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Hippocampal mossy fiber sprouting induced by forced and voluntary physical exercise
Physiology & Behavior 101 (2010) 302–308
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Hippocampal mossy fiber sprouting induced by forced and voluntary physical exercise Michelle Toscano-Silva a, Sérgio Gomes da Silva a, Fulvio Alexandre Scorza b, Jean Jacques Bonvent c, Esper Abrão Cavalheiro b, Ricardo Mario Arida a,⁎ a b c
Departamento de Fisiologia, Universidade Federal de São Paulo (UNIFESP), Rua Botucatu 862, Ed. Ciências Biomédicas, 5° andar. Vila Clementino, 04023-900, São Paulo (SP), Brazil Departamento de Neurologia e Neurocirurgia. Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil Núcleo de Pesquisas Tecnológicas. Universidade de Mogi das Cruzes (UMC), Mogi das Cruzes, Brazil
a r t i c l e
i n f o
Article history: Received 17 December 2009 Received in revised form 25 February 2010 Accepted 21 May 2010 Keywords: Physical exercise Hippocampus Mossy fiber sprouting Synaptogenesis Treadmill Voluntary wheel running
a b s t r a c t Alterations in the function and organization of synapses have been proposed to induce learning and memory. Previous studies have demonstrated that mossy fiber induced by overtraining in a spatial learning task can be related with spatial long-term memory formation. In this work we analyzed whether physical exercise could induce mossy fiber sprouting by using a zinc-detecting histologic technique (Timm). Rats were submitted to 3 and 5 days of forced or voluntary exercise. Rat brains were processed for Timm's staining to analyze mossy fiber projection at 7, 12 and 30 days after the last physical exercise session. A significant increase of mossy fiber terminals in the CA3 stratum oriens region was observed after 5 days of forced or voluntary exercise. Interestingly, the pattern of Timm's staining in CA3 mossy fibers was significantly altered when analyzed 12 days after exercise but not at 7 days post-exercise. In contrast, animals trained for only 3 days did not show increments of mossy fiber terminals in the stratum oriens. Altogether, these results demonstrate that sustained or programmed exercise can alter mossy fiber sprouting. Further Investigations are necessary to determine whether mossy fiber sprouting induced by exercise is also involved in learning and memory processes. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Plastic events in the central nervous system, particularly alterations in the function and organization of synapses have been proposed to induce learning and memory [1]. These alterations can produce adaptations in the parameters of transmission and permanent synaptic reorganization [2]. The hippocampus plays a crucial role in the performance of spatial tasks, and the activity of its cells has been suggested to be related with spatial representation [3]. In line with these findings, it has been suggested that morphological changes underlie memory formation [4]. One of the most dramatic plastic events that have been associated with learning and memory is mossy fiber sprouting. Alternatively, in the hippocampus, mossy fiber sprouting has also been observed after experimentally induced epilepsy [5–8] as well as after high-frequency stimulation-inducing LTP [9,10]. Within the hippocampus, the mossy fibers have been shown to be critical for spatial learning, as chelation
⁎ Corresponding author. Tel.: + 55 11 55764513; fax: + 55 11 55739304. E-mail address: [email protected] (R.M. Arida). 0031-9384/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2010.05.012
of zinc from hippocampal mossy fibers impairs spatial learning in rats [11]. Timm's staining reveals zinc and it has been used to study changes in the distribution of mossy fiber synapses [12]. Some elegant studies have shown that mossy fiber sprouting occurs in the CA3 hippocampus area after overtraining animals in a Morris water maze task using Timm's staining [13,14]. Recently, Holahan et al. [15,16] provided evidence of structural plasticity induced by learning showing that training rats to locate a hidden platform in a water maze induces growth of hippocampal granule cell mossy fiber terminal fields from the stratum lucidum of CA3 into the stratum oriens and stratum pyramidale. In addition, preliminary findings demonstrate that motor skill learning stimulates synaptogenesis in the cortex [17]. As reported above, the mossy fiber sprouting observed after spatial learning might also be attributed to the locomotor activity induced by the water maze test. In this regard, we analyzed whether physical exercise per se could induce mossy fiber sprouting using two different exercise protocols, voluntary and forced exercise. To establish the time course of mossy fiber sprouting induced by exercise, animals were examined at 7, 12 and 30 days after the last physical exercise session. Furthermore, mossy fiber sprouting distribution throughout the septotemporal axis of the dorsal hippocampus was analyzed in 4 serial hippocampal sections.
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2. Experimental procedures
2.3. Blood samples and lactate concentration analyses
2.1. Animals
Blood samples were collected from the tail vein at rest and immediately at the end of the first and last day of the physical training in the treadmill. All blood samples were immediately deposited in Eppendorf tubes (1.5 ml capacity) containing 50 µl sodium fluoride (1%) and kept in ice. Lactate concentration was determined by the electroenzymatic method using a lactate analyzer (Yellow Springs 1500 Sport, USA).
Male Wistar rats (250–300 g) were used in this experiment. Animals were group-housed (5/cage) with free access to food and water throughout the experiment and maintained on environmentally controlled conditions to 12 h light–dark cycle (light on from 07:00–19:00) and temperature at 22–24 °C. Animals submitted to physical exercise were divided into voluntary and forced exercise groups. Histological analyses were firstly performed in animals submitted to 3 days of exercise to verify whether a short period of exercise could induce mossy fiber sprouting. For this purpose, histological analysis was performed 7 and 12 days post-exercise (voluntary 3 days (n = 7); forced 3 days (n = 6) for histology 7 days post-exercise and voluntary 3 days (n = 7); forced 3 days (n = 8) for histology 12 days post-exercise). A group of animals were maintained in the treadmill for the same time of the training group without being submitted to physical exercise (SHAM, n = 6). This group was used to assess if the treadmill cage could have a stressful component. The last group served as control (control, n = 10). Considering that no evident mossy fiber sprouting was noted after 3 days of exercise, we performed a second set of experiments consisting of 5 days of physical exercise divided into the following groups: voluntary 5 days (n = 6) and forced 5 days (n = 6) for histology 7 days postexercise; voluntary 5 days (n = 10) and forced 5 days (n = 10) for histology 12 days post-exercise; voluntary 5 days (n = 6) and forced 5 days (n = 6) for histology 30 days post-exercise and SHAM (n = 6) (Fig. 1). 2.2. Physical training procedure Animals from forced training group were familiarized with the apparatus for one day by placing them on a treadmill (Columbus instruments) for 10 min at a speed of 8 m/min at 0% degree incline. To provide a measure of trainability, we rated each animal's treadmill performance on a scale of 1–5 according to the following anchors [1, refused to run; 2, before average runner (sporadic, stop and go, wrong direction); 3, average runner; 4, above average runner (consistent runner, occasionally fell back on the treadmill); and 5, good runner (consistently stayed at the front of the treadmill)] [18]. Animals with a mean rating of 3 or higher were included in the exercise groups and those with a mean rating of 1 or 2 were excluded from the experiment. This procedure was used to exclude possible different levels of stress between animals. Three animals were excluded from treadmill running (with a mean rating of 1 or 2) and were not included to the control group. Rats were submitted to a moderate exercise program of 3 or 5 sessions on a treadmill. In the first training sessions, an initial speed was of 12 m/min and gradually increased at 13–15 m/min during the subsequent days for 30 min. Animals submitted to the voluntary exercise were placed in a voluntary wheel running for 3 or 5 days with free access to food and water. All experimental protocols were conducted in accordance to ethical committee of the University.
