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The mechanism by which exercise modifies brain function
Physiology & Behavior, Vol. 60, No. 1, 177-181, 1996 Copyright © 1996 Elsevier Science Inc. Printed in the USA. All rights reserved I)031-9384/96 $15.00 + .00
ELSEVIER
PII 0031-9384(96)00011-2
The Mechanism by Which Exercise Modifies Brain Function D E N ' E T S U SUTOO 1 AND KAYO A K I Y A M A
Institute of Medical Science, University of Tsukuba, Tsukuba, Ibaraki 305, Japan Received 9 October 1995 SUTOO, D. AND K. AKIYAMA. The mechanism by which exercise modifies brain function. PHYSIOL BEHAV 60(1) 177-181, 1996.--The effect of exercise on central nervous system function was investigated in relation to the mechanism of calcium-calmodulin-dependent dopamine synthesis in the brain. It is shown here through animal experiments that exercise leads to an increase in the calcium level in the brain. This in turn enhances brain dopamine synthesis, and through this increased dopamine modifies and/or affects brain function, which might induce physiological, behavioral, and psychological changes. Exercise Calcium-calmodulin Dopamine synthesis Ethanol-induced sleep Microphotometry system Mice
Neostriatum
Nucleus accumbens septi
exposed to a 12-h light/dark cycle. Food and water were provided ad lib until the time of experiment and were not given during the exercise. All animals received humane care in compliance with the Guiding Principles for the Care and Use of Laboratory Animals formulated by the Japanese Pharmacological Society. For exercise, they were forced to run for 15-120 min at a speed of 20 m per min by using a programmed motor-driven wheel cage. Following the exercise, changes in the serum and brain calcium levels, brain dopamine level, and behavior were immediately analyzed. Experiments were carried out between 1000 and 1600 h.
ALTHOUGH exercise is known to produce a refreshing feeling, the mechanism by which exercise modifies brain function to produce this feeling has not been made clear. In previous investigations, our laboratory has examined the role of calcium ions in brain function. Studies demonstrated that calcium activates tyrosine hydroxylase (a rate-limiting enzyme for catecholamine synthesis) in the brain through a calmodulin-dependent system (16,20,22), and that an elevated dopamine regulates various functions, such as blood pressure, locomotor activity, and receptivity to drugs (16,19,21,24). This concept was developed in an attempt to elucidate the mechanism of convulsive seizures in epileptic mice, and it was demonstrated that convulsions are induced when dopamine synthesis in the brain is decreased, and that the convulsions increase the brain calcium level, which in turn rectifies disorders of brain dopamine synthesis and of brain function (17,23). Therefore, it was hypothesized that exercise may also affect brain function through action of calcium and biogenic amines such as dopamine, as has been shown in convulsive seizures in epileptic mice. This study was carried out to confirm this hypothesis.
Calcium Levels Calcium levels in the serum and brain were measured according to the method using an o-cresolphthalein complexone (13). Whole brain and blood were obtained from mice that had been forced to run for 15-120 min as well as from unexercised control mice. The serum was separated quickly and assayed. Brains were homogenized with saline at 4°C and centrifuged for 1 h at 25,000 × g (4°C), and after which the supernatant was assayed. Data were analyzed by an ANOVA and Dunnett's t-test for multiple comparisons between individual exercised groups and the unexercised control group.
METHOD
Animals Adult male mice of the ddY strain (20-25 g) were provided by Doken Co. Ltd. (Ibaraki, Japan). They were housed in groups of 8-10 in stainless steel cages at room temperature (22 + 2°C) for more than 1 week before use in the experiments and were
Dopamine Levels Brain dopamine levels were determined immunohistochemically and compared quantitatively. Three animal groups [10
i To whom requests for reprints should be addressed.
