat was removed from the pool. The proportion of time the rat spent searching for the platform in the training quadrant, i.e. the previous location of the platform, was recorded, and used as a measure of retention Radial arm maze A subset of animals (TRNX, n=14; SHAM, n=8) was tested on a radial arm maze task following completion of the watermaze test. Testing was carried out in an eight-arm radial maze made of wood. This apparatus was of standard design, but all of the arms had walls made of clear Perspex. The walls ensured that the rats could not cross from one to another without entering the central, octagonal arena. Each arm was 87 cm long and 10 cm wide, with the side walls being 24 cm high. At the end of each arm was a food well 2 cm in diameter and 0.5 cm deep. Each arm led through a clear Perspex guillotine door 12 cm high to a central octagonal arena, 24 cm in diameter. Each of the guillotine doors had strings attached, allowing the experimenter to open the doors either individually or simultaneously. The arms of the maze were mounted on a turntable so that they stood 53 cm off the ground. The turntable enabled the maze to be rotated through 360. The floor of the testing room was marked so that the position of the maze could be standardized in relation to the room cues. The radial arm maze was placed in a different room to that used for the previous experiments. Lighting was provided by fluorescent lights 165 cm above the maze. The animals were habituated for 3 days, and each animal was placed into the maze for 10 min. During this time, all the guillotine doors were raised, and the animals allowed to explore the maze throughout which food pellets had been scattered. Formal training, which consisted of two stages, lasted for 14 sessions. In Stage 1 (sessions 1 10), all eight arms were baited with two 45 mg reward pellets (Campden Instruments, Loughborough). The animal was placed in the central arena, and all the doors were simultaneously raised. When the animal had made a choice by entering an arm, all doors were lowered and the animal allowed to eat the pellets. An animal was considered to have made a choice when it placed a back foot in the arm. No retracing by the rats was allowed. Once the animal had eaten the pellets, the doors were again opened and the animal allowed to return to the central arena, where it was confined for 10 s, after which the doors were again opened, and another choice of arm was made. This procedure was repeated until all eight arms had been visited, or 10 min had elapsed. The final four sessions (Stage II) were designed to control for odour cues and to test for allocentric strategies. The start of each session was as before, but now, after the animal had made four choices, it was removed from the maze and returned to the carrying box for 25 min. During this period, the maze was rotated by 45, and the arms in those positions that had not been visited were baited. This meant that the baited (unvisited) arms were still in the same place with regard to room cues, but the actual arms had changed. As a consequence, the performance of a rat using allocentric cues would be unaffected by the rotation, but the performance of a rat using odour cues would be disrupted. Following the 25 min delay, the rat was placed back into the central arena and the standard testing procedure continued until the animal had visited all of the remaining baited arms Acti ity After completion of the above tests, all rats were placed in novel test cages (30 cm 52 cm 18.5 cm) for 2 h in a novel room. Total activity was measured using pairs of photobeams (Paul Fray Limited, Cambridge, UK).