Fig. 1. Experimental design of physical exercise and neo-Timm protocol.
2.4. Histology Animals were sacrificed either 7, 12 or 30 days after the last training session. The neo-Timm method is a modification of the traditional Timm's method in order to increase the specific staining of zinc and thereby produce a more distinct visualization of the mossy fiber system [19]. Within 7, 12 or 30 days after the completion of the last session of exercise, the animals were deeply anaesthetized by pentobarbital 50 mg/kg injected intraperitoneally. They were then perfused transcardially with 25 ml of Millonigs's buffer (MB), followed by 50 ml of 0.1% solution of sodium sulphide (Na2S solution) dissolved in MB (pH 7.4). After this process, the perfusion was followed with 100 ml of 3% solution of glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and 200 ml of 0.1% solution of sodium sulphide diluted in MB. The brains were carefully dissected and kept in 30% sucrose solution diluted in glutaraldehyde for overnight postfixation. Coronal brain sections (40 m-thick) were obtained with a cryostat and disposed in phosphate buffer. Region-representative sections were mounted in glass slides and dried before staining procedure. The brain sections were processed for Timm's heavy metal staining with a solution containing Arabic gum, hydroquinone, citric acid and silver nitrate. The bregmas of the CA3 were selections at four levels in the septotemporal axis of the hippocampus (−2.30, − 3.30, −3.80, and −4.25 mm from bregma: Paxinos and Watson [20]) for posterior analysis. Fluoro-Jade B is a fluorescent dye that can specifically stain degenerating neurons in the CNS. In order to verify whether mossy fiber sprouting could occur due to neuronal loss the Fluoro-Jade B staining was performed to animals from all groups. For Fluoro-Jade B staining, control (n = 4), SHAM (n = 4), trained voluntary (n = 4) and forced (n = 4) animals for 5 days and analyzed 24 h and 12 days postexercise. They were deeply anaesthetized by pentobarbital 50 mg/kg injected intraperitoneally and then perfused transcardially with phosphate buffer followed by phosphate-buffered 4% paraformaldehyde. After this period, the brains were carefully dissected and kept in paraformaldehyde during 4 h and in phosphate buffer for overnight post-fixation. Coronal brain sections (40 μm) were obtained and mounted in glass slides and dried before staining procedure. The glass slides were immersed for 5 min in a solution of 1% sodium hydroxide in 80% ethanol and for 2 min in 70% ethanol. After rinsing in distilled water, the glass slides were immersed in a solution of 0.06% potassium permanganate for 15 min, then rinsed for 2 min and stained for 30 min in a solution prepared from 0.0004% of stock solution FluoroJade B (0.01%) in 0.1% acetic acid. Slices were then washed for 1 min in each of three changes of distilled water, rapidly dried for 10 min, transferred to 100% ethanol for 3 min and then to xylol for 3 min. The presence of sprouting of mossy fibers in the stratum oriens was evaluated using optical density. The sections selected in both the stratum oriens and stratum lucidum was digitalized with the same parameters by a camera (Sony) connected to an optical microscope of polarized light (model DMLP, Leica), equalized by application of the same contrast and brightness and then analyzed by Image Pro Plus v6.0 software. The area of Timm's staining with an optical density range between 120 and 255 points visualized for histogram was measured for automatic model in each image according to the methodology used in a previous study [14]. The results in percentage
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of the density of mossy fibers in the CA3 region were obtained and compared between groups. The analyses were conducted using Minitab software and graphs were created using Excel Office (Microsoft). The data showed a normal distribution according to the Kolmogorov–Smirnov test and were compared using one-way ANOVA with Tukey post hoc. For comparison of running distance between groups and lactate blood analysis the Student t test was used. In all cases, the statistical significance was set at p b 0.05. Fluoro-Jade B materials were examined under fluorescence with excitation filters revealing Fluoro-Jade B and digitalized for a camera connected to a microscope. 3. Results Animals from forced exercise (treadmill running) presented similar performance during the exercise protocol (Fig. 2). Statistical analysis did not demonstrate significant changes in running distance between groups (p N 0.05). There were no significant differences among animals submitted to voluntary activity in running distance. Furthermore, when running distance was computed for voluntary and forced running, no significant changes were observed among groups. In this study, mossy fiber density was analyzed in the hippocampus of animals at different time points, i.e., 7, 12 and 30 days after the last physical exercise session; in two different exercise period durations, 3 and 5 consecutive days, and for two types of physical exercise, voluntary and forced exercise (Fig. 1). 3.1. Timm's staining in animals submitted to 3 consecutive days of physical exercise A normal pattern of Timm's staining was observed in control and SHAM animals where mossy fibers were present almost exclusively in the stratum lucidum. Three days of exercise (forced or voluntary) with a latent period of 7 and 12 days to Timm's staining did not induce significant changes in CA3 area (control = 0.87 ± 0.14, SHAM = 0.78 ± 0.09, voluntary 3 days = 0.94 ± 0.09, forced 3 days = 1.02±0.14 for histology 7 days post-exercise — p N 0.05; control = 0.87 ± 0.14, SHAM = 0.74 ± 0.07, voluntary 3 days = 1.01 ± 0.14, forced 3 days = 0.95 ± 0.14 for histology 12 days post-exercise — p N 0.05). 3.2. Timm's staining in animals submitted to 5 consecutive days of physical exercise The pattern of Timm's staining in CA3 mossy fibers was dramatically altered when analyzed 12 days after physical exercise (forced or voluntary) but not at 7 days of this latent period (control = 0.87 ± 0.14, SHAM = 0.74 ± 0.07, voluntary 5 days = 0.83 ± 0.04, forced 5 days = 0.87 ± 0.06 for histology 7 days post-exercise — p N 0.05). Timm's-stained granules were observed bilaterally in the CA3 stratum