177
178
mice/group: unexercised control mice, mice exercised for 120 min, and unexercised mice pretreated IP with 40 /zmol/kg of CaC12 (0.48-0.54 ml of saline solution was injected 1 h before sacrifice)] were anesthetized with pentobarbital sodium and perfused intracardially with 50-ml solution of 0.1 M cacodylate and 1% sodium metabisulfite (SMB) containing 2.5% glutaraldehyde (pH 7.5). The whole brain was removed and postfixed with the same solution for 30 min, then frozen on dry ice. The frozen brain was sectioned coronally and continuously at 20-/xm thickness in a cryostat. For analysis of the distribution and amount of dopamine, brain slices located approximately 5 mm rostral from the interaural line (Fig. 2) were chosen in light of a previous report (20) that dopamine levels in the neostriatum and nucleus accumbens sept± regions of this section were increased by the intracerebroventricular (ICV) administration of CaCI 2. Immunohistochemical procedure was performed according to a previous report (20). The brain slices were washed three times with Tris-SMB buffer (Tris 0.05 M and SMB 0.85%, pH 7.5). The dopamine antibody was diluted l:800 with Tris-SMB buffer and 50 /zl of antibody solution was applied to each brain slice, then incubated at 4°C for 12 h. After the reaction, the slices were washed three times with phosphate-buffered saline (PBS). The second antibody, anti-rabbit IgG goat serum labelled with fluorescein isothiocyanate, was diluted 1:100 with PBS and applied to the sections. It was incubated in the dark at 20°C for 3 h, and then washed with PBS. Nonspecific fluorescence with glutaraldehyde was reduced by 0.1 M NaBH 4 in PBS. After being rinsed three times with PBS, the stained sections were embedded in 10% glycerin PBS, and kept at 4°C in a dark room until the measurements, performed within 10 h, could be carried out. The distribution of dopamine was quantitatively analyzed using a fluorescence microphotometry system (Nikon, Japan) (18). This system is a measuring microscope for distribution of immunohistochemical fluorescence intensity in the tissue slice. The average fluorescence intensity in a small brain region can be measured by a photomultiplier tube through a pinhole of a microscope. The brain slice is continuously moved in the x- or y-direction by means of a scanning stage under the objective lens of a microscope and the data in each brain region are collected. Fluorescence intensity values in each region can be indicated quantitatively in comparison with that of standard uranium glass. This system surpasses by two orders the quantitative linearity of image analyzers used with high-sensitivity TV cameras, and it also surpasses by three orders the sensitivity of high performance liquid chromatography with an electrochemical detector. The slices stained with the dopamine antibody were measured reticulately at 20-/xm intervals. The total fluorescence intensity value in each measuring point had the background value subtracted. The background value was obtained from slices that were treated with the same procedure without dopamine antibody. The average fluorescence intensity in each region was analyzed by an ANOVA and Newman-Keuls t-test for subsequent comparisons of three groups (20 slices/group).
Ethanol-Induced Sleeping Time When ethanol is injected IP at a dose of 3.6 g / k g , mice lose their righting reflex and lapse into a behavior resembling sleep for approximately 20 rain. The duration of this ethanol-induced sleep is regulated by brain biogenic amines and can be prolonged by ICV administration of dopamine or other biogenic amines (5,14,16). Therefore, it is possible to monitor the change in the brain dopamine level elicited by exercise in live mice through measurement of the duration of ethanol-induced sleep. We have already observed, using this measurement, that pretreatment of
SUTOO AND AKIYAMA
mice with CaC12 increases the brain dopamine level in a dose-dependent manner (16). In the first step of the investigation, ethanol (3.6 g / k g ) was injected IP into unexercised control mice and mice exercised for 15-120 rain, and the duration of sleep was compared. Sleeping time duration is defined as the time from the loss of the righting reflex to the time when the righting reflex occurs twice within 30 s. As demonstrated in the Results section, the duration of ethanol-induced sleep in mice was increased by exercise. In the next step of the investigation, therefore, the effects of pretreatment with EDTA or a-methyltyrosine (a-MPT, an inhibitor of tyros±he hydroxylase) on the prolongation of ethanol-induced sleep following exercise were analyzed. EDTA (1 /xmol/mouse), a-MPT (100 mg/kg), or saline was injected IP 1, 24, or 1 h, respectively, before exercise. Mice were forced to run for 60 min, and the duration of ethanol-induced sleep was measured. The data were analyzed using an ANOVA and Dunnett's t-test for multiple comparisons between individual test groups and the control group. RESULTS
As shown in Fig. 1, serum and brain calcium levels increased during exercise. Serum calcium level increased rapidly and temporarily, and then returned to the original level during exercise. Serum calcium level in mice exercised for 15-60 min was higher by 7-18% ( p < 0.01-0.05) when compared to the control level. Brain calcium level increased more slowly during exercise. Brain calcium level in mice exercised for 15-30 min was not significantly changed, and in mice exercised for 60-120 min it was higher by 8% ( p < 0.05) when compared to the control level. Table 1 and Fig. 2 show measurement results of brain dopamine level. The neostriatum and nucleus accumbens sept± showed the highest immunohistochemical fluorescence intensity of dopamine in control mice. In these regions, the dopamine levels were increased following exercise. The fluorescence intensities of dopamine in the neostriatum and nucleus accumbens sept± in mice exercised for 120 min were higher by 31% ( p < 0.01) and 28% ( p < 0.01), respectively, than those in the brains of the control mice. As previously reported (21), the dopamine levels in the neostriatum and nucleus accumbens sept± were increased by 22% ( p < 0.01) and 21% ( p < 0.01), respectively, following IP administration of CaC12. The dopamine levels in those regions were not changed by administration of control vehicle (saline, data are not shown). The changes in duration of ethanol-induced sleep in mice elicited by exercise are shown in Fig. 3. The duration of ethanolinduced sleep was increased following exercise; the longer the
TABLE 1 THE EFFECTOF EXERCISE OR CaCl2 ADMINISTRATIONON THE BRAIN DOPAMINELEVELS IN MICE Fluorescence Intensity (Measured Value × 10) Brain Region
Control
Exercise
CaCI 2
Cortex cerebri Neostriatum Nucleus accumbenssept± Nucleus sept±lateralis Tractus diagonalis
0.64 ± 0.09 4.62 ± 0.18 4.11 ± 0.17 0.66 ± 0.13 0.80 ± 0.11
0.68 ± 0.10 6.05 ± 0.22* 5.26 _+0.18" 0.60 ± 0.08 0.96 ± 0.ll
0.65 ± 0.11 5.62 ± 0.19" 4.97 ± 0.16" 0.67 ± 0.10 0.89 ± 0.12
The immunohistochemicalfluorescenceintensitiesof dopaminein the brain are shown for the unexercisedcontrol mice, mice exercised for 120 rain, and unexercised mice pretreated IP with 40 /xmol/kg of CaCIz (injected 1 h before sacrifice). Each value represents the mean ± SEM of relative fluorescence intensityper 6 ,u.mdiameter area of 20 slices. Uranium glass was used as the fluorescenceintensity standard (18). * p < 0.01 compared with control group by the Newman-Keuls t-test.
