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Time course of the changes of catecholamine levels in rat brain during swimming stress
372
Brain Research. 27~ t 1983 ) 372--.374 [ilsevier
Time course of the changes of catecholamine levels in rat brain during swimming stress AYAKO SUDO Department of Industrial Physiology, National Institute of lndustrial Health, Nagao 6-chome, Tama-ku, Kawasaki 214 (Japan)
(Accepted June 7th, 1983) Key words': adrenaline - - noradrenaline -- dopamine -- stress -- rat brain
The time course of the changes in the levels of adrenaline, noradrenaline and dopamine was investigated in rat hypothalamus, ponsmedulla and midbrain during 4-h swimmingat 35 °C. The adrenaline levels showed gradual decrease whereas the noradrenaline and dopamine levels were kept in almost steady-state during the swimming. These findingssuggest that brainstem adrenaline levels are not so fast adaptive as the noradrenaline and dopamine levels under the stress. Many workers have reported that acute stress changes the levels of noradrenaline and dopamine in the rat brainl. Kvetnansky et al. 3 observed marked decrease of adrenaline contents in some hypothalamic nuclei of rats under immobilization stress. Electric foot-shock and cold swimming were also shown to produce the decrease of adrenaline in hypothalamus and pons-medulla of rats 5-~. Some 5.~ of these studies described that the degree of the stress-induced decrease in the adrenaline contents is greater than that of noradrenaline. However, little is known about the time course of the changes in brain adrenaline, noradrenaline and dopamine levels during stress. In this study, the levels of the 3 catecholamines were measured in the brains of rats exposed to swimming stress for various periods. Seven-week-old SD male rats, kept in 12-h lightdark cycle with light on at 10.30 h, were put in water individually for 0.25, 0.5, 1, 2 and 4 h. The experiments were performed during the last 5 h of the dark periods to minimize the effects of sleep deprivation during swimming periods, and the temperature of the water was maintained within the range of 34 and 37 °C. Six or 7 rats were used for each swimming period. Immediately after the swimming stress, the rectal temperature of the rats was measured by a thermister thermometer, and the animals were killed by decapitation. The brain was quickly removed and dissected into 7 parts on ice according to the method of Glowinski and lversen2. Dissected brain samples 0006-8993/83/$03.00 © 1983Elsevier Science Publishers B.V.
were weighed, homogenized in 10 vols. of 0.1 N HCIO4-5 mM EDTA.2 Na with a Polytron homogenizer and centrifuged at 4 °C and 20,000 g. After isolation on alumina columns 10, adrenaline and noradrenaline concentrations in hypothalamus, pons-medulla and midbrain, and dopamine concentrations in striatum and midbrain were determined by means of a high-performance liquid chromatograph connected with an autoanalyzer and a fluorometer using the THI method 1°. Dopamine in hypothalamus and pons-medulla could not be detected by the method used. The amine contents were expressed as nmol or pmol per g wet tissue. The results of the present work are summarized in Table 1. Noradrenaline levels in hypothalamus, pons-medulla and midbrain slightly decreased at the beginning of swimming, and then remained almost unchanged. Dopamine in midbrain and striatum did not show any significant change throughout the period of stress. In contrast, the level of adrenaline in hypothalamus showed a gradual decrease under the swimming stress and after 4 h of swimming had declined to 60% of the level of control rats. Similar changes were observed in ports-medulla and midbrain. The degree of the stress-induced decrease of adrenaline in pons-medulla (25%) was less than that in hypothalamus (40%), which is possibly attributable to higher activity of phenylethanolamine-Nmethyltransferase (PNMT), an adrenaline-forming enzyme, in pons-medulla than in hypothalamus 7.
373 TABLE I
Changes of brain catecholamine levels in rats under the stress of swimming Each value represents the mean and S.E. of 6--7 rats.
Control group
Duration of swimming (h) 0.25
0.5
230 + 12 35.1 + 1.7 16.6 + 0.5
195 + 15 30.8 + 2.0 14.7 + 0.9
Adrenaline (pmol/g wet tissue) Hypothalamus Pons-meduUa Midbrain
234 + 11 38.8 + 2.9 18.4 + 1.5
167 + 10"* 31.8 + 2.6 13.8 + 1.6
200 _+ 15 33.8 + 2.1 15.5 +_0.5
136 _+ 8** 28.6 + 1.3" 11.8 + 0.5**
Noradrenaline (nmol/g wet tissue) Hypothalamus Pons-medulla Midbrain
12.61 + 0.16 3.71 + 0.10 2.92 + 0.06
11.27 + 0.43* 10.83 + 0.54** 11.63 + 0.42* 3.20 + 0.09** 3.55 + 0.12 3.46 + 0.09 2.55 + 0.04** 2.84 + 0.09 2.69 +_0.29*
10.90 + 0.50** 11.31 + 0.22** 3.34 + 0.08* 3.36 + 0.07* 2.70 + 0.04* 2.86 + 0.07
Dopamine (nmol/g wet tissue) Striatum Midbrain
58.3 + 2.0 1.62 + 0.15
59.6 + 2.1 1.59 + 0.04
55.0 + 1.8 1.33 + 0.05
59.0 _+ 1.3 1.41 + 0.04
58.2 + 1.7 1.52 + 0.24
57.8 + 1.8 1.39 + 0.03
Difference from the value of control group was tested by the Student's t-test: * P < 0.05; ** P < 0.01.
