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Involvement of the mammillary bodies in spatial working memory revealed by cytochrome oxidase activity
Brain Research 1011 (2004) 107 – 114 www.elsevier.com/locate/brainres
Research report
Involvement of the mammillary bodies in spatial working memory revealed by cytochrome oxidase activity Ne´lida M. Conejo a,*, He´ctor Gonza´lez-Pardo a, Guillermo Vallejo b, Jorge L. Arias a a
Laboratory of Psychobiology, Faculty of Psychology, University of Oviedo, Plaza Feijoo, s/n E-33003, Oviedo, Asturias, Spain b Methodology Area, Faculty of Psychology, University of Oviedo, Plaza Feijoo, s/n E-33003, Oviedo, Asturias, Spain Accepted 23 March 2004 Available online 27 April 2004
Abstract In view of the inconclusive findings relating the nuclei of the mammillary bodies (MB) with spatial memory, we evaluated the oxidative metabolic activity of the medial and lateral nuclei of the mammillary bodies (MB) after training young rats (30 days) of both sexes in the Morris water maze. Different groups were trained in spatial working (WM) or reference memory (RM) tasks, respectively. The corresponding naı¨ve groups swam for the same amount of time as the trained groups but without the escape platform. Control groups were added that had not been manipulated in any way. No sex-related differences were detected in the working memory task although males exhibited better reference memory than females. Cytochrome oxidase (CO) activity, an endogenous metabolic marker for neuronal activity, was measured in all the groups. CO activity increased significantly in both MB nuclei of male and female rats only in the spatial working memory group. In addition, high CO activity in the lateral nucleus of the MB was linearly correlated with lower escape latencies in both sexes after training in the working memory task. No CO activity changes were found in the basolateral amygdala (BL) in any of the experimental groups. This nucleus was used as a control brain region because of its participation in emotional behavior. The results suggest a specific role of the MB nuclei in spatial working memory in both sexes. D 2004 Elsevier B.V. All rights reserved. Theme: Neural basis of behavior Topic: Learning and memory: systems and functions Keywords: Spatial memory; Mammillary body; Basolateral amygdala; Cytochrome oxidase; Neuronal metabolism
1. Introduction The mammillary bodies (MB) have traditionally been associated with memory processes. In humans, the amnesic nature of Korsakoff syndrome is often related with damage to the MB [26,40,65,70]. However, some clinical studies have demonstrated that amnesic symptoms in patients with this syndrome must also be due to damage in other diencephalic regions [36,38,39,70,76]. In fact, the specific role of the MB in memory and learning is still controversial in spite of the amount of research done on experimental models. Abbreviations: BL, basolateral amygdala; CO, cytochrome oxidase; f, fornix; LM, lateral mammillary; MB, mammillary bodies; MM, medial mammillary; mt, mammillothalamic tract; mtg, mammillotegmental tract; OD, optical density * Corresponding author. Tel.: +34-985-103212; fax: +34-985-104144. E-mail address: [email protected] (N.M. Conejo). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.03.025
Owing to the direct anatomical relationship between the MB and the hippocampus via the fornix, most research has focused on the association between these and spatial memory [19,55,64,68]. The MB have been involved in the delayed alternance in the T maze [27,37,56], place preference [1,45], working memory (WM) in radial maze [56,62,64] and Morris water maze [55,68]. The MB are composed of several well-defined nuclei. The largest of these are the medial mammillary (MM) and lateral mammillary (LM) nuclei. Current evidence points to a probable differential participation of these nuclei in spatial memory. In particular, the MM nucleus has been shown to be directly involved in spatial working memory tasks by lesion techniques and methods measuring c-fos protein expression [17,54,63,68]. Electrophysiological data have shown that the cells of this nucleus fire in bursts synchronized with the theta rhythm of the hippocampus
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[3,8,33 –35] and that this rhythmic activity is dependent upon the action of the hippocampus on this nucleus [8,33]. It is thought that the theta frequency could modulate the spatial memory processes [33,46,69]. In contrast, this same activity was only detected in a small proportion of cells of the LM nucleus [60]. On the other hand, neurons of this nucleus are specifically activated in relation to the position of the animals head irrespective of its location and are referred to as ‘‘head direction cells’’ [6,60]. Similarly, theoretical models have been proposed that specifically implicate neurons of the LM nucleus in spatial working memory tasks [24,58]. However, in spite of these models, it has not yet been clearly confirmed that the LM nucleus plays a role in spatial learning. On the other hand, the biological determinant, sex, is known to clearly affect performance in spatial learning tasks. Hence, in the Morris water maze, male rats usually perform reference memory (RM) tasks better than females [5,12,52, 53,61,72]. Reference memory refers to memory for spatial information that does not change with time, while working memory requires retention of information that is only useful for short intervals of time [47]. However, sex differences have not been consistently detected in reference memory tasks [30,66,67]. Sex differences in performance in these spatial tasks seem to be restricted to the early postnatal development and may reflect a difference in maturation rate together with an important role of sex hormones on brain development [11,12,51]. Conversely, in spatial working memory tasks assessed by the Morris water maze, no differences have been reported between rats of both sexes [25]. The aim of our work was to study the different involvement of the MM and LM nuclei in these two types spatial memory and the relevance of these nuclei in the learning of these tasks in both sexes. In order to avoid the influence of the estrous cycle of these females in this type of task [14], here, we have used 30-day-old male and female rats. Particularly, sex-related differences have been previously reported in spatial learning at this age [12,13]. Accordingly, if an MB nucleus would be effectively required for spatial memory, then we would expect its activation in both sexes after learning the task. In order to evaluate the participation of MB nuclei in spatial memory, we used cytochrome oxidase (CO) histochemistry. CO is a key enzyme in cellular oxidative energy metabolism because it participates in the mitochondrial electron transport chain involved in the direct production of energy as ATP using most of the available oxygen. Local CO activity is considered to be a reliable marker of brain oxidative metabolic potential, due to the close relationship between energy metabolism and neuronal activity [74]. In contrast to other metabolic markers, CO activity can be used as an index of the sustained energy requirements of nervous tissue associated with brain function [20,22]. Moreover, this technique has been shown to be sufficiently sensitive to detect changes in local cerebral oxidative metabolism induced after learning [43,48,50].