EXERCISE AND BRAIN FUNCTION
179
(ng/mg wet wt.) 62
(ug/rnl) 130 E
E
120
58
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54 .~ n'~
100-
(min) Q._ 800~ 60o "~ 40c" t
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9'0
120
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Exercise (rain) FIG. 1. Change in calcium levels in the serum and brain of mice elicited by exercise. Serum calcium level increased rapidly and brain calcium level increased slowly in mice that were forced to run (20 m/rain) in a motor-driven wheel cage. Results are expressed as mean + SEM of 10-18 data. * p < 0.05, ** p < 0.01 compared with unexercised control mice (Dunnett's t-test).
duration of exercise, the greater was the duration of sleep. In mice exercised for 60 min, the ability of exercise to increase the duration of ethanol-induced sleep was significantly inhibited by pretreatment IP with E D T A or c~-MPT (Fig. 4). By the administration of these drugs, the duration of ethanol-induced sleep in mice exercised for 60 min was decreased and approached that in unexercised control mice. Previous studies have demonstrated that EDTA prevents the increase in calcium and dopamine levels in the brain after treatment with CaC12 (19,24).
6'0
90
120
Exercise (min) FIG. 3. Change in duration of ethanol-induced sleep in mice elicited by exercise. Ethanol (3.6 g/kg) was injected IP following exercise. The duration of ethanol-induced sleep increased in a duration of exercise-dependent manner. Results are expressed as mean_+SEM of 13-15 data. * p < 0.01 compared with unexercised mice (Dunnett's t-test).
Saline
(1 O)
EDTA ctMPT I
i
i
i
0
30
60
90
Ethanol-induced sleep (min)
DISCUSSION In this study, serum calcium level in mice was significantly increased after running for 1 5 - 6 0 min. An increase in blood calcium level after exercise has been reported by a number of investigators (1,4,6,15), although some reports describe conflicting observations, probably due to differences in experimental conditions such as the type of exercise, method of chemical
.... :?:,
FIG. 4. Effects of pretreatment with EDTA or a-MPT on the prolongation of ethanol-induced sleep following exercise. EDTA (1 /zmol/mouse) or a-MPT (100 mg/kg) was injected IP 1 or 24 h, respectively, before exercise. Pretreatment with EDTA or a-MPT significantly inhibited the prolongation of ethanol-induced sleep following exercise for 60 rain. Results are expressed as mean+SEM (number of mice). *p < 0.01 compared with saline-injected control mice (Dunnett's t-test).
i'" i
FIG. 2, Changes in brain dopamine level in mice elicited by exercise or by the administration of calcium. (I) Left side of coronal section approximately 5 mm rostral from the interaural line. The data of dopamine distribution in framed area are shown in II-IV, which were obtained stepwise at 20-/xm intervals through a pinhole (6 p,m in diameter) using a fluorescence microphotometry system. A, nucleus accumbens septi; CA, commissura anterior; CP, neostriatum. (II-IV) Examples of quantitative immunohistochemical distribution of dopamine in the unexercised control mice (II), mice exercised for 120 min (III), and unexercised mice pretreated IP with 40 /zmol/kg of CaC12 (IV). Immunohistochemical fluorescence intensities were classified into five ranks and indicated as sizes of dot.
