Supramammillary and adjacent nuclei lesions impair spatial working memory and induce anxiolitic-like behavior

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1 Behavioural Brain Research 167 (2006) Research report Supramammillary and adjacent nuclei lesions impair spatial working memory and induce anxiolitic-like behavior Lourdes Aranda a, Luis J. Santín a,, Azucena Begega b, José A. Aguirre c, Jorge L. Arias b a Dpto. Psicobiología y Metodología de las CC, Facultad de Psicología, Universidad de Málaga, Campus de Teatinos s/n, Málaga, Spain b Laboratorio de Psicobiología, Universidad de Oviedo, Oviedo, Spain c Dpto. Fisiología Humana, Universidad de Málaga, Málaga, Spain Received 18 February 2005; received in revised form 31 August 2005; accepted 5 September 2005 Available online 19 October 2005 Abstract The present study assesses the involvement of the supramammillary and adjacent nuclei in spatial memory and anxiety-like behaviors. Rats with electrolytic lesions in the supramammillary nucleus were pre- and post-operatively trained in two spatial memory tasks and two anxiety tasks. Spatial memory tasks were performed in an open field with seven different goal positions containing the reward. Anxiety-like behaviors were tested in the elevated T-maze. In the spatial reference memory task, neither lesioned nor sham-lesioned groups were impaired. In the working memory task, lesioned animals were permanently impaired in their ability to solve the delayed-matching-to-position task. This working memory deficit is not related to increased proactive interference. It could be related to impairment of the rats ability to reorganize spatial stimuli. Consequently, rats were not able to achieve an optimal performance level to solve spatial tasks with continuous changes in the place location. In the elevated T-maze, lesioned rats reduced passive avoidance response but no changes in the escape response were observed. These results suggest a clear involvement of the supramammillary nucleus in working memory and behavioral inhibition but not in either spatial reference memory or in escape responses Elsevier B.V. All rights reserved. Keywords: Spatial learning; Mammillary region; Electrolytic lesion; Sprague Dawley rats 1. Introduction Previous studies have shown the relevance of the mammillary region (MR) in both memory and emotion [2,12,29]. The mammillary region is located around the mammillary bodies, at the caudal end of the hypothalamus, and comprised of several adjacent nuclei and fibre systems [28]. The main nuclei of this hypothalamic region are the mammillary bodies, premammillary nuclei, supramammillary and ventral tegmental nuclei [27,28]. The principal fibre systems included in the MR are the fornix, mammillary peduncle, mammillothalamic and mammillotegmental tracts [5,6,11,19]. The mammillary bodies (MB) have been mainly associated to learning and memory. In this sense, it has been shown that MB lesions impair memory functions in both animals and humans [32]. Most behavioral studies about the effects of MB lesions in memory functions have involved other MR or adjacent nuclei. Corresponding author. Tel.: ; fax: address: luis@uma.es (L.J. Santín). The supramammillary nucleus (SuM) is located dorsally to the MB and has been included in the majority of these lesion studies [28,29]. To date, few experiments have studied directly the SuM involvement in memory, independently of MB. Increased c-fos immunoreactivity in SuM has also been related to spatial memory but no clear relationship was found between SuM and spatial working memory in the study of Santín et al. [22]. Vann et al. also reported an increase of c-fos immunoreactivity in SuM neuronal nuclei related to radial-arm maze training [33,34]. Notwithstanding, chlordiazepoxide microinfusions in SuM and adjacent regions were found to reduce theta hippocampal frequency and impair spatial memory. However, the spatial memory deficit was very small and was not observed when microinfusions were introduced in the hypothalamic nuclei located 500 m outside the SuM [16,17,38,39]. More recently, Shahidi et al. have reported a clear involvement of SuM in spatial memory [25]. The role of MR in emotion has been less studied. Moreover, several studies have shown the importance of some MR nuclei such as MMn and SuM in several behaviors related to emotion [16,37]. In general, it has been suggested that anxiety /$ see front matter 2005 Elsevier B.V. All rights reserved. doi: /j.bbr