2. Material and methods 2.1. Animals Wistar rats aged 30 days were obtained from the University of Oviedo central vivarium. They were housed under standard conditions (12 h light– dark cycle with lights on from 8.00 to 20.00 h), at constant room temperature of 21 F 2 jC with ad lib access to food and water. All experimental procedures carried out with animals were approved by a local veterinary committee from the University of Oviedo vivarium and subsequent handling strictly followed the European Communities Council Directive 86/609. 2.2. Apparatus Animals were trained in a Morris water escape task using a 1.5-m-diameter black fiberglass pool, 75 cm high and 50 cm above the floor. The pool was filled with tap water to a height of 32 cm and a black escape platform was placed 2 cm beneath the water surface. The water temperature was kept at 23 jC during the entire test period. The experimental room had numerous visual cues such as colored maps, posters and plastic dishes fixed on the walls, a shelf, covered windows and a table. Lighting was provided by two halogen spotlights (500 W) placed on the floor and facing the walls. Animal cages were kept outside the experimental room to avoid odor cues and the bucket where the rats remained between consecutive trials was placed randomly around the pool during each trial. All swim paths of the animals were recorded live and analyzed later using a computerized video-tracking system (Ethovision Pro, Noldus, The Netherlands). 2.3. Behavioral procedures The pool was divided virtually into four quadrants, according to the cardinal points (N, S, E, W). During the habituation day, rats were released facing the pool wall from the central border of each quadrant following a pseudorandom sequence, four times each session. Each rat received two daily sessions spaced 1 h apart. Rats were returned to their home cages between sessions. The escape platform used on the first day was painted white and stood up 2 cm above the water surface. Rats were allowed to swim to locate the escape platform or placed on it after 60 s, where they remained for 15 s before they were placed in a black plastic bucket for 30 s. A standard protocol for spatial learning in the Morris water maze was used for the spatial reference memory (RM) female and male groups. After habituation to the maze, animals were trained daily using a single four-trial session. In each trial, the rats were released randomly from one of four start locations and had to search for a hidden escape platform beneath the water surface. The
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platform was located in the same position during the training days. To test spatial working memory (WM) in female and male groups, two sessions were performed daily and each session consisted of two consecutive trials (sample and choice). During sample trials, a visible platform was randomly located in one quadrant, and the rat was released from a randomly chosen start point. When the rat located the platform or when 60 s had elapsed, it remained on the platform for 15 s and was then returned to the bucket for 30 s. The platform location was the same for the next trial (choice) but was hidden beneath the water surface. In addition, animals were released from a different randomly chosen point in the choice trial. In the second daily session, the platform location changed randomly. Therefore, in the WM task, the position of the escape platform changed not only between both daily sessions but also between training days. In both spatial memory tasks, training ended when the mean escape latencies reached an asymptotic level. The naı¨ve animals used to compare with the WM groups swam four times a day during 1 min with no escape platform present (similar to four trials divided into two sessions of the WM groups: sample/choice, sample/ choice). In these naı¨ve groups, same intersession and intertrial intervals of the WM groups were used. The number of training days of the naı¨ve groups matched those required by WM groups. The naı¨ve groups used to compare with the RM groups swam also four times a day during 1 min with no escape platform (similar to the four consecutive trials of the RM groups in a single daily session). The number of training days of the naı¨ve groups matched those required by both the male and female RM groups, respectively. Finally, control groups without swimming experience were included to account for the effects of stress, handling and learning. 2.4. Tissue preparation Animals from naı¨ve and trained groups were deeply anaesthetized by intraperitoneal injection of sodium pentobarbital (100 mg/kg) 1 h after finishing the behavioral procedure in each group and immediately in control groups. Rats were transcardially perfused with a cold phosphate buffer solution (pH 7.4; 0.1 M). Their brains and medial hepatic lobes were quickly removed and immersed in a 20% sucrose buffer solution at 5 jC for 24 h. Tissues were embedded in a cryoprotective gel (OCT Compound; Miles USA) frozen in freon-22 cooled by liquid nitrogen and stored at 70 jC. Next, 20-Am-thick sections from the brains were obtained at 20 jC with a cryostat microtome (Microm, Heidelberg, Germany). Sets of rat liver sections were used as cytochrome oxidase (CO) activity standards because the hepatic tissue is histologically uniform and has an elevated intrinsic CO activity. Cryostat slides containing sections of different
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thickness (20, 40, 60 and 80 Am) were obtained from the medial hepatic lobe of intact adult male rats. 2.5. Cytochrome oxidase histochemistry Sections from the brains together with a complete set of standards with different CO activity were used to perform CO histochemistry. We used a modified version of the method originally described by Wong-Riley [73], based on the quantitative CO histochemical method developed by Gonzalez-Lima and Jones [22]. In brief, slides were lightly fixed for 5 min with a 1.5% glutaraldehyde solution, rinsed three times in phosphate buffer and incubated at 37 jC for 1 h in the dark and with continuous stirring in a solution containing 50 mg 3,3V-diaminobenzidine, 15 mg cytochrome c (Sigma, USA), 4 g sucrose per 100 ml phosphate buffer (pH 7.4; 0.1 M). The slides were rinsed three times with cold phosphate buffer, dehydrated and coverslipped with Entellan (Merck, USA). CO histochemical staining intensity was measured by densitometric analysis using a computer-assisted image analysis workstation (Leica Q550IW, Germany) composed of a computer-controlled microscope and light source (Leica DM-RHC), CCD camera and image analysis software (Leica Q-Win). A minimum of seven measurements were taken bilaterally in the mammillary bodies (MM and LM nuclei) from each animal and expressed as arbitrary units of optical density (OD). The basolateral nucleus of the amygdala (BL) was also measured, included as a control brain region to take into account the effects of fear or anxiety associated with the behavioral test. Delimitation of the mammillary nuclei and basolateral amygdala (BL) was performed according to the Paxinos and Watson’s [49] atlas. In order to establish comparisons and count for possible staining variations between brain sections from different staining baths, measurements were taken from CO-stained liver standards. Regression curves and coefficients (R2) between section thickness and CO activity measured from each set of standards were calculated for each incubation bath, and further calculations were performed to compare the regression plots obtained from each set of CO activity standards. Finally, OD values measured for the brain regions selected were put on the same level using the OD differences calculated from the regression plots of the liver standards. 2.6. Statistical analysis Escape latencies or path lengths in each trained group (WM males and females, RM males and females) were analyzed using one-way repeated measures ANOVA, with training day as the repeated-measures factor. Student’s ttests were used to compare escape latencies or path lengths between sexes on the last day of WM or RM training, respectively. Group differences for each sex (RM, WM,
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naı¨ve and control; n = 10 per group) in CO activity measured as relative OD values from each brain region (MM, LM and BL) were assessed by one-way ANOVAs. Post hoc tests were used to assess differences between means when ANOVA indicated significant overall differences. To evaluate differences in CO activity between the MM and LM nuclei, paired t-tests were used for each sex in the different groups. Pearson’s product – moment correlation coefficients (r) were calculated between behavioral scores and CO activity in the selected regions for each trained group.