oriens in all voluntary and forced animals (Fig. 3), but not in control or SHAM animals (Fig. 3). Timm's staining in the dentate gyrus apparently remained unchanged. One-way ANOVA of Timm's positive surface area obtained by quantitative densitometric analysis of the stratum oriens revealed significant group differences (Fig. 4). Tukey post hoc analysis revealed increased CA3 Timm's positive area in stratum oriens in forced and voluntary animals compared with control and SHAM animals (control = 0.87 ± 0.14, SHAM = 0.74 ± 0.07, voluntary 5 days = 1.11 ± 0.11 — p = 0.002, forced 5 days = 1.09 ± 0.13 — p = 0.001 for histology 12 days post-exercise). No statistical difference was observed among exercise groups. Depending of the intensity of effort, physical exercise can be a stressful component, and for this purpose lactate concentration was determined. A significant reduction of blood lactate was observed immediately post-exercise in the first day of exercise program (before = 2.1 ± 1.3 mmol/L and after = 1.0 ± 0.2 mmol/L; p b 0.01). No significant changes were noted before and immediately postexercise in the last day of physical training (before = 1.9 ± 0.7 mmol/L and after = 1.7 ± 0.5 mmol/L; p N 0.05). To this point, all lactate concentrations remained bellow from 2 mmol after physical exercise. Thus, Fluoro-Jade B staining, a fluorescent marker that binds to irreversibly damaged neurons was performed 24 h and 12 days postexercise. No neuronal degeneration was observed in all groups, 24 h and 12 days post-exercise (Fig. 5). Concerning the distribution of mossy fiber sprouting throughout the rat hippocampus, Timm's-stained area was analyzed by the septotemporal axis. The analysis revealed significant differences between septotemporal measures, that is, Timm's-stained area in the stratum oriens decrease throughout the septotemporal measures (Fig. 3). Animals submitted to physical exercise presented more Timm's-stained granules throughout the septotemporal hippocampus (−2.30 to −3.30 mm) than the control and SHAM groups. In sum, mossy fiber sprouting was observed mainly in the septal portion of dorsal hippocampus and gradually decreases towards the temporal pole. Thus, Timm's staining for each bregma demonstrated significant differences in voluntary and forced groups when compared to control and SHAM groups (− 2.30 = voluntary (p = 0.03) and forced (p = 0.01), − 3.30 = voluntary (p = 0.001) and forced (p = 0.001); − 3.80 = voluntary (p = 0.004) and forced (p = 0.04); − 4.25 = voluntary (p = 0.01) and forced (p = 0.04)). 3.3. Timm's staining 30 days after the last exercise session in animals submitted to 5 consecutive days of physical exercise The pattern of Timm's staining in CA3 mossy fibers 30 days after the last exercise session was similar to the control and SHAM groups, i.e., mossy fibers were present almost exclusively in the stratum lucidum (control = 0.87 ±0.14, SHAM=0.74 ±0.07, voluntary 5 days = 0.79 ±0.13, forced 5 days= 0.9± 0.06 — p N 0.05). 4. Discussion
Fig. 2. Mean running distance (metres) in animals submitted to wheel and treadmill for 5 days during 30 min. No significant changes in running distance were noted between groups (p N 0.05).
Several researchers have shown a general correlation between mossy fiber sprouting (revealed by Timm histochemistry) and learning [13–16]. The mossy fiber sprouting observed in animals after several learning training sessions could also be related to a motor ability produced by physical training. In the present work, it was evidenced that both forced and voluntary exercise induced a remarkable increase of mossy fiber projection to the stratum oriens in rats. Mossy fiber density was analyzed in the hippocampus at different time points after the last physical exercise session (7, 12 and 30 days) and in two different exercise period durations (3 and 5 consecutive days). The pattern of Timm's staining in CA3 mossy fibers was dramatically altered in animals trained for 5 consecutive days of forced or voluntary exercise. Although previous studies have
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Fig. 3. Representative sections from control, SHAM, voluntary and forced groups through the dorsal hippocampal axis. Arrows highlight Timm's staining in CA3 mossy fibers. Magnification 50×. Scale bar = 100 μm.
Fig. 4. Timm's staining analysis. The graph shows the mean Timm-positive area of stratum oriens and stratum lucidum from control, SHAM, forced and voluntary groups. Forced and voluntary groups trained for 5 days. *Different from control and SHAM groups.
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Fig. 5. Representative sections of the Fluoro-Jade B (FJB) staining in hippocampal CA3 region from control (A), positive control (B), voluntary (C,D) and forced (E,F) exercise. No neuronal degeneration was observed in all groups, 24 h and 12 days post-exercise (C–F). Note the presence of FJB-positive neurons from positive control (post-status epilepticus). Magnification 100×. Scale bar = 100 μm.