180
SUTOO AND A K I Y A M A
analysis, or time of blood collection (11,12,25). Also, brain calcium level was increased after exercise, and this increase in the brain occurred later and more gradually than that in the serum. These results indicate that the increased serum calcium may be transported to the brain, where it may act to affect various central nervous system functions. We have previously determined that serum calcium is transported to the brain where it stimulates dopamine synthesis, as does calcium injected directly into the brain (16,19,24). Also, this ability of calcium was abolished by the calmodulin antagonist (20). Thus, it could be inferred based on our previous studies that brain dopamine level is increased after exercise through the calmodulin-dependent system. In the immunohistochemical analysis, in fact, an increase in dopamine levels in the neostriatum and nucleus accumbens septi in exercised mice was confirmed. This finding was in agreement with previous reports that levels of dopamine and its metabolites and tyrosine hydroxylase activity in the brain were enhanced following exercise (3,8,10). Furthermore, the duration of ethanol-induced sleep in mice was prolonged following exercise as well as after calcium or dopamine administration. The ability of exercise to enhance the effect of ethanol was inhibited by pretreatment with a calcium-chelating agent or an inhibitor of tyrosine hydroxylase. Therefore, it is suggested that exercise leads to an increase in the calcium level, which in turn leads to an increase in dopamine synthesis in the brain through the calmodulin-dependent system and that the subsequent increase in dopamine induces behavioral changes. It must be noted that synthesis of other catecholamines (e.g., norepinephrine) and serotonin is also activated by the calcium-
calmodulin-dependent system in the brain as described previously (2,9,16), and that calcium also affects various functions via other pathways. Moreover, it was reported that levels of opioid peptides such as fl-endorphin are increased following exercise (7). Thus, exercise-dependent changes in other pathways must be investigated. However, we believe that the increase in calciumdependent dopamine synthesis is very important in elucidating the mechanism of the ability of exercise to modify brain function. To confirm the effect of exercise on the brain dopamine level, the neostriatum and nucleus accumbens septi regions were chosen, because the amount of dopamine in these regions was increased by the ICV administration of CaC12 in a previous report (20). However, it has not been made clear as yet which compartments of the brain become enriched with calcium during exercise. Finally, the obtained results on calcium and dopamine levels in the exercised mouse brains were the same as those obtained in cold-stressed mice in our previous study; that is, cold stress led to increases in brain calcium and dopamine levels, and the increased dopamine induced behavioral changes (2l). Therefore, we think that exercise and cold stress have similar actions on brain function and induce physiological, behavioral, and psychological changes through actions on central nervous system neurotransmitters. ACKNOWLEDGEMENTS We thank Professor R. D. Myers for advice, and Professor G. R. Breese for critical review of the manuscript. This study was supported by a grant (07922014) from the Ministry of Education, Science, and Culture of Japan.
REFERENCES 1. Aloia, J. F.; Rasulo, P.; Deftos, L. J.; Vaswani, A.; Yeh, J. K. Exercise-induced hypercalcemia and the calciotropic hormones. J. Lab. Clin. Med. 106:229-232; 1985. 2. Boadle-Biber, M. C. Further studies on the role of calcium in the depolarization-induced activation of tryptophan hydroxylase. Biochem. Pharmacol. 31:2495-2503; 1982. 3. Chaouloff, F. Physical exercise and brain monoamines: A review. Acta Physiol. Scand. 137:1-13; 1989. 4. Cordova, A.; Gimenez, M.; Escanero, J. F. Effect of swimming to exhaustion, at low temperatures, on serum Zn, Cu, Mg and Ca in rats. Physiol. Behav. 48:595-598; 1990. 5. Cott, J. M.; Breese, G. R.; Cooper, B. R.; Barlow, T. S.; Prange, A. J., Jr. Investigations into the mechanism of reduction of ethanol sleep by thyrotropin-releasing hormone (TRH). J. PharmacoL Exp. Ther. 196:594-604; 1976. 6. Cunningham, J.; Segre, G. V.; Slatopolsky, E.; Avioli, L. V. Effect of heavy exercise on mineral metabolism and calcium regulating hormones in humans. Calcif. Tissue Int. 37:598-601; 1985. 7. Farrell, P. A. Exercise and endorphins--male responses. Med. Sci. Sports Exerc. 17:89-93; 1985. 8. Freed, C. R.; Yamamoto, B. K. Regional brain dopamine metabolism: A marker for the speed, direction, and posture of moving animals. Science 229:62-65; 1985. 9. Hamon, M.; Bourgoin, S.; Artaud, F.; Glowinski, J. The role of intraneuronal 5-HT and of tryptophan hydroxylase activation in the control of 5-HT synthesis in rat brain slices incubated in K+-enriched medium. J. Neurochem. 33:1031-1042; 1979. 10. Heyes, M. P.; Garnett, E. S.; Coates, G. Nigrostriatal dopaminergic activity is increased during exhaustive exercise stress in rats. Life Sci. 42:1537-1542; 1988. 11. Ljunghall, S.; Joborn, H.; Roxin, L.-E.; Skaffors, E. T.; Wide, L. E.; Lithell, H. O. Increase in serum parathyroid hormone levels after prolonged physical exercise. Med. Sci. Sports Exerc. 20:122-125; 1988.