3. Results 3.1. Behavioral tests 3.1.1. Reference memory task As shown in Fig. 1, escape latencies significantly decreased during training days both in males [ F(7,72) = 10.78, P < 0.001] and females [ F(9,80) = 12.53, P < 0.001]. Similarly, mean path lengths measured significantly decreased during training days in males [ F(7,72) = 11.45, P < 0.001] and females [ F(9,80) = 13.17, P < 0.001]. No sex differences were found in escape latencies or path lengths recorded on the last training day. 3.1.2. Working memory task Analysis of the escape latencies corresponding to the retention trial (choice) during the second daily session over the training days, revealed a significant decrease in the time used to find the hidden platform in males [ F(7,72) = 5.72, P < 0.001] and females [ F(7,72) = 2.68, P = 0.01] (Fig. 2). Path lengths measured in the retention trials also were significantly shorter with training in males [ F(7,72) = 3.14, P < 0.01] and females [ F(7,72) =
Fig. 1. Female rats showed significantly higher latencies to find the hidden escape platform as compared to males (Student’s t-test; P < 0.001) during training in the reference memory task. Mean escape latencies ( F S.E.M.) measured during 8 training days in males and 10 days in females.
Fig. 2. Mean escape latencies ( F S.E.M.) to reach the hidden platform during the choice trial of the second daily session over 8 days in male and female rats. No sex differences were found in the spatial working memory task (Student’s t-test).
2.72, P < 0.01]. No sex differences were found in escape latencies or path lengths recorded on the last training day. 3.2. Cytochrome oxidase activity CO staining of the selected nuclei from the mammillary body nearly overlaps the boundaries of the regions described in Paxinos and Watson’s atlas (Fig. 3). Regression analyses applied to the CO activity standards obtained from the liver tissue showed high correlation coefficients between CO activity and section thickness in all cases (R2>0.9; P < 0.05). Because the naı¨ve groups corresponding to the
Fig. 3. Photomicrograph of a coronal section from a control male rat at the level of the mammillary bodies, histochemically stained for CO activity. It is remarkable the high CO activity of the MB nuclei as shown by its darker staining as compared with the surrounding brain tissue. LM: lateral mammillary nucleus; MM: medial mammillary nucleus; f: fornix; mt: mammillothalamic tract; mtg: mammillotegmental tract. Scale bar = 600 Am.
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Fig. 4. Mean relative optical density of cytochrome oxidase staining measured in the MM nucleus of males and females. Significance of group differences for each sex: *P < 0.05 and **P < 0.01, one-way ANOVA. WM: working memory group; RM: reference memory group; CTR: control or untrained group.
WM and RM groups had a similar swimming experience, preliminary statistical analyses of CO activity in the selected brain regions were performed for each sex. No statistically significant differences between both naı¨ve groups (WM and RM) of each sex were found regarding CO activity in the MB nuclei and basolateral amygdala. Therefore, in order to simplify the experimental design and statistical analyses of CO activity data, we decided to use CO data of one naı¨ve group of each sex. Significantly higher CO activity was found in WM males compared to the other male groups in the MM nucleus [ F(3,33) = 15.21, P < 0.001]. In female rats, CO activity was significantly different among the experimental groups [ F(3,32) = 3.34, P < 0.05]. Post hoc analysis using Tukey’s tests showed higher CO activity in the WM
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Fig. 6. Mean relative optical density of cytochrome oxidase staining measured in the BL amygdala of males and females. No statistically significant group differences were found for each sex. See Fig. 4 for abbreviations used.
females as compared to both the naı¨ve and control groups ( P < 0.05; Fig. 4). Similar results were found for the LM nucleus because WM males showed the highest CO activity compared to the other groups [ F(3,33) = 9.90, P < 0.001]. Moreover, WM females had higher CO activity levels in this nucleus compared to the other groups [ F(3,32) = 3.89, P < 0.05]. Further analysis demonstrated that WM females had higher CO activity compared to the naı¨ve and control groups ( P < 0.05; Fig. 5). No significant CO activity differences were detected in the BL amygdala in groups of both sexes (Fig. 6). Analysis of differences between the MM and LM nuclei in each group showed significantly higher values in the LM nucleus in males (paired t-test, P < 0.01) and females (paired t-test, P < 0.01) trained in the WM task. Furthermore, CO activity levels measured in this nucleus correlated negatively with escape latencies recorded the last training day in the choice trial of the WM task in males (r = 0.74, P < 0.01) and females (r = 0.68, P < 0.05).