demonstrated that overtraining in a spatial learning task induced mossy fiber sprouting [13,14], animals just allowed to swim did not show increments of mossy fiber terminals in the stratum oriens, that is, swim controls, that were allowed to swim for the same amount of time with no platform present, did not show mossy fiber sprouting. These animals were introduced in the water maze tank containing no platform, no spatial cues, and were allowed to swim for 1 min, 3 times per day, over 3 days. In accordance, another study showed that Timm's staining in the CA3 area was significantly greater in rats trained to find a hidden platform than rats trained to locate a visible platform and swim control rats [21]. The discrepancy of findings could be attributed to the exercise paradigm employed in our study. Firstly, the amount of time performed in the water maze was shorter than in animals submitted to treadmill or voluntary running (30 min per day in the treadmill and free access to voluntary running during 24 h). The type of exercise could also interfere in this process. Swimming has often confounded
the exertional stress of physical activity with the emotional stress of coercion. Therefore, it is difficult to distinguish general stress effects from effects exclusive to exercise. One study has revealed that repeated brief swims associated with learning (including the use of an escape platform) up-regulate hippocampal BDNF mRNA [22]. On the other hand, in another study, a paradigm involving prolonged time in room temperature water with no escape platform imposed stress on the animals rather than simply providing another means of physical activity [23]. This fact adds to the difficulty of interpreting brain changes in animals submitted to the swim model of exercise. The great limitation of swimming studies is the lack of knowledge of effort intensity performed by the rat. Some authors criticize researches using this type of exercise, i.e., influence of water temperature, stress caused by exercise (some researchers believe that animals struggle to survive in the water instead of performing a mere physical effort) and difficulty in the accuracy of the effort overload. However, if exercise training is applied adequately, positive effects can be found in the
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swim protocol. For instance, Ra et al. [24] demonstrated that both treadmill running and swimming increase the number of BrdU + cells in the dentate gyrus. Considering that treadmill running has the propensity, “if not adequately programmed” to induce both psychological and physical stress such as adrenal hypertrophy, elevated basal corticosterone levels or immunosuppression [25], care must be taken to provide a beneficial effect on brain plasticity. Therefore, the stress-evoking effects of treadmill training can be minimized if the intensity of effort is adequate. For this purpose, lactate concentration determined before and immediately post-exercise did not increase in animals before and after training. As previously reported, hippocampal learning can produce spatial expansions of mossy fiber projection, whereas chronic stress produces global reductions in postsynaptic spine densities [14,26–28]. Stress causes atrophy and in severe cases, death of hippocampal neurons [29–32]. Represa and Ben-Ari [33] stated that the abnormal activity related to kindling may be responsible for the morphologic changes observed in the stratum oriens. Excitotoxic treatments, which have been used as animal models for epilepsy, induce sprouting of the granule cell mossy fiber pathway in the hippocampus and lead to the occurrence of spontaneous seizures [5,34]. In this regard, to discard the possibility of association among mossy fiber sprouting and hippocampal cell death, a fluorescent marker (Fluoro-Jade B staining) that binds to irreversibly damaged neurons was performed 24 h and 12 days post-exercise. No neuronal degeneration was observed in all groups, suggesting that sprouting presented in trained animals was not related to a stress component, but to the exercise per se. The pattern of Timm's staining in CA3 mossy fibers was altered in animals trained for 5 consecutive days of exercise. These changes were observed 12 days but not after 7 days of physical exercise. Thus, 3 days of exercise, was not able to induce significant changes in the stratum oriens in both 7 and 12 days after the last exercise session. Our findings are in accordance to Ramírez-Amaya and collaborators [14] who demonstrated that mossy fiber sprouting occurred mainly in animals that were overtrained in the Morris water maze for 4 or 5 days and not in animals that received less training. Concerning the timing of mossy fiber sprouting occurrence in stratus oriens induced by exercise, Rekart et al. [21] reported that mossy fiber sprouting did not appear neither during the 5 days training period nor 2 days after training. In their investigation, only 7 days after training did the growth of the axon terminals become evident. In our study, mossy fiber sprouting was visible after a period of 12 days, in contrast to 7 days in previous studies [14,21]. The delay to this occurrence might be attributed to the different mechanisms of stimuli, i.e., learning versus exercise. Certainly, other stimuli following periods of strong activity, such as epilepsy induced by kainate injection or kindling [12,33], could also explain different time points of mossy fiber sprouting growth. For instance, Vaidya and collaborators [35] showed that the pattern of Timm staining in chronic electroconvulsive seizure treated animals was significantly different from SHAM animals six days following the last seizure treatment. Thus, Timm granules along the supragaranular zone of the dentate gyrus continued to increase and reached maximal levels 12 days after the last electroconvulsive seizure treatment. Interestingly, administration of a single electroconvulsive seizure did not alter the pattern of Timm staining, but five daily electroconvulsive seizures led to a significant increase in mossy fiber sprouting. These results indicate that mossy fiber sprouting requires more than one treatment and that the degree of sprouting induced in the mossy fiber pathway is dependent on the number of treatments. In accordance, 3 days of exercise in our study was not able to induce sprouting, however, 5 days was sufficient to observe significant Timm staining in the stratum oriens. Thus, the growth process occurs after learning and as previously shown, persists for at least 30 days [14]. In this line, to establish whether of mossy fiber sprouting persisted after physical
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exercise, Timm's staining pattern was also analyzed 30 days after the last exercise session. The pattern of Timm's staining in CA3 mossy fibers was similar to the control and SHAM groups, suggesting that mossy fiber sprouting induced by exercise, contrary to learning stimuli, is not long-lasting. Some hypotheses are raised to justify the nature of the exerciseinduced sprouting. An increased survival of new neurons in the dentate gyrus that are likely to project to CA3 cells has been demonstrated after spatial training [36–38]. In the same way, exposure to other novel environments increases granule cell proliferation [39]. If we assume that the newly formed granule cells also generate mossy fiber axons that form synapses on CA3 pyramidal cells, then the mossy fiber pathway also may be undergoing continuous remodeling. However, not all the mossy fiber sprouting is due to the generation of new granule cells [40], suggesting that existing granule cells can each innervate more CA3 pyramidal cells. In this regard, there are several evidences of mossy fiber sprouting induced both by learning and physical exercise. For instance, voluntary physical activity is known to enhance the performance of spatial learning [41] and passive avoidance memory in experimental animals [42]. Thus, van Praag and coworkers [43] verified whether physical activity, be it voluntary, forced or in combination with a learning task, would be involved in the increased adult hippocampal neurogenesis. Cell proliferation was only increased in mice housed with free access to the running wheel. Moreover, both voluntary physical activity and enrichment doubled the total number of surviving newborn cells in the dentate gyrus. In contrast, mice trained in the water maze and yoked-swim controls showed no change in BrdU-positive cell number, suggesting that this type of learning or activity alone is not an adequate stimulus for adult hippocampal neurogenesis. Based on the information above, we can suggest that swimming using the water maze protocol may not be adequate to produce mossy fiber sprouting, but programmed (treadmill) or sustained exercise for longer periods of time (wheel running) may induce mossy fiber sprouting. In this work, we also found evidence that increased mossy fiber projection to CA3 occurs mainly in the septal region of the dorsal hippocampus. Our data are consistent with other works which observed that mossy fiber sprouting occurs in the more septal area [14,21]. For instance, the restriction of growth to the rostral third of the septal hippocampus of hidden platform-trained rats observed in the Rekart and collaborators study [21] is consistent with findings that lesions of the septal but not the temporal hippocampus produce specific impairments on tasks requiring spatial processing [44–47]. In addition, the Ramírez-Amaya group [14] suggests the septal part of the hippocampus as of great relevance for spatial memory formation. In conclusion, studies in mice and rats have reported correlations between the extent of the mossy fiber projection and behaviors mediated by the hippocampal formation, showing that larger mossy fiber projections are frequently associated with superior performance. Thus, the significance of mossy fiber variations in real-life situations of mice and other species has been proposed. In this regard, Pleskacheva et al. [26] have proposed that size variations of mossy fibers can be related to habitat and lifestyle. The functional significance of the new mossy fibers is not yet clear, considering different forms of stimuli, i.e., learning, lesion or exercise to induce mossy fiber sprouting; however, different stimuli or altered lifestyle, such as physical exercise may alter mossy fiber sprouting and probably exert some involvement in the learning and memory process. Acknowledgments The authors would like to thank Hilda S. Reis for help with histological techniques. Research supported by CAPES, FAPESP, CNPq, INNT and CInAPCe (Brazil).