12. Nosaka, K.; Clarkson, P. M.; Apple, F. S. Time course of serum protein changes after strenuous exercise of the forearm flexors. J. Lab. Clin. Med. 119:183-188; 1992. 13. Ray Sarkar, B. C.; Chauhan, U. P. S. A new method for determining micro quantities of calcium in biological materials. Anal. Biochem. 20:155-166; 1967. 14. Rosenfeld, G. Potentiation of the narcotic action and acute toxicity of alcohol by primary aromatic monoamines. Q. J. Stud. Alcohol 21:584-596; 1960. 15. Ruben, J. A.; Bennett, A. F. Intense exercise, bone structure and blood calcium levels in vertebrates. Nature 291:411-413; 1981. 16. Sutoo, D.; Akiyama, K.; Iimura, K. Effect of calmodulin antagonists on calcium and ethanol-induced sleeping time in mice. Pharmacol. Biochem. Behav. 23:627-631; 1985, 17. Sutoo, D.; Akiyama, K.; Takita, H. The relationship between metal ion levels and biogenic amine levels in epileptic mice. Brain Res. 418:205-213; 1987. 18. Sutoo, D.; Akiyama, K.; Maeda, I. The development of a high sensitivity and high linearity fluorescence microphotometry system for distribution analysis of neurotransmitter in the brain. Folia Pharmacol. Jpn. 9l:173-180; 1988. 19. Sutoo, D.; Matsukura, T.; Akiyama, K. The mechanism by which calcium reduces blood pressure. Eur. J. Phamlacol. 155:189-192; 1988. 20. Sutoo, D.; Akiyama, K.; Geffard, M. Central dopamine-synthesis regulation by the calcium-calmodulin-dependent system. Brain Res. Bull. 22:565-569; 1989. 21. Sutoo, D.; Akiyama, K.; Takita, H. Behavioral changes in coldstressed mice related to a central calcium-dependent-catecholamine synthesizing system. Pharmacol. Biochem. Behav. 40:423-428; 1991. 22. Sutoo, D.; Akiyama, K.; Yabe, K.; Kohno, K. Multiple analysis of tyrosine hydroxylase and calmodulin distributions in the forebrain of the rat using a microphotometry system. Brain Res. Bull. 26:973-982; 1991.
EXERCISE AND BRAIN FUNCTION
23. Sutoo, D.; Akiyama, K.; Takita, H. The effect of convulsions on the rectification of central nervous system disorders in epileptic mice. Physiol. Behav. 52:865-872; 1992. 24. Sutoo, D. Disturbances of brain function by exogenous cadmium. In: Isaacson, R. L.; Jensen, K. F., eds. The vulnerable brain and environ-
181
mental risks, vol. 3: Toxins in air and water. New York: Plenum Press; 1994:281-300. 25. Yeh, J. K.; Aloia, J. F. Effect of physical activity on calciotropic hormones and calcium balance in rats. Am. J. Physiol. 258: E263E268; 1990.
ELSEVIER
PII 0031-9384(96)00011-2
The Mechanism by Which Exercise Modifies Brain Function D E N ' E T S U SUTOO 1 AND KAYO A K I Y A M A
Institute of Medical Science, University of Tsukuba, Tsukuba, Ibaraki 305, Japan Received 9 October 1995 SUTOO, D. AND K. AKIYAMA. The mechanism by which exercise modifies brain function. PHYSIOL BEHAV 60(1) 177-181, 1996.--The effect of exercise on central nervous system function was investigated in relation to the mechanism of calcium-calmodulin-dependent dopamine synthesis in the brain. It is shown here through animal experiments that exercise leads to an increase in the calcium level in the brain. This in turn enhances brain dopamine synthesis, and through this increased dopamine modifies and/or affects brain function, which might induce physiological, behavioral, and psychological changes. Exercise Calcium-calmodulin Dopamine synthesis Ethanol-induced sleep Microphotometry system Mice
Neostriatum
Nucleus accumbens septi
exposed to a 12-h light/dark cycle. Food and water were provided ad lib until the time of experiment and were not given during the exercise. All animals received humane care in compliance with the Guiding Principles for the Care and Use of Laboratory Animals formulated by the Japanese Pharmacological Society. For exercise, they were forced to run for 15-120 min at a speed of 20 m per min by using a programmed motor-driven wheel cage. Following the exercise, changes in the serum and brain calcium levels, brain dopamine level, and behavior were immediately analyzed. Experiments were carried out between 1000 and 1600 h.
