The Association Between Effective Dose of

Biol Trace Elem Res DOI 10.1007/s12011-016-0916-8

The Association Between Effective Dose of Magnesium and Mild Compulsive Exercise on Spatial Learning, Memory, and Motor Activity of Adult Male Rats Shahnaz Hajizade Ghonsulakandi 1 & Mahmuod Sheikh 1 & Marzieh Dehghan Shasaltaneh 2 & Samira Chopani 3 & Nasser Naghdi 3

Received: 18 July 2016 / Accepted: 14 December 2016 # Springer Science+Business Media New York 2017

Abstract One of the most important survival mechanisms is learning and memory processes. To emphasize the role of physical exercises and magnesium (Mg) in improvement of cognitive performance, we planned to investigate the effect of Mg and mild compulsive exercise on spatial learning and memory of adult male rats. Accordingly, we divided male Wistar rats into four groups: (I) control, (II) Mg treatment, (III) exercise, and (IV) Mg-exercise in the different dosages of Mg (0.5, 1, 1.5, and 2 mmol/kbw) were injected in the form of gavage during 1 week. Also, 1-week mild running on treadmill was used for exercise treatment. The Morris water maze (MWM) test and open field tool were used to evaluate spatial learning, memory, and motor activity, respectively. Our results clearly showed that 1 mmol/kbw Mg was applied as an effective dosage. Strikingly, 1-week mild exercise on treadmill had no significant effect on spatial motor activity, learning, and memory. Feeding 1 mmol/kbw Mg for a week showed a significant difference in learning and exploration stages. Compared to control animals, these results reveal exercise and Mg simultaneously had effect on learning and reminding. As a consequence, although mild exercise had no effect on motor activity and memory, Mg intake improved spatial learning, memory, and locomotor

* Nasser Naghdi [email protected] 1

Department of Physical Education, Sport Faculty, University of Tehran, Tehran, Iran

2

Laboratory of Neuro-organic Chemistry, Institute of Biochemistry and Biophysics (IBB), University of Tehran, Tehran, Iran

3

Physiology and Pharmacology Department, Pasteur Institute of Iran, Tehran, Iran

activity. The Mg feeding could be a promising supplemental treatment in the neurodegenerative disease. It is worthwhile to mention consumption of Mg leads to enhancement of memory, so animals find the hidden platform with the highest velocity. Keywords Memory . Learning . Motor activity . Magnesium . Treadmill . Rat

Introduction Learning and memory are fundamental functions of the brain which are affected by food and environmental factors [1]. Diet, in combination with environmental factors, plays a crucial role in shaping of the brain’s cognitive capacity [2]. Minerals especially cations are the effective factors on memory and learning. Magnesium (Mg) is one of the four main cations in the body with two positive charges, and the second intracellular cation which plays a major role in different metabolism processes and accelerates over 300 enzymatic reactions. Mg activates reactions of amino acids and protein synthesis. Furthermore, it plays a key role in many biological processes, such as muscle contraction, enzyme activity, blood clotting and nerve stimulation, ribosomes evolution, DNA and RNA reactions [3]. Mg deficiency causes specific behavioral effects that are related to a specific recipient or a particular region of the brain. Long-term of oral administration enhance the ability to store data in memory caches and increase the power to recall stored information. Recently, investigations show that Mg supplements improve short-term, long-term, and functional memory, but the difficulty of

Ghonsulakandi et al.