5 L.A.K. Wilton et al. / Beha ioural Brain Research 120 (2001) Results 3.1. Histological analyses Seven animals were excluded from the reticular lesion group following histological analysis. One animal was excluded as it had bilateral anterior thalamic damage, and the other six were excluded as they had sparing of the rostral reticular nucleus either unilaterally or bilaterally. Fig. 1 illustrates the largest and smallest lesions included in the study. Of these 14 animals, there was unilateral damage to part of the anteroventral (AV) thalamic nucleus adjacent to TRN in two animals, and in two others, unilateral damage to the rostro-lateral portion of the lateral dorsal thalamic nucleus. There was ventral anterior complex damage in four animals, including the two with AV damage. In three animals, there was slight bilateral sparing at the dorsal-most region of the TRN. In three others, similar sparing was seen unilaterally, with one animal showing contralateral sparing at the caudal extent of the rostral TRN. The final groups for behavioural analysis were Group SHAM (n=15) and Group TRNX (n=14) Fluorogold staining Following fluorogold injections into the anterior thalamus, fluorescence microscopy revealed retrograde staining of many neurons in the rostral TRN in both animals from Group SHAM (see Fig. 2). Of the three animals from group TRNX that were injected with fluorogold, two were amongst those rejected, as one had unilateral sparing, and the second had bilateral TRN sparing. However, in the remaining TRNX animal, fluorogold staining was largely absent from the rostral TRN area, consistent with the loss of those neurons that project to the anterior thalamic nuclei. The lesion in this case was typical of those in the final TRNX group. This striking difference in the number of labelled cells in the TRN in the SHAM and TRNX animals is shown in Fig Beha iour T-maze Spatial forced alternation: acquisition. Fig. 3 illustrates the performance of the two groups on acquisition of the T-maze spatial forced-alternation task. Analysis of variance (ANOVA) revealed that there was no significant difference between the groups [F(1,27) = 2.01, P 0.1]. There was a significant effect of session [F(11,297)=1.84, P 0.05], as animals improved on Fig. 1. Diagrammatic reconstructions showing the cases with the largest (grey) and smallest (black) lesions in the reticular lesion group (TRNX) plotted onto standard coronal sections. The numbers correspond to the distance (mm) from bregma [13].

6 182 L.A.K. Wilton et al. / Beha ioural Brain Research 120 (2001) Fig. 2. Photomicrographs of representative examples of fluorogold staining in the region of the rostral reticular nucleus in the SHAM group (A) and in the TRNX group (reticular lesions; B) following injections of fluorogold into the anterior thalamic nucleus. Sections are cut coronally at 60 m, and the scale bar corresponds to 200 m. the task, but there was no significant interaction between these factors [F(11,297)=1.67, P 0.05]. Although there was no significant interaction, inspection of the data suggested a transient deficit at the onset of acquisition (Fig. 3), and simple effects indicated group differences for sessions 1 and 4. In view of previous experiments that have shown impaired performance of spatial tasks following TRN lesions, this possible transient effect was investigated. Post-hoc comparisons of performance were conducted on the first four sessions and revealed a significant main effect of group [F(1,27)=7.97, P 0.01]. To investigate this effect further, the TRNX group was sub-divided into those with the largest lesions, and those with the smallest lesions (n=7 for both groups). This analysis revealed that there was no significant difference between the two TRNX groups [F(1,12)=3.57, P 0.08], although the animals with larger lesions tended to perform more poorly Spatial forced alternation: massed trials. The performance of the animals on the spatial alternation task run with massed trials over three sessions (12 trials per session) did not differ between the two groups (mean number of trials correct per session: TRNX= 10.4; SHAM=10.4). ANOVA confirmed that there was no significant difference between the groups (F 1), no significant effect of session (F 1), and no significant interaction between group and session (F 1). The interpolation of delays, as illustrated in Fig. 4, did not result in any significant differences in perfor- Fig. 3. T-maze performance: mean number of trials correct in each session for the SHAM and reticular lesioned (TRNX) groups. Each session was composed of six trials, and chance is three correct. Error bars indicate the S.E.M.