4. Discussion
Fig. 5. Mean relative optical density of cytochrome oxidase staining measured in the LM nucleus of males and females. Significance of group differences for each sex: *P < 0.05 and **P < 0.01, one-way ANOVA. See Fig. 4 for abbreviations used.
Our results showed an increase in CO activity in the MB of male and female rats after performing a spatial working memory task. However, changes in CO activity of these regions were not observed after training in the group of animals performing the spatial reference memory task. The differences in CO activity found in these two spatial learning tasks could be explained by a differential participation of the MB in the working memory task. Our results agree with previous studies based on MB lesions in which specific deficits in spatial working memory have been consistently reported, studied by different experimental procedures [44,55,62,64]. Furthermore, other studies that
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used the 2-[3H] deoxyglucose uptake technique in rats [57], [14C] glucose in mice [9] and 2-[14C] deoxyglucose in rhesus monkeys [18,19] revealed increases in cerebral energy metabolism in the region of the MB in working memory tasks. Nevertheless, it is worth noting that qualitative differences exist between the techniques used to measure CO activity or to measure incorporation of glucose or radioactive analogs in their application to behavioral tests. More specifically, the CO technique shows long-term changes in endogenous metabolic potential of nervous tissue resulting from the whole training period, whereas techniques based on glucose incorporation are limited to analyzing short-term cellular energetic demands evoked at specific times during the training period [21]. We can, therefore, interpret this result functionally as the continual participation of the MB particularly in this working memory task. In fact, this working memory task involves remembering the location of the platform, which is only useful for a short-time period because the location is changed between the two daily sessions. Therefore, learning a new location of the platform would require sustained activity of the MB over the whole training period, in accordance with the nature of the CO technique. Another interpretation of the results obtained could be based on the role of the MB in emotional behavior. The MB are considered to be involved in controlling anxiety because they are among the main sites of action of anxiolytic agents [31,75]. Lesion of the MB reduces anxiety expressed as increased exploration of the open arms in the elevated plus-maze [4]. Hence, the increased activity of the MB associated with performance of the working memory task can be explained in relation to the anxiety generated by the high demands of this task. However, changes were not detected in the oxidative metabolism in the BL amygdala in any of the learning groups compared to the control and naı¨ve groups. The BL amygdala is more closely related to emotional behavior than the MB, especially with fear and anxiety [15]. In contrast with the MB, lesions of the BL amygdala do not affect spatial learning in the water maze [16]. This is also a preferential site of action of anxiolytic drugs and administration of these has been shown to reduce the energy metabolism of this brain region [32]. Other recent studies show that the BL amygdala present increased c-fos expression associated with drug-induced anxiety [59]. Moreover, increased CO activity is observed in older compared to young rats due to stress associated with learning in the water maze [71]. Therefore, the increase in CO activity detected in the MB in the group that performed the spatial memory task seems to be more associated with the mnemonic rather than the emotional components of the task. In the reference memory task, there are several possible explanations for the lack of changes in CO activity in the MB after training. On the one hand, this result could suggest that the MB participate during a
short period of time in performance of this task and that the CO technique would not detect these transient changes in MB activity. On the other hand, as suggested by other authors, it is also possible that the MB are not involved in this type of task [2,54,64]. Thus, rats with lesions in the medial mammillary region have only modest impairments on a spatial reference memory task in the Morris water maze [54]. Current research suggests that the MB specifically contribute to the learning of new locations in a familiar environment [68], as occurs in spatial working memory tasks. In addition, our results indicate that not only the medial mammillary nucleus but also the lateral nucleus seem to participate in spatial working memory tasks in both sexes. It should be taken into account that the LM nucleus presents head direction cells that would be associated with the spatial navigation required for this type of learning [6,60]. It has been proposed that the LM nucleus can be the main source of generation of the head direction signal [7,58]. Therefore, the higher CO activity found in LM nucleus as compared to the MM nucleus could reflect the differential participation of these two MB nuclei in this type of spatial memory task. In this regard, the inverse correlation found between CO activity of the LM nucleus and escape latencies measured in the WM task could be associated with its specific recruitment for this task. On the other hand, the specific role of the MM nucleus in spatial memory still remains to be determined. One possible hypothesis to explain our results would be that changes in the synchronization of the theta rhythm between the MM nucleus and other limbic structures in response to environmental changes, such as the frequent change in position of the escape platform, could be related with the increased metabolic demands of this nucleus. Hence, in the case of new sensory stimuli, when the theta frequency is more regular, synchronization of this rhythm between the MM nucleus and other structures involved in spatial learning is high [34]. The parallel activation of the LM nucleus could be associated with its involvement in updating the head direction cells, the activity of which is modulated by several sensory signals including vestibular, visual and proprioceptive information [10,23,58]. Therefore, MM and LM nuclei would have complementary roles in the mnemonic and navigational aspects of spatial learning, respectively. In summary, these results suggest an important differential role of the MB and their nuclei in spatial memory tasks. CO histochemistry appears to be sensitive at detecting changes in prolonged neuronal activity as a consequence of relatively more complex tasks compared to traditional conditioning paradigms in which this technique was used previously [50]. Taking into account the value of CO histochemistry in this type of spatial learning study, future works could also focus on other cerebral regions in an attempt to gain more knowledge of the cerebral systems involved in spatial memory.
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Acknowledgements We thank the laboratory technicians Piedad Burgos and Begon˜a Valde´s for their essential contribution to this work. This work was supported by grants MCT BSO 2001 –2757 (Ministry of Science and Technology, Spain) and PR-01GE-2 (Principado de Asturias, Spain).