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Physiology & Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h b
Hippocampal mossy fiber sprouting induced by forced and voluntary physical exercise Michelle Toscano-Silva a, Sérgio Gomes da Silva a, Fulvio Alexandre Scorza b, Jean Jacques Bonvent c, Esper Abrão Cavalheiro b, Ricardo Mario Arida a,⁎ a b c
Departamento de Fisiologia, Universidade Federal de São Paulo (UNIFESP), Rua Botucatu 862, Ed. Ciências Biomédicas, 5° andar. Vila Clementino, 04023-900, São Paulo (SP), Brazil Departamento de Neurologia e Neurocirurgia. Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil Núcleo de Pesquisas Tecnológicas. Universidade de Mogi das Cruzes (UMC), Mogi das Cruzes, Brazil
a r t i c l e
i n f o
Article history: Received 17 December 2009 Received in revised form 25 February 2010 Accepted 21 May 2010 Keywords: Physical exercise Hippocampus Mossy fiber sprouting Synaptogenesis Treadmill Voluntary wheel running
a b s t r a c t Alterations in the function and organization of synapses have been proposed to induce learning and memory. Previous studies have demonstrated that mossy fiber induced by overtraining in a spatial learning task can be related with spatial long-term memory formation. In this work we analyzed whether physical exercise could induce mossy fiber sprouting by using a zinc-detecting histologic technique (Timm). Rats were submitted to 3 and 5 days of forced or voluntary exercise. Rat brains were processed for Timm's staining to analyze mossy fiber projection at 7, 12 and 30 days after the last physical exercise session. A significant increase of mossy fiber terminals in the CA3 stratum oriens region was observed after 5 days of forced or voluntary exercise. Interestingly, the pattern of Timm's staining in CA3 mossy fibers was significantly altered when analyzed 12 days after exercise but not at 7 days post-exercise. In contrast, animals trained for only 3 days did not show increments of mossy fiber terminals in the stratum oriens. Altogether, these results demonstrate that sustained or programmed exercise can alter mossy fiber sprouting. Further Investigations are necessary to determine whether mossy fiber sprouting induced by exercise is also involved in learning and memory processes. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Plastic events in the central nervous system, particularly alterations in the function and organization of synapses have been proposed to induce learning and memory [1]. These alterations can produce adaptations in the parameters of transmission and permanent synaptic reorganization [2]. The hippocampus plays a crucial role in the performance of spatial tasks, and the activity of its cells has been suggested to be related with spatial representation [3]. In line with these findings, it has been suggested that morphological changes underlie memory formation [4]. One of the most dramatic plastic events that have been associated with learning and memory is mossy fiber sprouting. Alternatively, in the hippocampus, mossy fiber sprouting has also been observed after experimentally induced epilepsy [5–8] as well as after high-frequency stimulation-inducing LTP [9,10]. Within the hippocampus, the mossy fibers have been shown to be critical for spatial learning, as chelation
⁎ Corresponding author. Tel.: + 55 11 55764513; fax: + 55 11 55739304. E-mail address: [email protected] (R.M. Arida). 0031-9384/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2010.05.012
of zinc from hippocampal mossy fibers impairs spatial learning in rats [11]. Timm's staining reveals zinc and it has been used to study changes in the distribution of mossy fiber synapses [12]. Some elegant studies have shown that mossy fiber sprouting occurs in the CA3 hippocampus area after overtraining animals in a Morris water maze task using Timm's staining [13,14]. Recently, Holahan et al. [15,16] provided evidence of structural plasticity induced by learning showing that training rats to locate a hidden platform in a water maze induces growth of hippocampal granule cell mossy fiber terminal fields from the stratum lucidum of CA3 into the stratum oriens and stratum pyramidale. In addition, preliminary findings demonstrate that motor skill learning stimulates synaptogenesis in the cortex [17]. As reported above, the mossy fiber sprouting observed after spatial learning might also be attributed to the locomotor activity induced by the water maze test. In this regard, we analyzed whether physical exercise per se could induce mossy fiber sprouting using two different exercise protocols, voluntary and forced exercise. To establish the time course of mossy fiber sprouting induced by exercise, animals were examined at 7, 12 and 30 days after the last physical exercise session. Furthermore, mossy fiber sprouting distribution throughout the septotemporal axis of the dorsal hippocampus was analyzed in 4 serial hippocampal sections.
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2. Experimental procedures
2.3. Blood samples and lactate concentration analyses
2.1. Animals
Blood samples were collected from the tail vein at rest and immediately at the end of the first and last day of the physical training in the treadmill. All blood samples were immediately deposited in Eppendorf tubes (1.5 ml capacity) containing 50 µl sodium fluoride (1%) and kept in ice. Lactate concentration was determined by the electroenzymatic method using a lactate analyzer (Yellow Springs 1500 Sport, USA).
Male Wistar rats (250–300 g) were used in this experiment. Animals were group-housed (5/cage) with free access to food and water throughout the experiment and maintained on environmentally controlled conditions to 12 h light–dark cycle (light on from 07:00–19:00) and temperature at 22–24 °C. Animals submitted to physical exercise were divided into voluntary and forced exercise groups. Histological analyses were firstly performed in animals submitted to 3 days of exercise to verify whether a short period of exercise could induce mossy fiber sprouting. For this purpose, histological analysis was performed 7 and 12 days post-exercise (voluntary 3 days (n = 7); forced 3 days (n = 6) for histology 7 days post-exercise and voluntary 3 days (n = 7); forced 3 days (n = 8) for histology 12 days post-exercise). A group of animals were maintained in the treadmill for the same time of the training group without being submitted to physical exercise (SHAM, n = 6). This group was used to assess if the treadmill cage could have a stressful component. The last group served as control (control, n = 10). Considering that no evident mossy fiber sprouting was noted after 3 days of exercise, we performed a second set of experiments consisting of 5 days of physical exercise divided into the following groups: voluntary 5 days (n = 6) and forced 5 days (n = 6) for histology 7 days postexercise; voluntary 5 days (n = 10) and forced 5 days (n = 10) for histology 12 days post-exercise; voluntary 5 days (n = 6) and forced 5 days (n = 6) for histology 30 days post-exercise and SHAM (n = 6) (Fig. 1). 2.2. Physical training procedure Animals from forced training group were familiarized with the apparatus for one day by placing them on a treadmill (Columbus instruments) for 10 min at a speed of 8 m/min at 0% degree incline. To provide a measure of trainability, we rated each animal's treadmill performance on a scale of 1–5 according to the following anchors [1, refused to run; 2, before average runner (sporadic, stop and go, wrong direction); 3, average runner; 4, above average runner (consistent runner, occasionally fell back on the treadmill); and 5, good runner (consistently stayed at the front of the treadmill)] [18]. Animals with a mean rating of 3 or higher were included in the exercise groups and those with a mean rating of 1 or 2 were excluded from the experiment. This procedure was used to exclude possible different levels of stress between animals. Three animals were excluded from treadmill running (with a mean rating of 1 or 2) and were not included to the control group. Rats were submitted to a moderate exercise program of 3 or 5 sessions on a treadmill. In the first training sessions, an initial speed was of 12 m/min and gradually increased at 13–15 m/min during the subsequent days for 30 min. Animals submitted to the voluntary exercise were placed in a voluntary wheel running for 3 or 5 days with free access to food and water. All experimental protocols were conducted in accordance to ethical committee of the University.