2 L. Aranda et al. / Behavioural Brain Research 167 (2006) like behaviors decrease in animals with MR lesions and this effect has been reported in both conditioned and spontaneous responses. To our knowledge, Pan and McNaughton research has been the first to directly study the role of SuM and surrounding nuclei in emotional behavior [16]. In this sense, SuM lesion induces anxiolytic-like behaviors such as decreased contextual fear evaluated in the conditioning chamber and an increase in both ambulation in the open field and behavioral inhibition in the operant chamber. These behavioral results have led Pan and McNaughton to suggest that SuM (and adjacent nuclei) would be more involved in emotional behaviors than in cognitive processes [16]. The aim of our study is to clarify the role of the SuM and surrounding nuclei in spatial memory and emotional behavior. This was assessed avoiding the concommitant MMn and MLn involvement in these psychological processes because the lesions did not reach these ventral MR nuclei. Behavioral experiments were designed to study the performance of the rats in two spatial memory tasks and two anxiety-like behaviors pre- and post-surgery. 2. Material and methods 2.1. Animals Twenty-four male Sprague Dawley rats ( g) were obtained from Criffa (Barcelona, Spain). All rats were maintained in groups of four at a temperature of 22 ± 2 C and on a constant light dark cycle (08:00 20:00 h). Water was available ad libitum. Food was available ad libitum at the beginning of the experiments. A week before the behavioral assessment, the food was restricted to achieve a 20% weight loss. During the restriction process, pieces of biscuit were mixed in with the food to accustom animals to the reward that would later be used in the behavioral study. The care and use of the animals were in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC), and were conducted with approval of the Animal Care and Use Committee of the University of Malaga Surgery The day after finishing the pre-surgery behavioral tasks, the animals were assigned randomly to one of two groups: supramammillary and adjacent (SuM- ADJ) nuclei lesioned group (n = 14) and sham-lesioned group (n = 10). During surgery one animal died from each group. The electrolytic lesions were done stereotaxically (Kopf, USA) under anaesthesia equitexin (3 ml/kg). The coordinates were AP 4.6 mm, DV 8 mm and at midline from the skull [18] with bregma and lambda at the same level. A 2 ma direct current was applied for 10 s. The lesion was made by passing a direct current through an electrode that had a non-insulated end 1 mm in length. Sham-lesioned rats were operated but were not administered the electrical discharge. Post-operative behavioral tests were carried out 10 days after surgery. Animals received paracetamol in the drinking water for 3 days after surgery Apparatus and behavioral procedures Spatial memory A75cm 150 cm 75 cm open field was used to train and test the rats in the memory tasks. In the open field, seven containers of equal size, shape and color were used. All of them contained sand and the reward was placed on top. The animals were introduced manually in the maze from seven different starting locations (around the edge of the maze). When the rat obtained the reward from one of the containers, it was manually removed from the maze and returned to its homecage. All the animals were trained in the following behavioral tasks: Habituation: All the animals explored the maze for 3 consecutive days. Each day, four trials were performed each of which lasted for 5 min. In the habituation, the animals acquired the reward from each of the seven containers in the maze. The habituation phase finished when each of the animals had eaten the reward from inside the seven containers. Spatial reference memory task: Training was carried out for 4 consecutive days with 10 trials per day (both pre- and post-operatively). There was an intertrial interval of 5 min. The reward was always placed in the same container and the rats were both manually and randomly released in the maze, from one of the seven start positions. Solution of this task requires application of a reference memory rule since the information presented remains constant over the training period [9,23]. The time the animals took to reach the reward (escape latencies) and the containers they visited before reaching the reward (errors) were recorded. Spatial working memory task: Training in the spatial working memory task was carried out on 3 consecutive days. The task is based on the delayed-matchingto-position paradigm (DMTP). This