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Research report
Involvement of the mammillary bodies in spatial working memory revealed by cytochrome oxidase activity Ne´lida M. Conejo a,*, He´ctor Gonza´lez-Pardo a, Guillermo Vallejo b, Jorge L. Arias a a
Laboratory of Psychobiology, Faculty of Psychology, University of Oviedo, Plaza Feijoo, s/n E-33003, Oviedo, Asturias, Spain b Methodology Area, Faculty of Psychology, University of Oviedo, Plaza Feijoo, s/n E-33003, Oviedo, Asturias, Spain Accepted 23 March 2004 Available online 27 April 2004
Abstract In view of the inconclusive findings relating the nuclei of the mammillary bodies (MB) with spatial memory, we evaluated the oxidative metabolic activity of the medial and lateral nuclei of the mammillary bodies (MB) after training young rats (30 days) of both sexes in the Morris water maze. Different groups were trained in spatial working (WM) or reference memory (RM) tasks, respectively. The corresponding naı¨ve groups swam for the same amount of time as the trained groups but without the escape platform. Control groups were added that had not been manipulated in any way. No sex-related differences were detected in the working memory task although males exhibited better reference memory than females. Cytochrome oxidase (CO) activity, an endogenous metabolic marker for neuronal activity, was measured in all the groups. CO activity increased significantly in both MB nuclei of male and female rats only in the spatial working memory group. In addition, high CO activity in the lateral nucleus of the MB was linearly correlated with lower escape latencies in both sexes after training in the working memory task. No CO activity changes were found in the basolateral amygdala (BL) in any of the experimental groups. This nucleus was used as a control brain region because of its participation in emotional behavior. The results suggest a specific role of the MB nuclei in spatial working memory in both sexes. D 2004 Elsevier B.V. All rights reserved. Theme: Neural basis of behavior Topic: Learning and memory: systems and functions Keywords: Spatial memory; Mammillary body; Basolateral amygdala; Cytochrome oxidase; Neuronal metabolism
1. Introduction The mammillary bodies (MB) have traditionally been associated with memory processes. In humans, the amnesic nature of Korsakoff syndrome is often related with damage to the MB [26,40,65,70]. However, some clinical studies have demonstrated that amnesic symptoms in patients with this syndrome must also be due to damage in other diencephalic regions [36,38,39,70,76]. In fact, the specific role of the MB in memory and learning is still controversial in spite of the amount of research done on experimental models. Abbreviations: BL, basolateral amygdala; CO, cytochrome oxidase; f, fornix; LM, lateral mammillary; MB, mammillary bodies; MM, medial mammillary; mt, mammillothalamic tract; mtg, mammillotegmental tract; OD, optical density * Corresponding author. Tel.: +34-985-103212; fax: +34-985-104144. E-mail address: [email protected] (N.M. Conejo). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.03.025
Owing to the direct anatomical relationship between the MB and the hippocampus via the fornix, most research has focused on the association between these and spatial memory [19,55,64,68]. The MB have been involved in the delayed alternance in the T maze [27,37,56], place preference [1,45], working memory (WM) in radial maze [56,62,64] and Morris water maze [55,68]. The MB are composed of several well-defined nuclei. The largest of these are the medial mammillary (MM) and lateral mammillary (LM) nuclei. Current evidence points to a probable differential participation of these nuclei in spatial memory. In particular, the MM nucleus has been shown to be directly involved in spatial working memory tasks by lesion techniques and methods measuring c-fos protein expression [17,54,63,68]. Electrophysiological data have shown that the cells of this nucleus fire in bursts synchronized with the theta rhythm of the hippocampus
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[3,8,33 –35] and that this rhythmic activity is dependent upon the action of the hippocampus on this nucleus [8,33]. It is thought that the theta frequency could modulate the spatial memory processes [33,46,69]. In contrast, this same activity was only detected in a small proportion of cells of the LM nucleus [60]. On the other hand, neurons of this nucleus are specifically activated in relation to the position of the animals head irrespective of its location and are referred to as ‘‘head direction cells’’ [6,60]. Similarly, theoretical models have been proposed that specifically implicate neurons of the LM nucleus in spatial working memory tasks [24,58]. However, in spite of these models, it has not yet been clearly confirmed that the LM nucleus plays a role in spatial learning. On the other hand, the biological determinant, sex, is known to clearly affect performance in spatial learning tasks. Hence, in the Morris water maze, male rats usually perform reference memory (RM) tasks better than females [5,12,52, 53,61,72]. Reference memory refers to memory for spatial information that does not change with time, while working memory requires retention of information that is only useful for short intervals of time [47]. However, sex differences have not been consistently detected in reference memory tasks [30,66,67]. Sex differences in performance in these spatial tasks seem to be restricted to the early postnatal development and may reflect a difference in maturation rate together with an important role of sex hormones on brain development [11,12,51]. Conversely, in spatial working memory tasks assessed by the Morris water maze, no differences have been reported between rats of both sexes [25]. The aim of our work was to study the different involvement of the MM and LM nuclei in these two types spatial memory and the relevance of these nuclei in the learning of these tasks in both sexes. In order to avoid the influence of the estrous cycle of these females in this type of task [14], here, we have used 30-day-old male and female rats. Particularly, sex-related differences have been previously reported in spatial learning at this age [12,13]. Accordingly, if an MB nucleus would be effectively required for spatial memory, then we would expect its activation in both sexes after learning the task. In order to evaluate the participation of MB nuclei in spatial memory, we used cytochrome oxidase (CO) histochemistry. CO is a key enzyme in cellular oxidative energy metabolism because it participates in the mitochondrial electron transport chain involved in the direct production of energy as ATP using most of the available oxygen. Local CO activity is considered to be a reliable marker of brain oxidative metabolic potential, due to the close relationship between energy metabolism and neuronal activity [74]. In contrast to other metabolic markers, CO activity can be used as an index of the sustained energy requirements of nervous tissue associated with brain function [20,22]. Moreover, this technique has been shown to be sufficiently sensitive to detect changes in local cerebral oxidative metabolism induced after learning [43,48,50].