entering Mg to blood-brain barrier has limited its usage [1, 3–5]. Mg acts as an important factor for neural activity and releasing neurotransmitters [4]. Participating in guided route of nervous system is one of the remarkable functions of Mg. Moreover, it is one of the most vital micronutrients for basic activity of the brain. Mg ions affect Nmethyl-D-aspartate (NMDA) receptor that has an active site in NMDA receptor channel and blocks this receptor in rest phase [2]. Increasing of Mg concentrations in the brain cause more blocking of mentioned receptors channel and regulate decreasing these receptors [4]. Blocked receptors induce disorders related to memory processes dysfunction. So, NMDA is a target for Mg in the central nervous system. The mentioned ion has a little ability to cross the blood-brain barrier and even intravenous injection of Mg causes a slight increase in Mg level of cerebrospinal fluid [2]. Restriction of crossing the blood-brain barrier is a basic problem for the treatment of Mg deficiency in the central nervous system [6]. In order to prevent or reduce the side effects of medications on memory and learning processes, various methods have been suggested in which exercise is the useful one [7–9]. Evidence implies that treadmill and merry-go-round enhanced the formation and survival of new cells in the rodent’s hippocampus by development of cognitive performance [10–12]. Since exercise is one of the most essential reasons to release hormones and they play a significant role in learning and memory. Neurologically, assessment and evaluation of hormone concentrations and neurotransmitters are the interesting investigations subjects for scientific research. Increment of endorphins, which is released through pain, are one of the positive effects of physical activity. Enkephalin, the happiness hormone, refreshes and delights athletes. Also, its secretion increasing is with jolly games and sports. Suitable physical activity can reduce anxiety in athletes. Researchers have confidence that brain-derived neurotrophic factor (BDNF) is a pivotal transmitters for cell proliferation of hippocampus which is caused by exercise [13, 14]. Disruption of hippocampal neurogenesis is closely related to the destruction of learning and memory processes [15, 16]. It is well known that exercise has improvement effects on memory loss due to aging and degradation caused by disease [15]. Clinically, the exercise reduces the risk of cognitive disorders and difference methods of exercise can show distinct results on spatial learning and memory [17]. As the abovementioned, the role of Mg and exercise simultaneously on learning and memory has not been determined; therefore, in this study, after definition

effective dosages of Mg on learning and memory, mild exercises as well as the impact of these factors were surveyed to enhance memory, learning, and locomotion activity.

Material and Methods Animals In present study, mature Wistar male rats with 10 weeks age and weight range of 200–250 g were purchased from laboratory animal breeding sector of Pasteur Institute of Iran, Tehran, Iran. The numbers of groups are seven and each group consisted of 10 rats [18]. All efforts were made to minimize animal suffering [19]. Animals were housed under a controlled environment condition with dark-light cycle of 12:12 h, mean temperature of 25 ± 2 °C, relative humidity of 40%. The adequate resources (food and water) were provided to rats. All procedures were conducted on the principles of the Ethics Committee of the Pasteur Institute of Tehran, Iran, and use of laboratory animals, published by the International Association for the Study of Pain and the National Institutes of Health. Before applying any manipulation and implementation of the protocol, rats have been kept for a week at the new location to adapt to conditions before they were divided into seven groups. After 2 weeks of acclimatization of animal with new environment, experimental protocols were initiated.

Exercise Protocol Exercise protocol was performed weekly on a motorized rodent treadmill. Experimental group and Mg receiving animals were trained on a treadmill 30 min per day. The period of implementation of this protocol was from 7 A.M. to 10 A.M. Exercise was including jogging on the slope at zero degrees in three different speeds. In the beginning, the first 10 min speed was set at 4 m/min, in second 10 min speed increased to 7 m/min. Finally, animals were ran the third 10 min at 10 m/min [18].

Magnesium Sulfate Injection Magnesium sulfate, 0.5, 1, 1.5, and 2 mmol/kbw dissolved in 5 ml distilled water. Magnesium sulfate solution was injected to animals via gavage method every day for 7 days, 20 min after exercise in Mg-exercise group and 7 days for Mg group.

The Association Between Effective Dose of Magnesium

Behavioral Tests Learning and memory of rats were evaluated by Morris water maze (MWM) test [20]. The movement of animals was recorded through a camera on the top of maze which was prepared with infrared radiation and software named Ethovision 1.6 Noldus, Waninggen. This complex be able to measured total swimming distance, average swimming speed, and time and distance of rats that take over in each quadrant of the maze. The Behavior of the Water Maze Consists of Several Stages Acquisition Test Training protocol includes a block of four trials per session for 4 days [20]. In each trial, the rats were dumped into the water in front of the maze wall, from one of the four starting points (North East, North West, South East and South West). After reaching the platform, rats stayed on it for 20 s. This time allows the rat to remember platform place according to the sign outside the maze. If the rats are not able to find the platform after 60 s, they will be guided to the stage by the tester and rest there for 20 s. Probe Test To measure the amount of storage memory, probe test was performed in day 5, which included a 60-s period swimming without platform. The percentage of time spent and traveled distance in the target quadrant were evaluated. Open Field Apparatus Locomotion activity of animals was evaluated by open field apparatus. Definitely, all rats to the environment, 1 h before the exercise program were transported to the lab. Then, the opportunity of half an hour was given to every rat to be able to go to open field device and move freely. The total latency of mobility and swimming speed were assessed. Statistical Analysis In this study, all statistical analyses were done by using SPSS software (version 16, SPSS Inc., Chicago, IL, USA). One-way repeated measures ANOVA and independent t tests were used to comparing groups under study variables and followed by Tukey’s HSD post hoc. The significance results between groups were analyzed by Graph Pad Prism 5 software. Statistically significant differences were accepted at

P < 0.05. All error bars in figures were resulting from mean ± standard deviation (SD).