task requires the use of working memory and the information available to solve the task is only valid for one trial [9,23]. Every day, 10 training sessions were carried out with a 5 min interval between sessions. Each session was comprised of two trials: an acquisition trial and a retention trial with an intertrial interval of 30 s. During the acquisition trial, the start position and the location of the rewarded container were randomized. During the retention trial, the start position and the location of the rewarded container were the same as those used for the acquisition trial. This procedure was repeated over the 10 training sessions for the 3 days of the experiment. The rats carried out these tasks using spatial cues in the experimental room. Previous data obtained in our laboratory, using this maze and the same spatial reference memory task, showed that when distal cues were changed by rearranging the experimental room, the errors increased (data not shown). These previous results, suggested that the rats were not able to use stereotyped behavior such as pattern of turns, to solve the spatial task. The escape latencies and the number of errors were recorded Anxiety-like behavior An elevated T-maze was used to test the rats in the emotional tasks. The T- maze was made of wood and had three similar arms (50 cm 10 cm). One arm was enclosed by walls 40 cm high and another two open arms were surrounded by a wooden rim 1 cm high. The apparatus was elevated 50 cm above the floor. Illumination was provided by a 60 W lamp located in the ceiling of the room. Environmental temperature was kept at 21 ± 2 C and an air conditioner provided a background noise. Passive avoidance and escape behaviors were assessed in the elevated T- maze. In the passive avoidance task, the rats are placed at the end of the enclosed arm and cannot see the open arm until they explore it. Exploration of the open arm is an aversive experience, because the rats show an innate fear of elevated, lighted and opened places. In a second training trial, the rats avoid the open arm and spend more time in the closed arm. In the escape task, the rats are placed at the end of one of the open arms. In this task, the animals escape from the open arm towards the closed arm [10]. The training in the passive avoidance task was divided into two trials, with an intertrial interval of 30 s. The rats were kept in the room for 30 min and handled by the researcher before the behavioral test. Briefly, in the passive avoidance task, the rats were placed in the bottom of the closed arm and the time spent to reach the open arm was recorded. The register finished when the rat was in the open arm with its four limbs. The register was cut short if the animal did not leave the closed arm in 120 s, and the rat was placed in its home cage. The escape behavior was studied 30 s after passive avoidance training. The experimenter registered the time spent by the animal to reach the closed arm from the end of the open arm (escape latencies) Experimental design Before surgery, all rats sequentially experienced the following tasks: habituation (days 1 3), spatial learning with reference memory demands (days 4 7), spatial learning with working memory demands (days 8 10) and anxiety tasks

3 158 L. Aranda et al. / Behavioural Brain Research 167 (2006) Fig. 1. Schematic representation of the experimental design used in our study. All the animals were trained in different tasks pre- and post-operatively. Over the pre-operative period, animals were habituated to the behavioral procedure for 3 days. They were trained in a spatial reference memory task on days 4 7. On days 8 10 were trained in a spatial working memory task and finally, on day 11 were tested in the elevated T-maze. Post-surgery, both lesioned and sham-lesioned animals were tested on the first day in the elevated T-maze and trained on days 2 4 in the spatial working memory task and on days 5 8 in the spatial reference memory task previously used during the pre-surgery registers. (day 11). In the 10 days post-surgery, the rats were tested on the anxiety-like behavior tasks (day 1), spatial learning with working memory demands (days 2 4) and later on the spatial learning with reference memory demands (days 5 8) (Fig. 1). Pre-operatively, the reference memory task permits animals to achieve a level of knowledge about their spatial environment before performing the spatial working memory task. Post-operatively, the working memory task was applied before the reference memory task, to directly observe