2. Material and methods 2.1. Animals Wistar rats aged 30 days were obtained from the University of Oviedo central vivarium. They were housed under standard conditions (12 h light– dark cycle with lights on from 8.00 to 20.00 h), at constant room temperature of 21 F 2 jC with ad lib access to food and water. All experimental procedures carried out with animals were approved by a local veterinary committee from the University of Oviedo vivarium and subsequent handling strictly followed the European Communities Council Directive 86/609. 2.2. Apparatus Animals were trained in a Morris water escape task using a 1.5-m-diameter black fiberglass pool, 75 cm high and 50 cm above the floor. The pool was filled with tap water to a height of 32 cm and a black escape platform was placed 2 cm beneath the water surface. The water temperature was kept at 23 jC during the entire test period. The experimental room had numerous visual cues such as colored maps, posters and plastic dishes fixed on the walls, a shelf, covered windows and a table. Lighting was provided by two halogen spotlights (500 W) placed on the floor and facing the walls. Animal cages were kept outside the experimental room to avoid odor cues and the bucket where the rats remained between consecutive trials was placed randomly around the pool during each trial. All swim paths of the animals were recorded live and analyzed later using a computerized video-tracking system (Ethovision Pro, Noldus, The Netherlands). 2.3. Behavioral procedures The pool was divided virtually into four quadrants, according to the cardinal points (N, S, E, W). During the habituation day, rats were released facing the pool wall from the central border of each quadrant following a pseudorandom sequence, four times each session. Each rat received two daily sessions spaced 1 h apart. Rats were returned to their home cages between sessions. The escape platform used on the first day was painted white and stood up 2 cm above the water surface. Rats were allowed to swim to locate the escape platform or placed on it after 60 s, where they remained for 15 s before they were placed in a black plastic bucket for 30 s. A standard protocol for spatial learning in the Morris water maze was used for the spatial reference memory (RM) female and male groups. After habituation to the maze, animals were trained daily using a single four-trial session. In each trial, the rats were released randomly from one of four start locations and had to search for a hidden escape platform beneath the water surface. The
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platform was located in the same position during the training days. To test spatial working memory (WM) in female and male groups, two sessions were performed daily and each session consisted of two consecutive trials (sample and choice). During sample trials, a visible platform was randomly located in one quadrant, and the rat was released from a randomly chosen start point. When the rat located the platform or when 60 s had elapsed, it remained on the platform for 15 s and was then returned to the bucket for 30 s. The platform location was the same for the next trial (choice) but was hidden beneath the water surface. In addition, animals were released from a different randomly chosen point in the choice trial. In the second daily session, the platform location changed randomly. Therefore, in the WM task, the position of the escape platform changed not only between both daily sessions but also between training days. In both spatial memory tasks, training ended when the mean escape latencies reached an asymptotic level. The naı¨ve animals used to compare with the WM groups swam four times a day during 1 min with no escape platform present (similar to four trials divided into two sessions of the WM groups: sample/choice, sample/ choice). In these naı¨ve groups, same intersession and intertrial intervals of the WM groups were used. The number of training days of the naı¨ve groups matched those required by WM groups. The naı¨ve groups used to compare with the RM groups swam also four times a day during 1 min with no escape platform (similar to the four consecutive trials of the RM groups in a single daily session). The number of training days of the naı¨ve groups matched those required by both the male and female RM groups, respectively. Finally, control groups without swimming experience were included to account for the effects of stress, handling and learning. 2.4. Tissue preparation Animals from naı¨ve and trained groups were deeply anaesthetized by intraperitoneal injection of sodium pentobarbital (100 mg/kg) 1 h after finishing the behavioral procedure in each group and immediately in control groups. Rats were transcardially perfused with a cold phosphate buffer solution (pH 7.4; 0.1 M). Their brains and medial hepatic lobes were quickly removed and immersed in a 20% sucrose buffer solution at 5 jC for 24 h. Tissues were embedded in a cryoprotective gel (OCT Compound; Miles USA) frozen in freon-22 cooled by liquid nitrogen and stored at 70 jC. Next, 20-Am-thick sections from the brains were obtained at 20 jC with a cryostat microtome (Microm, Heidelberg, Germany). Sets of rat liver sections were used as cytochrome oxidase (CO) activity standards because the hepatic tissue is histologically uniform and has an elevated intrinsic CO activity. Cryostat slides containing sections of different
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thickness (20, 40, 60 and 80 Am) were obtained from the medial hepatic lobe of intact adult male rats. 2.5. Cytochrome oxidase histochemistry Sections from the brains together with a complete set of standards with different CO activity were used to perform CO histochemistry. We used a modified version of the method originally described by Wong-Riley [73], based on the quantitative CO histochemical method developed by Gonzalez-Lima and Jones [22]. In brief, slides were lightly fixed for 5 min with a 1.5% glutaraldehyde solution, rinsed three times in phosphate buffer and incubated at 37 jC for 1 h in the dark and with continuous stirring in a solution containing 50 mg 3,3V-diaminobenzidine, 15 mg cytochrome c (Sigma, USA), 4 g sucrose per 100 ml phosphate buffer (pH 7.4; 0.1 M). The slides were rinsed three times with cold phosphate buffer, dehydrated and coverslipped with Entellan (Merck, USA). CO histochemical staining intensity was measured by densitometric analysis using a computer-assisted image analysis workstation (Leica Q550IW, Germany) composed of a computer-controlled microscope and light source (Leica DM-RHC), CCD camera and image analysis software (Leica Q-Win). A minimum of seven measurements were taken bilaterally in the mammillary bodies (MM and LM nuclei) from each animal and expressed as arbitrary units of optical density (OD). The basolateral nucleus of the amygdala (BL) was also measured, included as a control brain region to take into account the effects of fear or anxiety associated with the behavioral test. Delimitation of the mammillary nuclei and basolateral amygdala (BL) was performed according to the Paxinos and Watson’s [49] atlas. In order to establish comparisons and count for possible staining variations between brain sections from different staining baths, measurements were taken from CO-stained liver standards. Regression curves and coefficients (R2) between section thickness and CO activity measured from each set of standards were calculated for each incubation bath, and further calculations were performed to compare the regression plots obtained from each set of CO activity standards. Finally, OD values measured for the brain regions selected were put on the same level using the OD differences calculated from the regression plots of the liver standards. 2.6. Statistical analysis Escape latencies or path lengths in each trained group (WM males and females, RM males and females) were analyzed using one-way repeated measures ANOVA, with training day as the repeated-measures factor. Student’s ttests were used to compare escape latencies or path lengths between sexes on the last day of WM or RM training, respectively. Group differences for each sex (RM, WM,
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naı¨ve and control; n = 10 per group) in CO activity measured as relative OD values from each brain region (MM, LM and BL) were assessed by one-way ANOVAs. Post hoc tests were used to assess differences between means when ANOVA indicated significant overall differences. To evaluate differences in CO activity between the MM and LM nuclei, paired t-tests were used for each sex in the different groups. Pearson’s product – moment correlation coefficients (r) were calculated between behavioral scores and CO activity in the selected regions for each trained group.