ALTHOUGH exercise is known to produce a refreshing feeling, the mechanism by which exercise modifies brain function to produce this feeling has not been made clear. In previous investigations, our laboratory has examined the role of calcium ions in brain function. Studies demonstrated that calcium activates tyrosine hydroxylase (a rate-limiting enzyme for catecholamine synthesis) in the brain through a calmodulin-dependent system (16,20,22), and that an elevated dopamine regulates various functions, such as blood pressure, locomotor activity, and receptivity to drugs (16,19,21,24). This concept was developed in an attempt to elucidate the mechanism of convulsive seizures in epileptic mice, and it was demonstrated that convulsions are induced when dopamine synthesis in the brain is decreased, and that the convulsions increase the brain calcium level, which in turn rectifies disorders of brain dopamine synthesis and of brain function (17,23). Therefore, it was hypothesized that exercise may also affect brain function through action of calcium and biogenic amines such as dopamine, as has been shown in convulsive seizures in epileptic mice. This study was carried out to confirm this hypothesis.
Calcium Levels Calcium levels in the serum and brain were measured according to the method using an o-cresolphthalein complexone (13). Whole brain and blood were obtained from mice that had been forced to run for 15-120 min as well as from unexercised control mice. The serum was separated quickly and assayed. Brains were homogenized with saline at 4°C and centrifuged for 1 h at 25,000 × g (4°C), and after which the supernatant was assayed. Data were analyzed by an ANOVA and Dunnett's t-test for multiple comparisons between individual exercised groups and the unexercised control group.
METHOD
Animals Adult male mice of the ddY strain (20-25 g) were provided by Doken Co. Ltd. (Ibaraki, Japan). They were housed in groups of 8-10 in stainless steel cages at room temperature (22 + 2°C) for more than 1 week before use in the experiments and were
Dopamine Levels Brain dopamine levels were determined immunohistochemically and compared quantitatively. Three animal groups [10
i To whom requests for reprints should be addressed.
177
178
mice/group: unexercised control mice, mice exercised for 120 min, and unexercised mice pretreated IP with 40 /zmol/kg of CaC12 (0.48-0.54 ml of saline solution was injected 1 h before sacrifice)] were anesthetized with pentobarbital sodium and perfused intracardially with 50-ml solution of 0.1 M cacodylate and 1% sodium metabisulfite (SMB) containing 2.5% glutaraldehyde (pH 7.5). The whole brain was removed and postfixed with the same solution for 30 min, then frozen on dry ice. The frozen brain was sectioned coronally and continuously at 20-/xm thickness in a cryostat. For analysis of the distribution and amount of dopamine, brain slices located approximately 5 mm rostral from the interaural line (Fig. 2) were chosen in light of a previous report (20) that dopamine levels in the neostriatum and nucleus accumbens sept± regions of this section were increased by the intracerebroventricular (ICV) administration of CaCI 2. Immunohistochemical procedure was performed according to a previous report (20). The brain slices were washed three times with Tris-SMB buffer (Tris 0.05 M and SMB 0.85%, pH 7.5). The dopamine antibody was diluted l:800 with Tris-SMB buffer and 50 /zl of antibody solution was applied to each brain slice, then incubated at 4°C for 12 h. After the reaction, the slices were washed three times with phosphate-buffered saline (PBS). The second antibody, anti-rabbit IgG goat serum labelled with fluorescein isothiocyanate, was diluted 1:100 with PBS and applied to the sections. It was incubated in the dark at 20°C for 3 h, and then washed with PBS. Nonspecific fluorescence with glutaraldehyde was reduced by 0.1 M NaBH 4 in PBS. After being rinsed three times with PBS, the stained sections were embedded in 10% glycerin PBS, and kept at 4°C in a dark room until the measurements, performed within 10 h, could be carried out. The distribution of dopamine was quantitatively analyzed using a fluorescence microphotometry system (Nikon, Japan) (18). This system is a measuring microscope for distribution of immunohistochemical fluorescence intensity in the tissue slice. The average fluorescence intensity in a small brain region can be measured by a photomultiplier tube through a pinhole of a microscope. The brain slice is continuously moved in the x- or y-direction by means of a scanning stage under the objective lens of a microscope and the data in each brain region are collected. Fluorescence intensity values in each region can be indicated quantitatively in comparison with that of standard uranium glass. This system surpasses by two orders the quantitative linearity of image analyzers used with high-sensitivity TV cameras, and it also surpasses by three orders the sensitivity of high performance liquid chromatography with an electrochemical detector. The slices stained with the dopamine antibody were measured reticulately at 20-/xm intervals. The total fluorescence intensity value in each measuring point had the background value subtracted. The background value was obtained from slices that were treated with the same procedure without dopamine antibody. The average fluorescence intensity in each region was analyzed by an ANOVA and Newman-Keuls t-test for subsequent comparisons of three groups (20 slices/group).