Fig. 1. Experimental design of physical exercise and neo-Timm protocol.
2.4. Histology Animals were sacrificed either 7, 12 or 30 days after the last training session. The neo-Timm method is a modification of the traditional Timm's method in order to increase the specific staining of zinc and thereby produce a more distinct visualization of the mossy fiber system [19]. Within 7, 12 or 30 days after the completion of the last session of exercise, the animals were deeply anaesthetized by pentobarbital 50 mg/kg injected intraperitoneally. They were then perfused transcardially with 25 ml of Millonigs's buffer (MB), followed by 50 ml of 0.1% solution of sodium sulphide (Na2S solution) dissolved in MB (pH 7.4). After this process, the perfusion was followed with 100 ml of 3% solution of glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and 200 ml of 0.1% solution of sodium sulphide diluted in MB. The brains were carefully dissected and kept in 30% sucrose solution diluted in glutaraldehyde for overnight postfixation. Coronal brain sections (40 m-thick) were obtained with a cryostat and disposed in phosphate buffer. Region-representative sections were mounted in glass slides and dried before staining procedure. The brain sections were processed for Timm's heavy metal staining with a solution containing Arabic gum, hydroquinone, citric acid and silver nitrate. The bregmas of the CA3 were selections at four levels in the septotemporal axis of the hippocampus (−2.30, − 3.30, −3.80, and −4.25 mm from bregma: Paxinos and Watson [20]) for posterior analysis. Fluoro-Jade B is a fluorescent dye that can specifically stain degenerating neurons in the CNS. In order to verify whether mossy fiber sprouting could occur due to neuronal loss the Fluoro-Jade B staining was performed to animals from all groups. For Fluoro-Jade B staining, control (n = 4), SHAM (n = 4), trained voluntary (n = 4) and forced (n = 4) animals for 5 days and analyzed 24 h and 12 days postexercise. They were deeply anaesthetized by pentobarbital 50 mg/kg injected intraperitoneally and then perfused transcardially with phosphate buffer followed by phosphate-buffered 4% paraformaldehyde. After this period, the brains were carefully dissected and kept in paraformaldehyde during 4 h and in phosphate buffer for overnight post-fixation. Coronal brain sections (40 μm) were obtained and mounted in glass slides and dried before staining procedure. The glass slides were immersed for 5 min in a solution of 1% sodium hydroxide in 80% ethanol and for 2 min in 70% ethanol. After rinsing in distilled water, the glass slides were immersed in a solution of 0.06% potassium permanganate for 15 min, then rinsed for 2 min and stained for 30 min in a solution prepared from 0.0004% of stock solution FluoroJade B (0.01%) in 0.1% acetic acid. Slices were then washed for 1 min in each of three changes of distilled water, rapidly dried for 10 min, transferred to 100% ethanol for 3 min and then to xylol for 3 min. The presence of sprouting of mossy fibers in the stratum oriens was evaluated using optical density. The sections selected in both the stratum oriens and stratum lucidum was digitalized with the same parameters by a camera (Sony) connected to an optical microscope of polarized light (model DMLP, Leica), equalized by application of the same contrast and brightness and then analyzed by Image Pro Plus v6.0 software. The area of Timm's staining with an optical density range between 120 and 255 points visualized for histogram was measured for automatic model in each image according to the methodology used in a previous study [14]. The results in percentage
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of the density of mossy fibers in the CA3 region were obtained and compared between groups. The analyses were conducted using Minitab software and graphs were created using Excel Office (Microsoft). The data showed a normal distribution according to the Kolmogorov–Smirnov test and were compared using one-way ANOVA with Tukey post hoc. For comparison of running distance between groups and lactate blood analysis the Student t test was used. In all cases, the statistical significance was set at p b 0.05. Fluoro-Jade B materials were examined under fluorescence with excitation filters revealing Fluoro-Jade B and digitalized for a camera connected to a microscope. 3. Results Animals from forced exercise (treadmill running) presented similar performance during the exercise protocol (Fig. 2). Statistical analysis did not demonstrate significant changes in running distance between groups (p N 0.05). There were no significant differences among animals submitted to voluntary activity in running distance. Furthermore, when running distance was computed for voluntary and forced running, no significant changes were observed among groups. In this study, mossy fiber density was analyzed in the hippocampus of animals at different time points, i.e., 7, 12 and 30 days after the last physical exercise session; in two different exercise period durations, 3 and 5 consecutive days, and for two types of physical exercise, voluntary and forced exercise (Fig. 1). 3.1. Timm's staining in animals submitted to 3 consecutive days of physical exercise A normal pattern of Timm's staining was observed in control and SHAM animals where mossy fibers were present almost exclusively in the stratum lucidum. Three days of exercise (forced or voluntary) with a latent period of 7 and 12 days to Timm's staining did not induce significant changes in CA3 area (control = 0.87 ± 0.14, SHAM = 0.78 ± 0.09, voluntary 3 days = 0.94 ± 0.09, forced 3 days = 1.02±0.14 for histology 7 days post-exercise — p N 0.05; control = 0.87 ± 0.14, SHAM = 0.74 ± 0.07, voluntary 3 days = 1.01 ± 0.14, forced 3 days = 0.95 ± 0.14 for histology 12 days post-exercise — p N 0.05). 3.2. Timm's staining in animals submitted to 5 consecutive days of physical exercise The pattern of Timm's staining in CA3 mossy fibers was dramatically altered when analyzed 12 days after physical exercise (forced or voluntary) but not at 7 days of this latent period (control = 0.87 ± 0.14, SHAM = 0.74 ± 0.07, voluntary 5 days = 0.83 ± 0.04, forced 5 days = 0.87 ± 0.06 for histology 7 days post-exercise — p N 0.05). Timm's-stained granules were observed bilaterally in the CA3 stratum