the effect of the lesion in the working memory task. Moreover, subsequent training in the spatial reference memory task can reveal whether the lesion affects the animals ability to modify the memory rules required to solve spatial tasks and if it affects acquisition of spatial learning with reference memory demands. With relation to anxiety tasks, with this experimental design post-operative alterations can be assessed in an unconditioned fear task and in a conditioned fear task Histology After the last training day, rats were deeply anaesthetised (equitexin 4 ml/kg) and perfused transcardially with phosphate-buffered saline (PBS) 0.1 M, ph 7.4 and later with 10% formalin in PBS 0.1 M, ph 7.4. The brains were removed and stored in the same fixative solution for 2 weeks. Then, the brains were dehydrated and paraffin embedded. Coronal sections (15 m) were obtained and stained with toluidine blue. The location and extent of the electrolytic lesions were reconstructed according to the atlas of Paxinos and Watson [18] Data analysis The statistical analysis was performed on nine sham-lesioned rats and nine lesioned rats. Spatial learning was analyzed using two different analyses for both the mean escape latencies and errors Spatial reference memory This task was analyzed using an ANOVA with two repeated measures (first repeated measure with two levels: pre-surgery and post-surgery; second repeated measures with four levels: 4 training days) and two groups (lesioned group and sham-lesioned group). Green-Geisser epsilon was used when Mauchly s test was <0.05. In statistically significant cases (α < 0.05) trend analysis was carried out Spatial working memory A repeated measures ANOVA was used with the same groups but with two different repeated measures (first repeated measure with two levels: pre-surgery and post-surgery; second repeated measure with two levels: acquisition trial and retention trial). Post hoc comparisons were performed using the Tukey test. A second analysis was applied to the data obtained in the working memory task to determine the possible participation of proactive interference in the results found. For this analysis, data from retention trials were recorded. Mean errors and escape latencies were calculated using the data from the 3 training days. A single value was obtained for each of the 10 trials for each rat. The result of this recoding was analyzed by applying repeated measures ANOVA with two groups (lesioned and sham-lesioned group) and two repeated measures (first repeated measures: pre-surgery and post-surgery performance; second repeated measures: 10 trials). After this, trend analysis was applied to establish whether there was an increased probability that the earlier trials influenced the following trials, producing a proactive interference phenomenon Anxiety-like behavior The escape response and passive avoidance task were analyzed independently. Escape latencies were analyzed using ANOVA with one repeated measure (pre- and post-surgery) and two groups (SuM-ADJ lesion and sham-lesion groups). The latencies recorded in the passive avoidance task were analyzed using an ANOVA with two repeated measures and the same two groups previously described. The first repeated measure was pre- and post-surgery register, and the second one two passive avoidance trials. Post hoc comparisons were carried out with Fisher s test in the two anxiety behavior tasks. 3. Results 3.1. Histological results Six of the operated animals were excluded because the lesion was too lateralised and only about half of the lateral SuM was unilaterally lesioned. Histological analysis of the brains of the nine lesioned rats showed that the lesions did not damage either MB nuclei (medial and lateral nuclei) or the mammillothalamic tract (Fig. 2). In all the animals, the lesion damaged the supramammillary nucleus and to a variable degree, the region immediately above the SuM. In eight of the nine animals, the most ventral portion of the posterior hypothalamus was affected as well as a slight alteration of the lateral portion of the ventral tegmental area in two animals and the ventral portion of the lateral hypothalamus area in another two animals. The mammillotegmental tract was unilaterally lesioned in five rats and bilaterally damaged in two rats. In one of the nine rats lesioned, the lesion was limited to the medial SuM and to slight damage of the region immediately above the medial SuM. This selective and restricted lesion probably resulted from wearing the non-insulated end of the electrode.