Results Effect of Different Dosages of Magnesium on Spatial Learning and Memory of Rats by MWM Test Acquisition Test No statistically significant difference in distance was observed between Mg treated and control group (F4,779 = 0.876, P > 0.05). Escape latency was significantly decreased in Mg treated by 1 mmol/kbw compared with other dosages of Mg and control groups (F4,779 = 2.781, P < 0.01). Swimming speed was significant between rats treated by Mg (0.5 mmol/kbw, 1 mmol/kbw, 1.5 mmol/ kbw) and control group (F 4,779 = 2.781, P < 0.001) (Fig. 1a–c). The results of distance of different dosages of Mg during 4 days in acquisition stage showed no significant difference compared to control group (F 4,48 = 0.190, P > 0.05). Escape latency of days 1, 2, and 4 of treated rats with Mg had no significant difference (F4,48 = 1.039, P > 0.05), but there was a significant difference in escape latency of day 3 between different dosages of Mg and control group (F4,48 = 10.926, P < 0.001) (Fig. 2a, b). Probe Test The Btime spent^ (%) and Bcrossing numbers^ (sec) in the target and opposite (zone 3) quadrants were evaluated by probe trial [20]. As the Fig. 3a showed, there was a significant difference b etween Mg-treated animals (1.5 mmol/kbw) and control group in opposite zone (F4,44 = 3.496, P < 0.05). A remarkable enhancement of the percentage of Btime spent^ in the target zone was between Mg effective dose (1.5 mmol/kbw) and control group (F4,44 = 4.093, P < 0.05). Additionally, the results of the crossing numbers showed a significant difference between Mg-treated animals (1 mmol/kbw) compared to other dosages of Mg and control groups in target quadrant (F4,44 = 2.368, P < 0.05) (Fig. 3a, b). The Effect of Different Dosages of Mg on Locomotor Activity of Rats by Open Field Apparatus The results of total latency of mobile and the mobility of rats treated by different dosages of Mg showed 2 mmol/ kbw of Mg observed a significant increment comparison


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repeated t test (F3,2601 = 3.759, P > 0.05). The results of escape latency observed a significant reduction between Mg-treated animals and control group (F3,299 = 15.163, P < 0.05). Swimming speed was significantly increased between Mg and control group (F 3 , 2 2 7 0 = 4.252, P < 0.001) (Fig. 5a–c). The observed results of distance treated by Mg 1 mmol/ kbw and control group had no effective role on spatial memory in acquisition test in 4 days of the training test (F17,16.876 = 1.305, P > 0.05). All rats treated by an optimum dosage of Mg and control group showed a significant reduction in escape latency during 4 days of training trial test, especially in day 3 (F17,10.742 = 5.463, P < 0.05), but other days had no significant difference compared to control group (F17,16.967 = 3.562, P > 0.05). All days of training test except days 2 and 4 showed a significant difference in the swimming speed between Mg-treated animals and control group (F17,16.997, 3.765, P < 0.05) (Fig. 6a–c). Probe Test

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Furthermore, the effect of Mg in acquisition step displayed that Mg influences on the learning and memory in the probe test. The Btime spent^ (%) showed a significant reduction between Mg and control group in the opposite zone (F17,15.299 = 0.529, P < 0.05) and a significant enhancement in the target quadrant (F17,16.156 = 0.033, P < 0.05). Consistently, comparison between Bcrossing number^ in the target and opposite zones showed a significant increase in Mg treated group in target quadrant rather than control in terms of the mentioned parameter (F17,16.332 = 1.539, P < 0.05) (Fig. 7a, b).