7 L.A.K. Wilton et al. / Beha ioural Brain Research 120 (2001) analyses revealed that the amount of time spent in the training quadrant was significantly different from the other three quadrants (P 0.01). There was no significant interaction between group and quadrant (F 1). Fig. 4. Massed trials spatial forced alternation with delays: mean number of trials correct for the SHAM and reticular lesioned (TRNX) groups out of a maximum of 18. Error bars indicate the S.E.M Radial maze. A subgroup of animals (eight SHAM and nine TRNX after histological confirmation) were trained on this test of spatial working memory. Analysis of performance on Stage I, as measured by the number of correct choices in the first eight (acquisition; Fig. 6A), revealed an effect of session [F(9,135)=8.36, P ] but no significant difference between the two groups [F(1,15) =2.10, P 0.1] and no interaction between session and group (F 1). Similarly, an analysis of performance mance of the two groups (F 1), although there was a highly significant effect of delay [F(1,27) =14.8, P=0.0007]. There was no significant interaction between group and delay (F 1) Continuous alternation. This condition did not appear to distinguish the two groups (mean number of trials correct from a maximum of 24: TRNX= 17.6; SHAM=17.4). ANOVA confirmed the impression that there was no significant difference between the groups (F 1), no significant effect of session (F 1), and no significant interaction between these factors (F 1) Water maze. The performance of animals on the water maze task is illustrated in Fig. 5A and B. ANOVA confirmed that there were no significant overall differences between the escape latencies of the two groups for acquisition [F(1,27)=1.41, P 0.2]. While there was the expected effect of session (F(5, 135)=76.9, P ), there was no interaction between session and group [F(5,135)=1.40, P 0.2]. Further analyses of the path lengths and swim speeds revealed no differences between the groups [path length, F(1,27)=1.57, P 0.2; swim speed, F(1,27)= 1.25, P 0.2]. Analysis of the performance on the probe trial (Fig. 5B), comparing the percentage of time spent swimming in the training quadrant, again revealed no significant differences between the groups (F 1). There was, however, a significant effect of quadrant [F(3,81)=25.5, P ], with both groups spending a considerably greater proportion of the probe trial searching in the training quadrant. Post-hoc Fig. 5. Water maze performance. (A) Acquisition: mean escape latencies for the SHAM and reticular lesioned (TRNX) groups. Each session was composed of four trials. Error bars indicate the S.E.M. (B) Probe trial performance: mean percentage time of probe trial spent swimming in the training quadrant for the SHAM and reticular groups. Error bars indicate the S.E.M. Abbreviations: Adj L, quadrant adjacent to the training quadrant on the left; Adj R, adjacent right quadrant; Tr, Training quadrant; Opp, opposite quadrant.

8 184 L.A.K. Wilton et al. / Beha ioural Brain Research 120 (2001) Acti ity. Analysis of the total activity revealed no significant differences between the two groups (F 1). Fig. 6. Radial arm maze performance. (A) Acquisition: mean number of correct choices in the first eight choices for the SHAM and reticular lesioned (TRNX) groups. Error bars indicate the S.E.M. (B) Stage II, with delays: mean number of errors following a 25 min delay for the two groups (SHAM and TRNX). Error bars indicate the S.E.M. on Stage II of this task, measured by the number of errors made after the delay (25 min delay after choice 4), again revealed no significant difference between the two groups [F(1,15)=3.654, P 0.07; see Fig. 6B], and no effect of session [F(3,45)=1.2, P 0.3], or group by session interaction (F 1). In view of the suggestion of a transient deficit on the T-maze alternation task, performance on the first day of Stage II (maze rotation and delay) was examined. Comparisons were made between the number of arm choices required to enter the final four arms on the last session of Stage I and the first session of Stage II. There was no overall group difference [F(1,15)=1.44, P 0.1] and no group-by-stage interaction (F 1). Interestingly, there was also no stage effect [F(1,15) =1.44] showing that the rats performance levels were not affected when they were forced to rely on allocentric cues Comparisons with pre ious studies. Previous studies in our laboratory have shown that lesions of the adjacent anterior thalamic nuclei can impair all of the spatial tasks used in the present study. This