4 L. Aranda et al. / Behavioural Brain Research 167 (2006) Fig. 2. Reconstruction of the maximum and minimum size of the electrolytic lesion made in the study. The upper images show reconstruction of the size of the lesion (grey: maximum size; black: minimum size). The lower images show the normal structure of the supramammillary nucleus and the most important nuclei and adjacent tracts. Adapted from Paxinos and Watson [18] (PM: medial mammillary nucleus, posterior; VTA: ventral tegmental area; IF: interfascicular nucleus; Rli: rostral linear nucleus raphe; fr: fasciculus retroflesus; 3V: 3rd ventricle; LH: lateral hypothalamus; PH: posterior hypothalamus area; SuM: supramammillary nucleus; mp: mammillary peduncle; MM: medial mammillary nucleus, medial; LM: lateral mammillary nucleus; MRe: mammillary recess 3rd ventricle; mtg: mammillotegmental tract; sumx: supramammillary decussation; mt: mammillothalamic tract; f: fornix). Graphical reconstructions of the largest and smallest SuM-ADJ lesions are represented in Fig Spatial reference memory No statistically significant differences were found between the two groups or in the mean errors (F1/16 = 3.93; p 0.065) or the escape latencies (F1/16 = 1.72; p 0.208). Animals in both groups reduced mean errors (F1.22/19.55 = 140.3; p ) and escape latencies (F1.22/19.55 = 41.38; p 0.000) with training in the spatial task. These differences were not related either with the time of registry (pre- and post-surgery) or with the group (p 0.05). The only significant difference observed in the escape latencies was due to the time of registry (pre or post-surgery) (F1/16 = ; p 0.05). Nonetheless, this difference was not related either with group or with training day (p 0.05) (Fig. 3) Spatial working memory Analysis of the main effects revealed statistically significant differences between pre- and post-surgery registers both in the mean errors (F1/16 = ; p 0.000) and in the ecape latencies (F1/16 = ; p 0.001). Both variables were also different in the acquisition trial and the retention trial (F1/16 = ; p and F1/16 = 17.33; p 0.001, respectively) (Figs. 4 and 5). All the first-order interactions were statistically significant in the variable number of mean errors (group pre- and post-surgery: F1/16 = 8.777; p 0.009; group acquisition-retention trial: F1/16 = ; p 0.001; and pre- and post-surgery acquisition-retention trial: F1/16 = ; p 0.000). For the escape latencies, the only statistically first-order interaction was: group acquisitionretention trial (F1/16 = 5.156; p 0.037). The second-order interaction (group acquisition-retention trial pre- and post-surgery) was statistically significant both in the variable mean errors (F1/16 = ; p 0.000) and in the escape latencies (F1/16 = 6.591; p ). The post hoc analysis showed that the two groups make similar mistakes and have similar escape latencies during the acquisition trial, and there are no differences in the pre- and post-surgery register (p 0.05). Moreover, in each of the two groups and in the two variables a similar pre- and post-surgery performance was observed (p 0.05). In the retention trial, the animals in both groups made a similar mean number of errors and had similar mean escape latencies during the pre-operative registry (p 0.05). However, differences were observed between the two groups both in the number of errors made and also in the escape latencies in the post-surgery register (p 0.05). More specifically, the SuM-ADJ lesion group made significantly more errors (p 0.05) and presented longer escape latencies than the sham-lesion group. In a complementary manner, the two groups of animals presented longer escape latencies and a greater number of mean errors in the pre-surgery register, when the acquisition trial was compared with the retention trial (p 0.05). Nevertheless, during the post-surgery register this difference was observed in the sham-lesion group (p 0.05) but not in the SuM-ADJ lesion group (p 0.05) (Figs. 4 and 5).

5 160 L. Aranda et al. / Behavioural Brain Research 167 (2006) Fig. 3. Graphical representation of the behavioral registers in the spatial reference memory task. Reduction in mean errors (A) and escape latencies (B) were observed in both pre- and post-operative training. Knowledge of the procedure and pre-operative training increase the animals speed at solving the task as can be observed in the post-operative registers (B), but does not affect spatial learning with reference memory demands (A). Fig. 4. Graphical representation of the results obtained in the acquisition assay in the spatial working memory task. Statistically significant differences were not observed between the lesioned group and the sham-lesioned group or in the mean errors (A) or the escape latencies (B), at either of the two recording times (pre-surgery, post-surgery). The results obtained by recoding the data to study the phenomenon of proactive inteference revealed that the two groups differed post-operatively in their escape latencies and in the mean number of errors during the retention trial (F1/16 = ; p and F1/16 = 36.87; p 0.000, respectively). Nevertheless, no increasing linear trend was observed in either of the two groups or in the pre- or post-surgery performance (p 0.05) (Fig. 6). Fig. 5. Graphical representation of the results obtained in the retention trial in the spatial working memory task. Statistically significant differences were observed between the lesioned and the sham-lesioned group neither in the mean errors (A) nor in the escape latencies (B), during the pre-surgery register. Nonetheless, an increase in the errors (A) and in the escape latencies (B) was recorded in the post-operative registers of animals that had received a SuM-ADJ lesion. * p 0.05.