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Influence of an Optimum Dosage of Mg on Locomotor Activity of Rats by Open Field Apparatus

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Fig. 1 Effect of different doses of Mg treatment on the performance of training includes a distance, b escape latency, and c swimming speed (velocity) in a MWM test. All error bars in figures were resulting from mean ± SD. (b **P < 0.01 significant different doses of Mg and control group in escape latency; c ***P < 0.001 significant different doses of Mg and control group in swimming speed)

with other groups (F3,38 = 4.577, P < 0.001; F3,38 = 8.270, P < 0.001, respectively) (Fig. 4a, b). Effect of an Optimum Dosage of Mg on Spatial Learning and Memory During 4 Days of Acquisition Step Acquisition Test All rats treated by Mg (1 mmol/kbw) and control group showed no significant difference in distance during 4 days of training trial test which were evaluated according to

Our finding clearly showed that the total latency and time of motility (%) of treated rats by Mg (1 mmol/kbw) observed non-significant difference in comparison with control group (F17,16.581 = 0.676, P > 0.05) (data not shown). Effect of Exercise on the Improving of Spatial Learning and Memory of Rats by MWM Test Acquisition Test All treated rats by exercise has no significant difference in distance during 4 days of training trial test as well as control which were evaluated according to repeated t test (F317, 318 = 0.547, P > 0.05). The observed results of escape latency treated by exercise and control group have no significant difference (F317, 315 = 1.979, P > 0.05). No significantly statistical in swimming speed during 4 days (F317, 315 = 0.911, P > 0.05).


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Fig. 2 Trials performed in four continues days in different doses of Mg. a The mean value of distance. b The mean value of escape latency. Values are mean ± SD. (b ***P < 0.001 significant difference between day 3 of control and 1 mmol/kbw of Mg in escape latency. c **P < 0.01 significant difference between day 1 of control and 1.5 mmol/kbw of Mg in swimming speed, ***P < 0.001 significant difference between day 4 of control and 1 mmol/kbw of Mg in swimming speed)

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Fig. 3 Effect of different doses of Mg treatment in target and opposite quadrant. a The time spent in the target quadrant (%). b Crossing numbers (times). (a *P < 0.05 significant difference between control and 1.5 mmol/ kbw of Mg in zone 3 (opposite zone); # significant difference between control and 1 mmol/kbw of Mg in target zone; b *p < 0.05 significant difference between control and 1 mmol/kbw of Mg in target zone)

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The average of distance for locating to the hidden platform showed no significant difference between rats training with exercise and control group (F18,17.928 = 0.007, P > 0.05). Escape latency for locating to the hidden platform had no significant difference (F18,16.515 = 1.715, P > 0.05). No significant difference was observed in swimming speed during 4 days (F 18,14.054 = 1.067, P > 0.05) (data not shown). Probe Test The results of the comparison between treated rats by exercise and control group showed a significant reduction in the opposite quadrant (F18, 11.331 = 2.721, P < 0.05), but there was no significant difference in the crossing numbers between the mentioned groups in the both quadrants (F18,11.031 = 0.093, P > 0.05) (Fig. 8a, b).

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Influence of Exercise on Locomotor Activity of Rats by Open Field Apparatus The results showed total latency and time of motility (%) of treated rats by exercise comparison to control group were not observed a significant difference (F16, 15.99 = 0.350, P > 0.05, F16,15.806 = 0.081, P > 0.05, respectively) (data not shown). Effect of Mg and Exercise Simultaneously on the Improving of Spatial Learning and Memory of Rats by MWM Test Acquisition Test Distance for locating the platform below the water surface was significantly decreased between exercise-Mg and control group in the training trial test (F318, 310 = 7.082, P < 0.05). In addition, there was a significant reduction between the


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Fig. 4 Open field locomotor activity in different doses of Mg. a Total duration of highly walk. b The percentage time of highly walk for 30 min. (a ***P < 0.001 significant difference between control and 2 mmol/kbw of Mg. b ***P < 0.001 significant difference between control and 2 mmol/kbw of Mg)

mentioned groups in the escape latency in the acquisition test (F318, 306 = 14.432, P < 0.001). Swimming speed for locating the platform below the water surface was significant in comparison between exercise-Mg and control group (F318,317 = 0.0308, P < 0.001) (Fig. 9a–c). The effect of Mg-exercise simultaneously during 4 days showed a significance decreased in the distance of days 1 and 2 (F18,14.029 = 4.756, P < 0.05), but there was no significance difference in days 3 and 4 (F18,13.917 = 2.238, P > 0.05). Escape latency had a significance reduction in days 1 and 2 between Mg-exercise and control groups (F18,17.173 = 2.442, P < 0.001); nonetheless, no significance difference showed in days 3 and 4 (F18,17.173 = 0.054, P > 0.05). Interestingly, there was no significance difference during 4 days except in swimming speed had (F18,14.255 = 2.637, P > 0.05; F18,17.992 = 0.018, P < 0.001, respectively) (Fig. 10a–c).