made it possible to make direct comparisons between the effects of reticular nucleus lesions (present study) and anterior thalamic nucleus lesions (past studies) made by injecting NMDA. These additional analyses were confined to the T-maze alternation task, as in both of the comparison studies [3,20], the same protocol and the same apparatus had been used. Furthermore, the T-maze task was always the first after surgery, and so there were no confounds arising from transfer effects. The two comparison studies are highly appropriate as the anterior thalamic lesions were especially discrete. Of the seven animals in one study [3], only two showed any involvement of the reticular nucleus, and this was unilateral and confined to a very restricted region immediately adjacent to the anterior ventral nucleus. In the other study [20], the lesions were centred in the anterior dorsal nuclei and involved the anterior ventral nucleus and rostral lateral dorsal nucleus. In none of the 12 cases did the lesions involve the reticular nucleus. In both studies [3,20], the surgical sham procedures matched those used in the present study. For one of these studies [3], data are available for 12 sessions of T-maze alternation. Confirmation that the test conditions were equivalent came from the very similar scores of the two sets of surgical control groups (maximum score 72; mean correct, reticular SHAM= 62.9, anterior thalamic shams=63.8; t 1, Fig. 7). In contrast, the mean score of the animals with anterior thalamic lesions (44.9) was lower than that of the TRNX group (59.5), and this difference was significant [t(19)=5.52, P ; Fig. 7]. A similar pattern of results was found with the second comparison study [20]. Once again, the two sets of surgical controls did not differ [maximum 36; mean correct SHAM=31.8, anterior dorsal shams=30.1; t(29)=1.67, P 0.1, Fig. 7], but the animals with lesions centred in the anterior dorsal nucleus (mean= 20.6) were significantly worse [t(24)=4.89, P ] than those with reticular lesions (mean=28.6; Fig. 7). 4. Discussion The present study investigated the role of the rostral pole of the TRN in the performance of tests of spatial learning and memory. Interest in this most rostral portion of the TRN stems from its extensive reciprocal connections with the anterior thalamic nuclei [9], which

9 L.A.K. Wilton et al. / Beha ioural Brain Research 120 (2001) have been shown to be involved in spatial memory [2 4,11,12,15,18]. For this reason, all of the behavioural tasks chosen are known to be sensitive to lesions of the anterior thalamic nuclei as well as of the hippocampus [3,18]. This sensitivity extends to the effects of lesions restricted to those portions of the anterior thalamic nuclei closest to the TRN [2,4,20]. Our results suggest that excitotoxic lesions of the rostral pole of the TRN have little, if any, effect on the performance of tests of spatial learning and memory. Furthermore, the relatively large numbers of subjects in each group make it less likely that the null hypothesis was not adequately tested. It is also unlikely that the lack of striking impairments in the TRNX group was due to sparing of the TRN connections with the anterior thalamic nuclei, as the fluorogold staining indicated that the large majority of projections from the TRN to the anterior thalamic nuclei were destroyed within the typical lesion placement (Fig. 2). Our findings appear to be in direct contrast to those of Collery et al. [5] and M Harzi et al. [10], who both reported that excitotoxic (ibotenate) lesions of the rostral TRN result in significant impairments on the performance of seemingly similar spatial memory tasks. Collery et al. [5] reported poor performance on delayed alternation and place recognition tasks in the T-maze. M Harzi et al. [10] examined performance on a radial arm maze task with both working memory and reference memory components, and reported that the TRN lesioned animals showed a significant impairment on the working memory component of the task. In contrast, rats with anterior thalamic lesions did not differ from controls in the performance of this task [10]. The latter result is surprising in view of the large preponderance of studies reporting clear deficits in a variety of spatial maze tasks following lesions of the anterior thalamic nuclei [2 4,14,17,18,20]. Indeed, statistical comparisons between animals with lesions of the anterior thalamic nuclei made in the same way as the TRN lesions [3,20] clearly showed that the effects of the former are far more disruptive on T-maze alternation (Fig. 7). Furthermore, in both comparisons, the anterior thalamic nucleus lesions were very discrete, and in one study [20], there was no evidence of reticular nucleus damage. In the other study [3], only two cases showed any evidence of unilateral reticular nucleus damage. As this damage was very restricted and could not be related to overall performance, it can be concluded that anterior thalamic nuclei damage is sufficient to induce the severe spatial working memory deficit associated with rostral thalamic lesions. In contrast, the effects of reticular lesions appear much more subtle, and are poorly revealed by standard tests of spatial memory. This conclusion still leaves unresolved the discrepancy between the present result and those of M Harzi et al. [10] and Collery et al. [5]. While both of these studies used ibotenic acid, and the current study used NMDA, it is unlikely that this difference is critical as injections of NMDA into adjacent thalamic nuclei will produce very clear deficits on spatial tasks [2,3,17,18]. One possible explanation could lie in the size of the lesions. In the studies of M Harzi et al. [10] and Collery et al. [5], the TRN lesions sometimes extended into the anterior regions of the adjacent anteroventral (AV) thalamic nucleus (in most cases in the study by M Harzi et al. [10]). In the present study, there was almost no anterior thalamic damage in the TRNX group (see Fig. 1). Given that the anterior thalamic nuclei are all thought to contribute to spatial learning and memory [15], and selective lesion studies have shown that AV lesions can result in impaired spatial memory task performance [2,4], significant AV damage may have contributed to the impairments observed by M Harzi et al. [10] and Collery et al. [5]. This must be balanced against the surprising result that anterior thalamic lesions themselves did not disrupt Fig. 7. Left: mean trials correct (maximum 72) over 12 sessions of T-maze alternation (chance=36). The data refer to the present study (TRNX) and to a previous study [3] in which the effects of anterior thalamic nucleus lesions were tested (ATX). Right: mean trials correct (maximum 36) over six sessions of T-maze alternation (chance=18). The data refer to the present study (TRNX) and to a previous study [20] in which the effects of lesions centred in the anterior dorsal thalamic nucleus were tested (ATDX). Error bars indicate the S.E.M.

10 186 L.A.K. Wilton et al. / Beha ioural Brain Research 120 (2001) performance of the radial arm maze task, suggesting that anterior thalamic damage could not have contributed to the TRN lesion effect observed by M Harzi et al [10]. The performance of the TRNX animals was not, however, completely normal. Evidence was found of a mild impairment in the initial sessions of acquisition in the T-maze alternation task. At no other stage of testing was there any impairment, even when task difficulty was increased. It remains, however, a possibility that this initial, transient impairment in the T-maze was exaggerated by the very limited anterior thalamic damage that occurred in some of the TRN group. Thus, although analysis of the smallest and largest TRN lesions on performance of T-maze acquisition did not reveal a significant difference, there was a trend for the larger TRN lesions both to produce the greatest transient impairment and to be more likely to involve very limited anterior thalamic damage. While the results of the water maze and radial arm maze tasks show more clearly that TRN lesions do not result in any persistent deficits in working or reference allocentric spatial memory, these results do not exclude a contribution to spatial memory processes. Indeed, recent studies using the immediate early gene c-fos have shown increased rostral reticular nucleus activity during radial arm maze tasks [15]. What the present results do show is that the rostral reticular nucleus contribution is not necessary for normal performance. Theories concerning the role of the TRN in general have centred on its possible role in attention. That so many thalamo-cortical projections pass through the TRN suggests that the latter may be crucial in the control of messages reaching the cortex [7]. In a recent study, Weese et al. [19] explored the effects of TRN lesions on covert orienting in the rat, and found that unilateral lesions of the TRN impaired a contralateral priming effect on a covert visual attention task. The lesions in that study [19] were confined to the visual sector of the TRN, located dorsally and caudally in the