6 L. Aranda et al. / Behavioural Brain Research 167 (2006) Fig. 6. Graphical representation of the recoding and analysis of the data obtained during training in the spatial working memory task, in the retention trial, to study the phenomenon of proactive interference. A progressive increase was not observed either related with training, in the mean errors (A) or in the mean escape latencies (B), either during pre or post-operative registers. This steady increase, related with training, in the difficulty to retain information, would reflect the presence of proactive interference. Nonetheless, a worse post-operative performance was observed in the SuM-ADJ lesion group compared with the sham-lesion group (note the differences between the mean errors (A) and the escape latencies of both groups (B)) Anxiety-like behavior The escape behavior was different in both groups (F1/16 = 4.879; p 0.042). However, this difference was not related to either pre- or post-surgery training (F1/16 = 0.713; p 0.401). Post hoc comparisons confirmed these results, and there were no statistically significant differences between groups in either presurgery or in post-surgery training (p 0.05) (Fig. 7). In relation to passive avoidance behavior, results have shown that pre- and post-surgery registers are different (F1/16 = ; p 0.000) and that this difference is related with group (F1/16 = 7.559; p 0.014). Fourteen post hoc comparisons showed that the SuM-ADJ lesion group had longer post-surgery avoidance latencies (p 0.05) but no pre-surgery ones (p 0.05). Nonetheless, a post-operative increase in avoidance latencies was observed Fig. 7. Graphical representation of the performance of the two groups during the escape trial in the elevated T-maze. The results show that animals from both groups have similar escape latencies in the open arm. Nonetheless, postoperatively there is a tendency in the SuM-ADJ lesion group to take longer to escape of the open arm. in both groups compared with their pre-operative peformance (p 0.05) (Fig. 8). 4. Discussion 4.1. Spatial reference and working memory The results indicate that SuM and adjacent nuclei lesions impair the rats capacity to solve spatial working memory tasks. The working memory task used in our study is DMTP. These tasks require short-term representational demands since the animals must retain in their working memory the active information during a brief time interval (30 s in our study) to make the correct choice in the retention trial. In this task, the places the animals must locate do not change from the acquisition to the retention trial but are changed in the following training sessions. Our results indicate that animals with SuM-ADJ lesions present a severe impairment solving this task and make significantly more errors and take longer compared to controls and to their preoperative performance (Figs. 4 and 5). Therefore, the deficit in our study was characterized by the animals locating the reward in the retention trial randomly or using non-effective strategies (i.e. non-spatial strategies) with similar performances to those shown in the acquisition trial. Since it is a sequential task, there could be an increase in proactive interference, which would explain the impaired performance [7,23]. Several studies suggest that the mammillary region (especially the more ventrally located nuclei (MB and SuM)) serves to maintain the episodes temporally different [3]. In this way, lesion of this region would precede the appearance of proactive interference and, consequently, a severely altered performance of sequential spatial memory tasks. Nevertheless, Santín et al. and Vann and Aggleton show that proactive interference does not necessarily have to be detected to observe amnesic effects of lesions of the mammillary region [24,31]. Similarly, the results obtained in our study indicate that the alterations observed in the spatial working memory task, cannot be explained by an increase in proactive interference, at least with the interses-

7 162 L. Aranda et al. / Behavioural Brain Research 167 (2006) Fig. 8. Graphical representation of the two groups performance during the two passive avoidance trials conducted in the elevated T-maze. The results indicate that sham-lesioned animals and those undergoing surgical intervention exhibit more avoidance behavior patterns (A and B). This increase in passive avoidance behavior is partially counteracted by the SuM-ADJ lesion during the two avoidance trials recorded (A and B). * p < sion interval of 5 min used in our experiment (Fig. 6). Therefore, our results suggest that the alterations observed in this task are not due to the animal s inability to temporally distinguish spatial episodes, since the proactive interference expected with this hypothesis was not observed. One possible