3. Results 3.1. Behavioral tests 3.1.1. Reference memory task As shown in Fig. 1, escape latencies significantly decreased during training days both in males [ F(7,72) = 10.78, P < 0.001] and females [ F(9,80) = 12.53, P < 0.001]. Similarly, mean path lengths measured significantly decreased during training days in males [ F(7,72) = 11.45, P < 0.001] and females [ F(9,80) = 13.17, P < 0.001]. No sex differences were found in escape latencies or path lengths recorded on the last training day. 3.1.2. Working memory task Analysis of the escape latencies corresponding to the retention trial (choice) during the second daily session over the training days, revealed a significant decrease in the time used to find the hidden platform in males [ F(7,72) = 5.72, P < 0.001] and females [ F(7,72) = 2.68, P = 0.01] (Fig. 2). Path lengths measured in the retention trials also were significantly shorter with training in males [ F(7,72) = 3.14, P < 0.01] and females [ F(7,72) =
Fig. 1. Female rats showed significantly higher latencies to find the hidden escape platform as compared to males (Student’s t-test; P < 0.001) during training in the reference memory task. Mean escape latencies ( F S.E.M.) measured during 8 training days in males and 10 days in females.
Fig. 2. Mean escape latencies ( F S.E.M.) to reach the hidden platform during the choice trial of the second daily session over 8 days in male and female rats. No sex differences were found in the spatial working memory task (Student’s t-test).
2.72, P < 0.01]. No sex differences were found in escape latencies or path lengths recorded on the last training day. 3.2. Cytochrome oxidase activity CO staining of the selected nuclei from the mammillary body nearly overlaps the boundaries of the regions described in Paxinos and Watson’s atlas (Fig. 3). Regression analyses applied to the CO activity standards obtained from the liver tissue showed high correlation coefficients between CO activity and section thickness in all cases (R2>0.9; P < 0.05). Because the naı¨ve groups corresponding to the
Fig. 3. Photomicrograph of a coronal section from a control male rat at the level of the mammillary bodies, histochemically stained for CO activity. It is remarkable the high CO activity of the MB nuclei as shown by its darker staining as compared with the surrounding brain tissue. LM: lateral mammillary nucleus; MM: medial mammillary nucleus; f: fornix; mt: mammillothalamic tract; mtg: mammillotegmental tract. Scale bar = 600 Am.
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Fig. 4. Mean relative optical density of cytochrome oxidase staining measured in the MM nucleus of males and females. Significance of group differences for each sex: *P < 0.05 and **P < 0.01, one-way ANOVA. WM: working memory group; RM: reference memory group; CTR: control or untrained group.
WM and RM groups had a similar swimming experience, preliminary statistical analyses of CO activity in the selected brain regions were performed for each sex. No statistically significant differences between both naı¨ve groups (WM and RM) of each sex were found regarding CO activity in the MB nuclei and basolateral amygdala. Therefore, in order to simplify the experimental design and statistical analyses of CO activity data, we decided to use CO data of one naı¨ve group of each sex. Significantly higher CO activity was found in WM males compared to the other male groups in the MM nucleus [ F(3,33) = 15.21, P < 0.001]. In female rats, CO activity was significantly different among the experimental groups [ F(3,32) = 3.34, P < 0.05]. Post hoc analysis using Tukey’s tests showed higher CO activity in the WM
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Fig. 6. Mean relative optical density of cytochrome oxidase staining measured in the BL amygdala of males and females. No statistically significant group differences were found for each sex. See Fig. 4 for abbreviations used.
females as compared to both the naı¨ve and control groups ( P < 0.05; Fig. 4). Similar results were found for the LM nucleus because WM males showed the highest CO activity compared to the other groups [ F(3,33) = 9.90, P < 0.001]. Moreover, WM females had higher CO activity levels in this nucleus compared to the other groups [ F(3,32) = 3.89, P < 0.05]. Further analysis demonstrated that WM females had higher CO activity compared to the naı¨ve and control groups ( P < 0.05; Fig. 5). No significant CO activity differences were detected in the BL amygdala in groups of both sexes (Fig. 6). Analysis of differences between the MM and LM nuclei in each group showed significantly higher values in the LM nucleus in males (paired t-test, P < 0.01) and females (paired t-test, P < 0.01) trained in the WM task. Furthermore, CO activity levels measured in this nucleus correlated negatively with escape latencies recorded the last training day in the choice trial of the WM task in males (r = 0.74, P < 0.01) and females (r = 0.68, P < 0.05).