nucleus, and, as such, might be expected to play a role in visual attention. It is possible that the rostral pole of the TRN may play a similar role in the focusing of attention with regards to the information processed in the anterior thalamic nuclei. The lack of a persistent effect in the present study is quite consistent with this as all three tasks tapped allocentric spatial processing. As this reflects the type of processing that animals spontaneously use, the tasks did not require the redirection of attention. This was most clearly demonstrated during the radial arm maze task as the switch from Stage I to Stage II did not disrupt performance, yet only Stage II was dependent on allocentric processes. At the same time, the evidence of a change in performance at the initial stage of the first task (T-maze) could be a manifestation of attentional difficulties, as there is the greatest likelihood that other classes of stimuli might intrude at this preliminary stage of testing. However, from the current study, it is clear that the rostral pole of the TRN is not necessary for the subsequent performance of a variety of spatial learning and memory tasks that are sensitive to anterior thalamic damage. Thus, TRN damage is not sufficient to account for the severe effects of rostral thalamic lesions on tests of spatial memory. Acknowledgements This research was supported by a grant from the Wellcome Trust (UK). The authors wish to thank the assistance of Seralynne Vann. References [1] Aggleton JP, Brown MW. Episodic memory, amnesia and the hippocampal anterior thalamic axis. Behav Brain Sci 1999;22: [2] Aggleton JP, Hunt PR, Nagle S, Neave N. The effects of selective lesions within the anterior thalamic nuclei on spatial memory in the rat. Behav Brain Res 1996;81: [3] Aggleton JP, Neave N, Nagle S, Hunt PR. A comparison of the effects of anterior thalamic, mammillary body and fornix lesions on reinforced spatial alternation. Behav Brain Res 1995;68: [4] Byatt G, Dalrymple-Alford JC. Both anteromedial and anteroventral thalamic lesions impair radial-maze learning in rats. Behav Neurosci 1996;110: [5] Collery M, M Harzi M, Delacour J. Lesions of reticularis thalamic nucleus impair spatial working memory in rats. Neurosci Res Commun 1992;12:41 9. [6] Gonzalez-Ruiz A, Morte L, Leiberman AR. Evidence for collateral projections to the retrosplenial granular cortex and thalamic reticular nucleus from glutamate and/or aspartate-containing neurons of the anterior thalamic nuclei in the rat. Exp Brain Res 1997;116: [7] Guillery RW, Feig SL, Lozsadi DA. Paying attention to the thalamic reticular nucleus. TINS 1998;21: [8] Lozsadi DA. Organization of cortical afferents to the rostral, limbic sector of the rat thalamic reticular nucleus. J Comp Neurol 1994;341: [9] Lozsadi DA. Organization of connections between the thalamic reticular and the anterior thalamic nuclei in the rat. J Comp Neurol 1995;358: [10] M Harzi M, Collery M, Delacour J. First evidence of a possible role of the reticular thalamic nucleus in working memory in rats. Neurosci Res Commun 1991;8: [11] Morris RGM, Garrud P, Rawlins JNP, O Keefe J. Place navigation impaired in rats with hippocampal lesions. Nature 1982;297: [12] Olton DS, Becker JT, Handelmann GE. Hippocampus, space and memory. Behav Brain Sci 1979;2: [13] Paxinos G, Watson C. The Rat Brain in Stereotaxic Co-ordinates. San Diego, CA: Academic Press, Harcourt Brace & Company, [14] Sutherland RJ, Rodriguez AJ. The role of the fornix/fimbria and some related subcortical structures in place learning and memory. Behav Brain Res 1989;32:

11 L.A.K. Wilton et al. / Beha ioural Brain Research 120 (2001) [15] Vann, SD, Brown, MW, Aggleton, JP. Fos expression in the rostral thalamic nuclei and associated cortical regions in response to different spatial memory tests, Neuroscience 2000;101: [16] Van der Werf YD, Witter MP, Uylings HBM, Jolles J. Neuropsychology of infarctions in the thalamus: a review. Neuropsychologia 2000;38: [17] 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: [18] Warburton EC, Baird AL, Aggleton JP. Assessing the magnitude of the allocentric spatial deficit associated with complete loss of the anterior thalamic nuclei in rats. Behav Brain Res 1997;87: [19] Weese GD, Phillips JM, Brown VJ. Attentional orienting is impaired by unilateral lesions of the thalamic reticular nucleus in the rat. J Neurosci 1999;19: [20] Wilton, LAK, Baird, AL, Muir, JL, Aggleton, JP, Loss of thalamic nuclei for head direction produces impaired spatial memory in rats, Behavioral Neuroscience, in press..