explanation is that the impairment was caused by an increase in the rate of forgetting the spatial information in short time periods [24] or to an alteration in the coding processes for new spatial information [31]. In the DMTP task used, the animals do not use new spatial information in each trial although they do have to continually modify the spatial information to effectively solve the task. Therefore, the memory demands in this task imply a new rearrangement of the spatial stimuli in each group of trials, since location of the reward changes. When working memory tasks are required with a temporal rearrangement of spatial stimuli, the SuM-ADJ lesion impairs correct performance of the task. This effect is long-lasting and does not improve with training (Fig. 6). Our results show a clear deficit for short-term learning of new locations similar to that described by Shahidi et al. in rats with reversible inactivation of the SuM with lidocaine [25]. On the other hand, the rat s performance in this task shows that they retain procedural aspects of the task since they look for the reward inside the containers, reflecting that they have acquired knowledge about location of the reward. In a complementary manner, post-operative peformance of the spatial reference memory task shows that the SuM-ADJ lesion does not affect the rat s ability to find the place where the reward is located when the reward remains in the same place (Fig. 3). This performance is the same when the rats are compared with the control group and a similar pre-operative learning pattern is observed. The ability of the SuM-ADJ animals to acquire a spatial reference memory independently of whether or not the working memory is affected, suggests that this region could only be important in maintaining and retaining spatial information for short time periods and is independent of long-term spatial memory. Santín et al. have reported similar results. In their study, no association was observed between c-fos immunoreactivity in the SuM and learning of a spatial reference memory task in the circular pool [22]. With the experimental design we used, the use of the reference memory task, after the working memory task post-operatively, allowed us to study if the SuM-ADJ lesion impairs the ability of the animals to change accurately from the spatial working memory rule to the spatial reference memory rule (i.e. assess the effect of the lesion on the behavioral flexibility). Several works have shown that animals with hippocampal lesions present great difficulties in changing the strategy and the memory rule to solve spatial tasks [20]. The fact that the SuM is profusely connected with the hippocampal formation [14,35] and modulates the frequency of the hippocampal theta rhythm [13,30], suggests that lesion of this region could induce a similar deficit to that observed in hippocampal animals. Nevertheless, the SuM-ADJ lesion does not seem to affect this capacity, since the ability of SuM-ADJ lesioned animals to effectively change from a working memory rule to a reference memory rule is not altered (Fig. 3). Therefore, in spite of the anatomical and functional relationship of both regions these seem to play a different role in spatial memory. Possibly, the SuM-ADJ lesion does not cause an alteration of the hippocampal function of sufficient intensity as to induce changes in the development of a spatial reference memory. More specifically, our results suggest that the SuM-ADJ region does not form part of the neurobiological system associated with the general processing of spatial information. Its participation is restricted to tasks in which spatial working memory demands are important to solve spatial tasks. Similar results have been reported by Pan and McNaughton, who observed a mild change in the rats ability to form a spatial short-term memory in the circular pool, inhibiting the function of the SuM using microinfusions of chlordiazepoxide [17]. They also observed that this effect was accompanied by a slight reduction in hippocampal theta frequency. Pan and McNaughton sustained that this mild alteration could be due to impaired spatial working memory processes [16,17]. Nonetheless, this study was not designed to study the participation of the SuM in spatial working memory but in spatial reference memory. Hence, the ab-