oriens in all voluntary and forced animals (Fig. 3), but not in control or SHAM animals (Fig. 3). Timm's staining in the dentate gyrus apparently remained unchanged. One-way ANOVA of Timm's positive surface area obtained by quantitative densitometric analysis of the stratum oriens revealed significant group differences (Fig. 4). Tukey post hoc analysis revealed increased CA3 Timm's positive area in stratum oriens in forced and voluntary animals compared with control and SHAM animals (control = 0.87 ± 0.14, SHAM = 0.74 ± 0.07, voluntary 5 days = 1.11 ± 0.11 — p = 0.002, forced 5 days = 1.09 ± 0.13 — p = 0.001 for histology 12 days post-exercise). No statistical difference was observed among exercise groups. Depending of the intensity of effort, physical exercise can be a stressful component, and for this purpose lactate concentration was determined. A significant reduction of blood lactate was observed immediately post-exercise in the first day of exercise program (before = 2.1 ± 1.3 mmol/L and after = 1.0 ± 0.2 mmol/L; p b 0.01). No significant changes were noted before and immediately postexercise in the last day of physical training (before = 1.9 ± 0.7 mmol/L and after = 1.7 ± 0.5 mmol/L; p N 0.05). To this point, all lactate concentrations remained bellow from 2 mmol after physical exercise. Thus, Fluoro-Jade B staining, a fluorescent marker that binds to irreversibly damaged neurons was performed 24 h and 12 days postexercise. No neuronal degeneration was observed in all groups, 24 h and 12 days post-exercise (Fig. 5). Concerning the distribution of mossy fiber sprouting throughout the rat hippocampus, Timm's-stained area was analyzed by the septotemporal axis. The analysis revealed significant differences between septotemporal measures, that is, Timm's-stained area in the stratum oriens decrease throughout the septotemporal measures (Fig. 3). Animals submitted to physical exercise presented more Timm's-stained granules throughout the septotemporal hippocampus (−2.30 to −3.30 mm) than the control and SHAM groups. In sum, mossy fiber sprouting was observed mainly in the septal portion of dorsal hippocampus and gradually decreases towards the temporal pole. Thus, Timm's staining for each bregma demonstrated significant differences in voluntary and forced groups when compared to control and SHAM groups (− 2.30 = voluntary (p = 0.03) and forced (p = 0.01), − 3.30 = voluntary (p = 0.001) and forced (p = 0.001); − 3.80 = voluntary (p = 0.004) and forced (p = 0.04); − 4.25 = voluntary (p = 0.01) and forced (p = 0.04)). 3.3. Timm's staining 30 days after the last exercise session in animals submitted to 5 consecutive days of physical exercise The pattern of Timm's staining in CA3 mossy fibers 30 days after the last exercise session was similar to the control and SHAM groups, i.e., mossy fibers were present almost exclusively in the stratum lucidum (control = 0.87 ±0.14, SHAM=0.74 ±0.07, voluntary 5 days = 0.79 ±0.13, forced 5 days= 0.9± 0.06 — p N 0.05). 4. Discussion
Fig. 2. Mean running distance (metres) in animals submitted to wheel and treadmill for 5 days during 30 min. No significant changes in running distance were noted between groups (p N 0.05).
Several researchers have shown a general correlation between mossy fiber sprouting (revealed by Timm histochemistry) and learning [13–16]. The mossy fiber sprouting observed in animals after several learning training sessions could also be related to a motor ability produced by physical training. In the present work, it was evidenced that both forced and voluntary exercise induced a remarkable increase of mossy fiber projection to the stratum oriens in rats. Mossy fiber density was analyzed in the hippocampus at different time points after the last physical exercise session (7, 12 and 30 days) and in two different exercise period durations (3 and 5 consecutive days). The pattern of Timm's staining in CA3 mossy fibers was dramatically altered in animals trained for 5 consecutive days of forced or voluntary exercise. Although previous studies have
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Fig. 3. Representative sections from control, SHAM, voluntary and forced groups through the dorsal hippocampal axis. Arrows highlight Timm's staining in CA3 mossy fibers. Magnification 50×. Scale bar = 100 μm.
Fig. 4. Timm's staining analysis. The graph shows the mean Timm-positive area of stratum oriens and stratum lucidum from control, SHAM, forced and voluntary groups. Forced and voluntary groups trained for 5 days. *Different from control and SHAM groups.
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Fig. 5. Representative sections of the Fluoro-Jade B (FJB) staining in hippocampal CA3 region from control (A), positive control (B), voluntary (C,D) and forced (E,F) exercise. No neuronal degeneration was observed in all groups, 24 h and 12 days post-exercise (C–F). Note the presence of FJB-positive neurons from positive control (post-status epilepticus). Magnification 100×. Scale bar = 100 μm.