Ethanol-Induced Sleeping Time When ethanol is injected IP at a dose of 3.6 g / k g , mice lose their righting reflex and lapse into a behavior resembling sleep for approximately 20 rain. The duration of this ethanol-induced sleep is regulated by brain biogenic amines and can be prolonged by ICV administration of dopamine or other biogenic amines (5,14,16). Therefore, it is possible to monitor the change in the brain dopamine level elicited by exercise in live mice through measurement of the duration of ethanol-induced sleep. We have already observed, using this measurement, that pretreatment of
SUTOO AND AKIYAMA
mice with CaC12 increases the brain dopamine level in a dose-dependent manner (16). In the first step of the investigation, ethanol (3.6 g / k g ) was injected IP into unexercised control mice and mice exercised for 15-120 rain, and the duration of sleep was compared. Sleeping time duration is defined as the time from the loss of the righting reflex to the time when the righting reflex occurs twice within 30 s. As demonstrated in the Results section, the duration of ethanol-induced sleep in mice was increased by exercise. In the next step of the investigation, therefore, the effects of pretreatment with EDTA or a-methyltyrosine (a-MPT, an inhibitor of tyros±he hydroxylase) on the prolongation of ethanol-induced sleep following exercise were analyzed. EDTA (1 /xmol/mouse), a-MPT (100 mg/kg), or saline was injected IP 1, 24, or 1 h, respectively, before exercise. Mice were forced to run for 60 min, and the duration of ethanol-induced sleep was measured. The data were analyzed using an ANOVA and Dunnett's t-test for multiple comparisons between individual test groups and the control group. RESULTS
As shown in Fig. 1, serum and brain calcium levels increased during exercise. Serum calcium level increased rapidly and temporarily, and then returned to the original level during exercise. Serum calcium level in mice exercised for 15-60 min was higher by 7-18% ( p < 0.01-0.05) when compared to the control level. Brain calcium level increased more slowly during exercise. Brain calcium level in mice exercised for 15-30 min was not significantly changed, and in mice exercised for 60-120 min it was higher by 8% ( p < 0.05) when compared to the control level. Table 1 and Fig. 2 show measurement results of brain dopamine level. The neostriatum and nucleus accumbens sept± showed the highest immunohistochemical fluorescence intensity of dopamine in control mice. In these regions, the dopamine levels were increased following exercise. The fluorescence intensities of dopamine in the neostriatum and nucleus accumbens sept± in mice exercised for 120 min were higher by 31% ( p < 0.01) and 28% ( p < 0.01), respectively, than those in the brains of the control mice. As previously reported (21), the dopamine levels in the neostriatum and nucleus accumbens sept± were increased by 22% ( p < 0.01) and 21% ( p < 0.01), respectively, following IP administration of CaC12. The dopamine levels in those regions were not changed by administration of control vehicle (saline, data are not shown). The changes in duration of ethanol-induced sleep in mice elicited by exercise are shown in Fig. 3. The duration of ethanolinduced sleep was increased following exercise; the longer the
TABLE 1 THE EFFECTOF EXERCISE OR CaCl2 ADMINISTRATIONON THE BRAIN DOPAMINELEVELS IN MICE Fluorescence Intensity (Measured Value × 10) Brain Region
Control
Exercise
CaCI 2
Cortex cerebri Neostriatum Nucleus accumbenssept± Nucleus sept±lateralis Tractus diagonalis
0.64 ± 0.09 4.62 ± 0.18 4.11 ± 0.17 0.66 ± 0.13 0.80 ± 0.11
0.68 ± 0.10 6.05 ± 0.22* 5.26 _+0.18" 0.60 ± 0.08 0.96 ± 0.ll
0.65 ± 0.11 5.62 ± 0.19" 4.97 ± 0.16" 0.67 ± 0.10 0.89 ± 0.12
The immunohistochemicalfluorescenceintensitiesof dopaminein the brain are shown for the unexercisedcontrol mice, mice exercised for 120 rain, and unexercised mice pretreated IP with 40 /xmol/kg of CaCIz (injected 1 h before sacrifice). Each value represents the mean ± SEM of relative fluorescence intensityper 6 ,u.mdiameter area of 20 slices. Uranium glass was used as the fluorescenceintensity standard (18). * p < 0.01 compared with control group by the Newman-Keuls t-test.
EXERCISE AND BRAIN FUNCTION
179
(ng/mg wet wt.) 62
(ug/rnl) 130 E
E
120
58
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Exercise (rain) FIG. 1. Change in calcium levels in the serum and brain of mice elicited by exercise. Serum calcium level increased rapidly and brain calcium level increased slowly in mice that were forced to run (20 m/rain) in a motor-driven wheel cage. Results are expressed as mean + SEM of 10-18 data. * p < 0.05, ** p < 0.01 compared with unexercised control mice (Dunnett's t-test).
duration of exercise, the greater was the duration of sleep. In mice exercised for 60 min, the ability of exercise to increase the duration of ethanol-induced sleep was significantly inhibited by pretreatment IP with E D T A or c~-MPT (Fig. 4). By the administration of these drugs, the duration of ethanol-induced sleep in mice exercised for 60 min was decreased and approached that in unexercised control mice. Previous studies have demonstrated that EDTA prevents the increase in calcium and dopamine levels in the brain after treatment with CaC12 (19,24).