4. Discussion
Fig. 5. Mean relative optical density of cytochrome oxidase staining measured in the LM nucleus of males and females. Significance of group differences for each sex: *P < 0.05 and **P < 0.01, one-way ANOVA. See Fig. 4 for abbreviations used.
Our results showed an increase in CO activity in the MB of male and female rats after performing a spatial working memory task. However, changes in CO activity of these regions were not observed after training in the group of animals performing the spatial reference memory task. The differences in CO activity found in these two spatial learning tasks could be explained by a differential participation of the MB in the working memory task. Our results agree with previous studies based on MB lesions in which specific deficits in spatial working memory have been consistently reported, studied by different experimental procedures [44,55,62,64]. Furthermore, other studies that
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used the 2-[3H] deoxyglucose uptake technique in rats [57], [14C] glucose in mice [9] and 2-[14C] deoxyglucose in rhesus monkeys [18,19] revealed increases in cerebral energy metabolism in the region of the MB in working memory tasks. Nevertheless, it is worth noting that qualitative differences exist between the techniques used to measure CO activity or to measure incorporation of glucose or radioactive analogs in their application to behavioral tests. More specifically, the CO technique shows long-term changes in endogenous metabolic potential of nervous tissue resulting from the whole training period, whereas techniques based on glucose incorporation are limited to analyzing short-term cellular energetic demands evoked at specific times during the training period [21]. We can, therefore, interpret this result functionally as the continual participation of the MB particularly in this working memory task. In fact, this working memory task involves remembering the location of the platform, which is only useful for a short-time period because the location is changed between the two daily sessions. Therefore, learning a new location of the platform would require sustained activity of the MB over the whole training period, in accordance with the nature of the CO technique. Another interpretation of the results obtained could be based on the role of the MB in emotional behavior. The MB are considered to be involved in controlling anxiety because they are among the main sites of action of anxiolytic agents [31,75]. Lesion of the MB reduces anxiety expressed as increased exploration of the open arms in the elevated plus-maze [4]. Hence, the increased activity of the MB associated with performance of the working memory task can be explained in relation to the anxiety generated by the high demands of this task. However, changes were not detected in the oxidative metabolism in the BL amygdala in any of the learning groups compared to the control and naı¨ve groups. The BL amygdala is more closely related to emotional behavior than the MB, especially with fear and anxiety [15]. In contrast with the MB, lesions of the BL amygdala do not affect spatial learning in the water maze [16]. This is also a preferential site of action of anxiolytic drugs and administration of these has been shown to reduce the energy metabolism of this brain region [32]. Other recent studies show that the BL amygdala present increased c-fos expression associated with drug-induced anxiety [59]. Moreover, increased CO activity is observed in older compared to young rats due to stress associated with learning in the water maze [71]. Therefore, the increase in CO activity detected in the MB in the group that performed the spatial memory task seems to be more associated with the mnemonic rather than the emotional components of the task. In the reference memory task, there are several possible explanations for the lack of changes in CO activity in the MB after training. On the one hand, this result could suggest that the MB participate during a
short period of time in performance of this task and that the CO technique would not detect these transient changes in MB activity. On the other hand, as suggested by other authors, it is also possible that the MB are not involved in this type of task [2,54,64]. Thus, rats with lesions in the medial mammillary region have only modest impairments on a spatial reference memory task in the Morris water maze [54]. Current research suggests that the MB specifically contribute to the learning of new locations in a familiar environment [68], as occurs in spatial working memory tasks. In addition, our results indicate that not only the medial mammillary nucleus but also the lateral nucleus seem to participate in spatial working memory tasks in both sexes. It should be taken into account that the LM nucleus presents head direction cells that would be associated with the spatial navigation required for this type of learning [6,60]. It has been proposed that the LM nucleus can be the main source of generation of the head direction signal [7,58]. Therefore, the higher CO activity found in LM nucleus as compared to the MM nucleus could reflect the differential participation of these two MB nuclei in this type of spatial memory task. In this regard, the inverse correlation found between CO activity of the LM nucleus and escape latencies measured in the WM task could be associated with its specific recruitment for this task. On the other hand, the specific role of the MM nucleus in spatial memory still remains to be determined. One possible hypothesis to explain our results would be that changes in the synchronization of the theta rhythm between the MM nucleus and other limbic structures in response to environmental changes, such as the frequent change in position of the escape platform, could be related with the increased metabolic demands of this nucleus. Hence, in the case of new sensory stimuli, when the theta frequency is more regular, synchronization of this rhythm between the MM nucleus and other structures involved in spatial learning is high [34]. The parallel activation of the LM nucleus could be associated with its involvement in updating the head direction cells, the activity of which is modulated by several sensory signals including vestibular, visual and proprioceptive information [10,23,58]. Therefore, MM and LM nuclei would have complementary roles in the mnemonic and navigational aspects of spatial learning, respectively. In summary, these results suggest an important differential role of the MB and their nuclei in spatial memory tasks. CO histochemistry appears to be sensitive at detecting changes in prolonged neuronal activity as a consequence of relatively more complex tasks compared to traditional conditioning paradigms in which this technique was used previously [50]. Taking into account the value of CO histochemistry in this type of spatial learning study, future works could also focus on other cerebral regions in an attempt to gain more knowledge of the cerebral systems involved in spatial memory.
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Acknowledgements We thank the laboratory technicians Piedad Burgos and Begon˜a Valde´s for their essential contribution to this work. This work was supported by grants MCT BSO 2001 –2757 (Ministry of Science and Technology, Spain) and PR-01GE-2 (Principado de Asturias, Spain).
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