8 L. Aranda et al. / Behavioural Brain Research 167 (2006) sence of any clear effect in the spatial memory task and the very slight alteration of the hyppocampal rhythm observed by Pan and McNaughton could be due to selection of a reference memory task instead of a working memory task [17]. In our study, we used a DMTP paradigm, which is useful to test participation of working memory [9]. The results obtained in this DMTP task suggest that participation of the SuM in spatial memory is restricted to tasks with working memory demands. Even when all the lesions include the SuM, other nuclei (Fig. 2) have also been associated directly or indirectly with memory [4,15,38,40] making it difficult to guarantee that our results are due to lesion of the SuM. Nevetheless, one of the nine animals present a lesion restricted to the medial portion of the SuM (Fig. 2) and the results in this animal showed an impairment in the spatial working memory task similar to that observed in more extensive lesions. In support of our results, alteration of the hippocampal theta rhythm and performance in memory tasks have only been observed by GABAergic inhibition of medial SUM neurons and of those around but no farther than 500 m from the medial SuM [16,17,38,39]. Nevertheless, further research is required to confirm whether alteration of the SuM is responsible for the change in spatial working memory observed in our study Anxiety-like behavior The behavior in the elevated T-maze showed that surgery induced an increase in anxiogenic behavior and that the SuM-ADJ lesion partially reduces this increased anxiety (Figs. 7 and 8). The elevated T-maze is an ethologically based animal model of anxiety where two types of anxiety behavior can be distinguished: passive avoidance and escape response [10]. The escape behavior in the elevated T-maze indicates the degree of exploration by the animal in the open arm. Nevertheless, different pharmacological treatments related with anxiety control have not been able to affect this behavior, suggesting that this response in the elevated T-maze is not sensitive to anxiety-associated manipulations [10,36]. Passive avoidance studies, evaluate the extent to which the rats avoid the open arm and remain in the closed arm or the safe area. This behavior also depends on the animal s degree of anxiety, such that increased anxiety increases the avoidance response and vice versa [38,41]. Only in the passive avoidance task has the SuM-ADJ lesion been found to facilitate the appearance of ansiolytic-like behaviors (Fig. 8). Animals from the SuM-ADJ lesion group, present increased exploration of the open arm during the escape behavior although this difference is not due to the lesion since it is not related with the time of register (pre- and post-surgery) (Fig. 7). However, post-operatively the SuM-ADJ lesion favours exploration of the open arm, indicating that this reduces the rat s anxiety, which explore the open arm of the maze for a longer time. The SuM-ADJ lesion only partially reduces the post-operative increase in avoidance response, showing that the SuM-ADJ nuclei are related with controlling the avoidance response but are not the only regions responsible for its control. Taken together, our results show that the SuM-ADJ lesion is associated with the appearance of ansiolytic-like behaviors such as avoidance but not reduced escape responses (Fig. 8). Our results agree with those published in previous studies. These observed an increase in c-fos immunoreactivity in SuM neurons with exposure to ethological anxiety tasks [26,37] and conditioned anxiety tasks [1]. More recently, Pan and Mc- Naughton found that neurotoxic lesion of the SuM and surrounding regions reduces the appearance of anxiogenic behaviors in the open field and in a fear-conditioning task [16]. These studies, together with our results, confirm the participation of the SuM and adjacent nuclei in emotional behaviors. These effects on emotional behaviors could be due to an increased aversive value of the environmental stimuli, which would facilitate appearance of an avoidance response. A reduction in this emotional valency would be reflected in reduced avoidance latencies as shown by the SuM-ADJ lesion group (Fig. 8). Nevertheless, the results could also be related not with the emotional valency of the stimuli but rather with reduced behavioral inhibition or increased hyperactivity induced by lesion of the SuM-ADJ. In this way, the SUM lesion [16], administration of the GABAergic agonist chordiacepoxide in the SuM [39] causes hyperactivity in operant tasks observing an increased leverpressing. In addition, the lesion of other nuclei and tracts included in the MR has been associated with increased hyperactivity [8,21]. However, our experiment was not designed to study this phenomenon and we cannot guarantee that our results are not caused by an increased locomotor activity or a reduced anxiety. Nonetheless, if hyperactivity affects latencies in the passive avoidance task, this would also be reflected in the latencies of the escape task. However, in the escape task, the SuM-ADJ lesioned group remains the same amount of time as the sham-lesioned group in the open arm (Fig. 7), in contrast to in the closed arm (Fig. 8). 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