6'0
90
120
Exercise (min) FIG. 3. Change in duration of ethanol-induced sleep in mice elicited by exercise. Ethanol (3.6 g/kg) was injected IP following exercise. The duration of ethanol-induced sleep increased in a duration of exercise-dependent manner. Results are expressed as mean_+SEM of 13-15 data. * p < 0.01 compared with unexercised mice (Dunnett's t-test).
Saline
(1 O)
EDTA ctMPT I
i
i
i
0
30
60
90
Ethanol-induced sleep (min)
DISCUSSION In this study, serum calcium level in mice was significantly increased after running for 1 5 - 6 0 min. An increase in blood calcium level after exercise has been reported by a number of investigators (1,4,6,15), although some reports describe conflicting observations, probably due to differences in experimental conditions such as the type of exercise, method of chemical
.... :?:,
FIG. 4. Effects of pretreatment with EDTA or a-MPT on the prolongation of ethanol-induced sleep following exercise. EDTA (1 /zmol/mouse) or a-MPT (100 mg/kg) was injected IP 1 or 24 h, respectively, before exercise. Pretreatment with EDTA or a-MPT significantly inhibited the prolongation of ethanol-induced sleep following exercise for 60 rain. Results are expressed as mean+SEM (number of mice). *p < 0.01 compared with saline-injected control mice (Dunnett's t-test).
i'" i
FIG. 2, Changes in brain dopamine level in mice elicited by exercise or by the administration of calcium. (I) Left side of coronal section approximately 5 mm rostral from the interaural line. The data of dopamine distribution in framed area are shown in II-IV, which were obtained stepwise at 20-/xm intervals through a pinhole (6 p,m in diameter) using a fluorescence microphotometry system. A, nucleus accumbens septi; CA, commissura anterior; CP, neostriatum. (II-IV) Examples of quantitative immunohistochemical distribution of dopamine in the unexercised control mice (II), mice exercised for 120 min (III), and unexercised mice pretreated IP with 40 /zmol/kg of CaC12 (IV). Immunohistochemical fluorescence intensities were classified into five ranks and indicated as sizes of dot.
180
SUTOO AND A K I Y A M A
analysis, or time of blood collection (11,12,25). Also, brain calcium level was increased after exercise, and this increase in the brain occurred later and more gradually than that in the serum. These results indicate that the increased serum calcium may be transported to the brain, where it may act to affect various central nervous system functions. We have previously determined that serum calcium is transported to the brain where it stimulates dopamine synthesis, as does calcium injected directly into the brain (16,19,24). Also, this ability of calcium was abolished by the calmodulin antagonist (20). Thus, it could be inferred based on our previous studies that brain dopamine level is increased after exercise through the calmodulin-dependent system. In the immunohistochemical analysis, in fact, an increase in dopamine levels in the neostriatum and nucleus accumbens septi in exercised mice was confirmed. This finding was in agreement with previous reports that levels of dopamine and its metabolites and tyrosine hydroxylase activity in the brain were enhanced following exercise (3,8,10). Furthermore, the duration of ethanol-induced sleep in mice was prolonged following exercise as well as after calcium or dopamine administration. The ability of exercise to enhance the effect of ethanol was inhibited by pretreatment with a calcium-chelating agent or an inhibitor of tyrosine hydroxylase. Therefore, it is suggested that exercise leads to an increase in the calcium level, which in turn leads to an increase in dopamine synthesis in the brain through the calmodulin-dependent system and that the subsequent increase in dopamine induces behavioral changes. It must be noted that synthesis of other catecholamines (e.g., norepinephrine) and serotonin is also activated by the calcium-
calmodulin-dependent system in the brain as described previously (2,9,16), and that calcium also affects various functions via other pathways. Moreover, it was reported that levels of opioid peptides such as fl-endorphin are increased following exercise (7). Thus, exercise-dependent changes in other pathways must be investigated. However, we believe that the increase in calciumdependent dopamine synthesis is very important in elucidating the mechanism of the ability of exercise to modify brain function. To confirm the effect of exercise on the brain dopamine level, the neostriatum and nucleus accumbens septi regions were chosen, because the amount of dopamine in these regions was increased by the ICV administration of CaC12 in a previous report (20). However, it has not been made clear as yet which compartments of the brain become enriched with calcium during exercise. Finally, the obtained results on calcium and dopamine levels in the exercised mouse brains were the same as those obtained in cold-stressed mice in our previous study; that is, cold stress led to increases in brain calcium and dopamine levels, and the increased dopamine induced behavioral changes (2l). Therefore, we think that exercise and cold stress have similar actions on brain function and induce physiological, behavioral, and psychological changes through actions on central nervous system neurotransmitters. ACKNOWLEDGEMENTS We thank Professor R. D. Myers for advice, and Professor G. R. Breese for critical review of the manuscript. This study was supported by a grant (07922014) from the Ministry of Education, Science, and Culture of Japan.
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