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Fig. 5 Effect of an optimum dose of Mg (1 mmol/kbw) treatment on the performance of training includes a distance, b escape latency, and c swimming speed (velocity) in a MWM test. All error bars in figures were resulting from mean ± SD. (b *P < 0.05 significant difference between control and Mg; c ***P < 0.001 significant difference between control and Mg)

exercise-Mg groups in the target zone (F18,14.741 = 4.397, P < 0.05) (Fig. 11a, b). Influence of Exercise-Mg on Locomotor Activity of Rats by Open Field Apparatus The results showed total latency and the time of motility (%) of treated rats by exercise-Mg groups comparison to control g r o u p w e r e ob s e r v e d n o n - s i gn i f i c an t d i ff e r e n c e (F18,17.95 = 0.001, P > 0.05, F18, 17.420 = 0.075, P > 0.05, respectively) (data not shown).

Discussion and Conclusion Probe Test The Btime spent^ (%) in the opposite quadrants was significant reduction between exercise-Mg groups (F18,16.505 = 0.190, P < 0.05). The results of the Bcrossing numbers^ showed a significant enhancement was between

Roles of Mg in the metabolism of carbohydrates, fats, proteins, and electrolytes made it one of the most important mineral resources in shaping the brain’s cognitive capacity and performances. Our study seeks to address the effect of mild exercise, intake of Mg on


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Fig. 7 Effect of an optimum dose of Mg treatment in target and opposite quadrant. a The time spent in the target quadrant (%). b Crossing numbers (times). (a *P < 0.05 significant difference between control and Mg in zone 3 (opposite zone); # significant difference between control and Mg in target zone. b *P < 0.05 significant difference between control and Mg in target zone)

3. The exercise-Mg group: there is the significant effect was observed on the process of learning, probe stage, and locomotion activity.

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The Effect of Magnesium on Spatial Learning, Memory, and Locomotor Activity

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Fig. 6 Trials performed in four continues days in optimum dose of Mg. a The mean value of distance. b The mean value of escape latency. c The mean value of swimming speed (velocity) in control and Mg-treated rats. Values are mean ± SD. (b *P < 0.05 significant difference between control and Mg in day 3. c *P < 0.05 significant difference between control and Mg in days 2 and 4)

improvement of spatial learning, memory, and motor activity. 1. The exercise group: 1-week moderate intensity exercise had no significant effect on the learning stage, retrieval, and motor activity comparison to control. 2. The Mg group: Mg intake treatment (1 mmol/kbw) for 1 week enhanced spatial learning, memory, and motor activity significantly.

Consumption of long-term and oral Mg lead to improve an ability of animals to save information, increment a power to recall in memory caches and also increased motor activity significantly. Data provided in this report identified that Mg ameliorates glucose transporter to the membrane space, so it is one of the most important cofactors in the enzymatic systems such as glucose oxidation. Mg, as a main complements, improve different forms of memory including short-term, long– term, and working memory. One the other hand, it acts as a vital factor for neuronal activity and release neurotransmitters [4]. One of the remarkable roles of Mg is participating in the conduction of nervous systems and it is as important micronutrients for the basic activity of the brain. Mg influences NMDA receptor and it has a binding site on the channel