demonstrated that overtraining in a spatial learning task induced mossy fiber sprouting [13,14], animals just allowed to swim did not show increments of mossy fiber terminals in the stratum oriens, that is, swim controls, that were allowed to swim for the same amount of time with no platform present, did not show mossy fiber sprouting. These animals were introduced in the water maze tank containing no platform, no spatial cues, and were allowed to swim for 1 min, 3 times per day, over 3 days. In accordance, another study showed that Timm's staining in the CA3 area was significantly greater in rats trained to find a hidden platform than rats trained to locate a visible platform and swim control rats [21]. The discrepancy of findings could be attributed to the exercise paradigm employed in our study. Firstly, the amount of time performed in the water maze was shorter than in animals submitted to treadmill or voluntary running (30 min per day in the treadmill and free access to voluntary running during 24 h). The type of exercise could also interfere in this process. Swimming has often confounded
the exertional stress of physical activity with the emotional stress of coercion. Therefore, it is difficult to distinguish general stress effects from effects exclusive to exercise. One study has revealed that repeated brief swims associated with learning (including the use of an escape platform) up-regulate hippocampal BDNF mRNA [22]. On the other hand, in another study, a paradigm involving prolonged time in room temperature water with no escape platform imposed stress on the animals rather than simply providing another means of physical activity [23]. This fact adds to the difficulty of interpreting brain changes in animals submitted to the swim model of exercise. The great limitation of swimming studies is the lack of knowledge of effort intensity performed by the rat. Some authors criticize researches using this type of exercise, i.e., influence of water temperature, stress caused by exercise (some researchers believe that animals struggle to survive in the water instead of performing a mere physical effort) and difficulty in the accuracy of the effort overload. However, if exercise training is applied adequately, positive effects can be found in the
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swim protocol. For instance, Ra et al. [24] demonstrated that both treadmill running and swimming increase the number of BrdU + cells in the dentate gyrus. Considering that treadmill running has the propensity, “if not adequately programmed” to induce both psychological and physical stress such as adrenal hypertrophy, elevated basal corticosterone levels or immunosuppression [25], care must be taken to provide a beneficial effect on brain plasticity. Therefore, the stress-evoking effects of treadmill training can be minimized if the intensity of effort is adequate. For this purpose, lactate concentration determined before and immediately post-exercise did not increase in animals before and after training. As previously reported, hippocampal learning can produce spatial expansions of mossy fiber projection, whereas chronic stress produces global reductions in postsynaptic spine densities [14,26–28]. Stress causes atrophy and in severe cases, death of hippocampal neurons [29–32]. Represa and Ben-Ari [33] stated that the abnormal activity related to kindling may be responsible for the morphologic changes observed in the stratum oriens. Excitotoxic treatments, which have been used as animal models for epilepsy, induce sprouting of the granule cell mossy fiber pathway in the hippocampus and lead to the occurrence of spontaneous seizures [5,34]. In this regard, to discard the possibility of association among mossy fiber sprouting and hippocampal cell death, a fluorescent marker (Fluoro-Jade B staining) that binds to irreversibly damaged neurons was performed 24 h and 12 days post-exercise. No neuronal degeneration was observed in all groups, suggesting that sprouting presented in trained animals was not related to a stress component, but to the exercise per se. The pattern of Timm's staining in CA3 mossy fibers was altered in animals trained for 5 consecutive days of exercise. These changes were observed 12 days but not after 7 days of physical exercise. Thus, 3 days of exercise, was not able to induce significant changes in the stratum oriens in both 7 and 12 days after the last exercise session. Our findings are in accordance to Ramírez-Amaya and collaborators [14] who demonstrated that mossy fiber sprouting occurred mainly in animals that were overtrained in the Morris water maze for 4 or 5 days and not in animals that received less training. Concerning the timing of mossy fiber sprouting occurrence in stratus oriens induced by exercise, Rekart et al. [21] reported that mossy fiber sprouting did not appear neither during the 5 days training period nor 2 days after training. In their investigation, only 7 days after training did the growth of the axon terminals become evident. In our study, mossy fiber sprouting was visible after a period of 12 days, in contrast to 7 days in previous studies [14,21]. The delay to this occurrence might be attributed to the different mechanisms of stimuli, i.e., learning versus exercise. Certainly, other stimuli following periods of strong activity, such as epilepsy induced by kainate injection or kindling [12,33], could also explain different time points of mossy fiber sprouting growth. For instance, Vaidya and collaborators [35] showed that the pattern of Timm staining in chronic electroconvulsive seizure treated animals was significantly different from SHAM animals six days following the last seizure treatment. Thus, Timm granules along the supragaranular zone of the dentate gyrus continued to increase and reached maximal levels 12 days after the last electroconvulsive seizure treatment. Interestingly, administration of a single electroconvulsive seizure did not alter the pattern of Timm staining, but five daily electroconvulsive seizures led to a significant increase in mossy fiber sprouting. These results indicate that mossy fiber sprouting requires more than one treatment and that the degree of sprouting induced in the mossy fiber pathway is dependent on the number of treatments. In accordance, 3 days of exercise in our study was not able to induce sprouting, however, 5 days was sufficient to observe significant Timm staining in the stratum oriens. Thus, the growth process occurs after learning and as previously shown, persists for at least 30 days [14]. In this line, to establish whether of mossy fiber sprouting persisted after physical
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exercise, Timm's staining pattern was also analyzed 30 days after the last exercise session. The pattern of Timm's staining in CA3 mossy fibers was similar to the control and SHAM groups, suggesting that mossy fiber sprouting induced by exercise, contrary to learning stimuli, is not long-lasting. Some hypotheses are raised to justify the nature of the exerciseinduced sprouting. An increased survival of new neurons in the dentate gyrus that are likely to project to CA3 cells has been demonstrated after spatial training [36–38]. In the same way, exposure to other novel environments increases granule cell proliferation [39]. If we assume that the newly formed granule cells also generate mossy fiber axons that form synapses on CA3 pyramidal cells, then the mossy fiber pathway also may be undergoing continuous remodeling. However, not all the mossy fiber sprouting is due to the generation of new granule cells [40], suggesting that existing granule cells can each innervate more CA3 pyramidal cells. In this regard, there are several evidences of mossy fiber sprouting induced both by learning and physical exercise. For instance, voluntary physical activity is known to enhance the performance of spatial learning [41] and passive avoidance memory in experimental animals [42]. Thus, van Praag and coworkers [43] verified whether physical activity, be it voluntary, forced or in combination with a learning task, would be involved in the increased adult hippocampal neurogenesis. Cell proliferation was only increased in mice housed with free access to the running wheel. Moreover, both voluntary physical activity and enrichment doubled the total number of surviving newborn cells in the dentate gyrus. In contrast, mice trained in the water maze and yoked-swim controls showed no change in BrdU-positive cell number, suggesting that this type of learning or activity alone is not an adequate stimulus for adult hippocampal neurogenesis. Based on the information above, we can suggest that swimming using the water maze protocol may not be adequate to produce mossy fiber sprouting, but programmed (treadmill) or sustained exercise for longer periods of time (wheel running) may induce mossy fiber sprouting. In this work, we also found evidence that increased mossy fiber projection to CA3 occurs mainly in the septal region of the dorsal hippocampus. Our data are consistent with other works which observed that mossy fiber sprouting occurs in the more septal area [14,21]. For instance, the restriction of growth to the rostral third of the septal hippocampus of hidden platform-trained rats observed in the Rekart and collaborators study [21] is consistent with findings that lesions of the septal but not the temporal hippocampus produce specific impairments on tasks requiring spatial processing [44–47]. In addition, the Ramírez-Amaya group [14] suggests the septal part of the hippocampus as of great relevance for spatial memory formation. In conclusion, studies in mice and rats have reported correlations between the extent of the mossy fiber projection and behaviors mediated by the hippocampal formation, showing that larger mossy fiber projections are frequently associated with superior performance. Thus, the significance of mossy fiber variations in real-life situations of mice and other species has been proposed. In this regard, Pleskacheva et al. [26] have proposed that size variations of mossy fibers can be related to habitat and lifestyle. The functional significance of the new mossy fibers is not yet clear, considering different forms of stimuli, i.e., learning, lesion or exercise to induce mossy fiber sprouting; however, different stimuli or altered lifestyle, such as physical exercise may alter mossy fiber sprouting and probably exert some involvement in the learning and memory process. Acknowledgments The authors would like to thank Hilda S. Reis for help with histological techniques. Research supported by CAPES, FAPESP, CNPq, INNT and CInAPCe (Brazil).
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