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Fig. 8 Effect of different doses of Mg treatment in target and opposite quadrant. a The time spent in the target quadrant (%). b Crossing numbers (times). (a *P < 0.05 significant difference between exercise and control in zone 3)

of the mentioned receptor, so Mg causes to block resting potential by taking place on the NMDA receptor [1]. A number of studies have suggested that enhancement of Mg in the brain leads to block of NMDA receptor channel. Thus, NMDA receptors are the purpose of Mg in the central nervous system. Mg has least ability to pass the blood-brain barrier [4]. According to this issue, environmental intake of Mg because of the limitation on crossing the blood brain-barrier consider as an necessary problem for treatment resulting from deficiency of Mg in the central nervous system. In terms of action mechanisms of Mg on memory, previous studies have shown that binding of ion to the specific site in the cationic channel of NMDA receptor which inhibits the mentioned receptor in the resting potential. It seems apparently contradictory with improvement characteristics of Mg on memory. In order to explain the contradiction, we could be said NMDA receptor is blocked during the resting potential by Mg and after depolarization of the post synaptic cells, the mentioned inhibition is removed. Blocking of the NMDA is not specific effect of Mg. On the other hand, it has been



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Fig. 9 Effect of optimum dose of Mg and exercise on the performance of training includes a distance, b escape latency, and c swimming speed (velocity) in a MWM test. All error bars in figures were resulting from mean ± SD. (a *P < 0.05 significance difference between Mg-exercise and control. b, c ***P < 0.001 significant difference between Mgexercise and control)

defined Mg leads to activate the receptor, enter Ca+2 and Na+, depolarization of post synaptic and activation of cells by increment release of glutamate from nerve terminal. The enhancement of calcium entry into the cell, the ion could create intracellular signaling cascades such as cAMP, PKA, and PKC which they control intracellular mechanisms to retain long-term memory [21]. The Effects of Exercise on Spatial Learning, Memory and Locomotor Activity In this study, a week mild exercise program running on a treadmill had no effect on the learning, memory, and locomotion activity [22]. Optional and mandatory exercises improve spatial learning, memory, and synaptic plasticity in the hippocampus. There is the contradictory due to differences in the length and intensity of exercise protocol [23]. Previous studies indicated that different types of exercise could regulate synaptic plasticity in the different areas of the brain, so they affected different influences on the learning and memory [24].

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Fig. 11 Effect of Mg-exercise in target and opposite quadrant. a The time spent in the target quadrant (%). b Crossing numbers (times). (a *P < 0.05 significant difference between control and Mg-exercise in zone 3 (opposite zone); b *P < 0.05 significant difference between control and Mgexercise in target zone)

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Therefore, exercise activates a number of factors which is effective in neurogenesis [25].

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Fig. 10 Trials performed in four continues days in Mg-exercise. a The mean value of distance. b The mean value of escape latency. c The mean value of swimming speed (velocity) in control and Mg-treated rats. Values are mean ± SD. (a *P < 0.05 significant difference between days 1 and 2 and control group. b ***P < 0.001 significant difference between days 1 and 2 and control group. c ***P < 0.001 significant difference between day 4 and control group)

On the other hand, some studies have also pointed out the kind of learning test could be impressed on the receiving different result [15]. Nevertheless, in this study, exercise showed no effects on learning and memory formation. The influence of sport on functions of some genes that encoding neurotrophins and other proteins support this point that exercise could provide structural alterations and neuronal plasticity in the brain. Several surveys have been shown short-term physical activity induced a number of new neurons cause to enhance BDNF in the hippocampus and dentate gyrus in the adult animals.

The Impact of Exercise and Magnesium on Learning, Spatial Memory, and Locomotor Activity There is a significant difference in learning and locomotion activity between Mg-exercise and control groups. According to the above results, we could be argued that 1-week mild exercise protocol unable to affect Mg individually effects. The effect of exercise during administrating is the same as Mg alone. But alone or combined with exercise could also have an effect on acquisition and probe stages. According to non-significant effect of 1-week compulsive exercise on learning and memory, it seems that high intensity protocol would be able to act as an effective factor on spatial learning, memory, and locomotion activity. In addition, the amount of administrated Mg affected spatial learning, memory, and motor activity in rats more significantly. The results confirmed that the remarkable effect of and Mg-exercise on learning and memory. In conclusion, Mg leads to enhance memory formation, so animals will find the hidden platform with the highest velocity.

The Association Between Effective Dose of Magnesium Acknowledgements The financial aspects of this study were supported by the University of Tehran and Pasteur Institute of Iran.

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Compliance with Ethical Standards All procedures were conducted on the principles of the Ethics Committee of the Pasteur Institute of Tehran, Iran, and use of laboratory animals, published by the International Association for the Study of Pain and the National Institutes of Health.

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Conflict of Interest The authors declare that they have no conflict of interest.

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