Cold swimming is often used as a stress 1. In the in-
study indicates that after the beginning of stress the
terpretation of the results about a stress of such type,
biosynthesis rates were quickly readjusted to main-
possible reduction of the amine synthesis rate caused
tain the normal catecholamine levels in these re-
by hy p o t h erm i a 9 must be considered. In the present
gions. In contrast, the adrenaline levels were not so
study, however, the t e m p e r a t u r e of the water in the
fast adaptive under the stress, suggesting insufficient
swimming bath was controlled at 35 °C, and the rec-
increase in P N M T activities or enhanced release of
tal t e m p e r a t u r e of the rats did not fall below 36 °C af-
this amine from the nerve terminals, c o m p a r e d with
ter swimming. W e can exclude, therefore, the effects
the cases of noradrenaline and dopamine.
of hypothermia as a cause of the catecholamine de-
Stress usually accompanies emotional responses
crease. Brain tyrosine hydroxylase 4 and P N M T 7 activities
and enhanced cardiovascular activities. A d r e n a l i n e neurons in hypothalamus and pons-medulla are as-
were found to increase under acute stress. Increased
sumed to be involved in central stress responses
amounts of m a j o r metabolites of noradrenaline and
probably relating to endocrinoiogical and autonomic
d o p a m i n e were observed in the brain areas under the
functionsT. Increase of these central activities under
stress1, 5. E l e v a t e d levels of 3,4-dihydroxyphenylgly-
the stress might have been a cause of the dramatic de-
col, a m a j o r metabolite of noradrenaline, were found
crease of adrenaline levels in these areas.
in the rat hypothalamus and pons-medulla after 4 h swimming at 35 °C (unpublished results). These find-
The author is grateful to Prof. M. Otsuka, To k y o
ings suggest increased turnover rates of brain nor-
Medical and D e n t a l University, and Dr. S. Y a m a m o -
adrenaline and d o p a m i n e u n d e r the stress. T h e time
to, National Institute of Industrial Health, for their
course of the two amines observed in the present
kind advice.
1 Anisman, H., Neurochemical changes elicited by stress: behavioral correlates. In H. Anisman and G. Bignami (Eds.),
Psychopharmacology of Aversively Motivated Behavior, Plenum Press, New York, 1978, pp. 119-172. 2 Glowinski, J. and Iversen, L. L., Regional studies of catecholamines in the rat brain. I. The distribution of H3-norephinephrine, H3-dopamine and H3-DOPA in various regions of the brain, J. Neurochem., 13 (1966) 655-669.
3 Kvetnansky, R., Kopin, I. L. and Saavedra, J. M., Changes in epinephrine in individual hypothalamic nuclei after immobilization stress, Brain Research, 155 (1978) 387-390. 4 Masserano, J. M., Takimoto, G. S. and Weiner, N., Electroconvulsive shock increases tyrosine hydroxylase activity in the brain and adrenal gland of the rat, Science, 214 (1981) 662-665. 5 Roth, K. A., Mefford, I. M. and Barchas, J. D., Epineph-
374 rine, norepinephrine, dopamine and serotonin: differential effects of acute and chronic stress on regional brain amines, Brain Research, 239 (1982) 417-424. 6 Saavedra, J. M., Kvetnansky, R. and Kopin, I. J., Adrenaline, noradrenaline and dopamine levels in specific brain stem areas of acutely immobilized rats, Brain Research, 160 (1979) 271-280. 7 Saavedra, J. M., Brain and pineal adrenaline: its level and possible function in stress. In E. Usdin, R. Kevemansky and I. k. Kopin (Eds.), Catecholamines and Stress: Recent Advances, Elsevier/North-Holland, Amsterdam, 1980, pp.
37-45. 8 Sauter, A. M., Baba, Y., Stone, E. A. and Goldstein, M., Effects of stress and of phenylethanolamine-N-methyltransferase inhibition on central norepinephrine and epinephrine levels, Brain Research, 144 (1978) 415-419. 9 Stone, E. A., Behavioral and neurochemical effects of acute swim stress are due to hypothermia, Life Sci., 9 (1970) 877-888. 10 Sudo, A., Measurement of adrenaline in the rat brain by high-performance liquid chromatography with fluorometric detection, Ind. Health, 20 (1982) 151-156.
Brain Research. 27~ t 1983 ) 372--.374 [ilsevier
Time course of the changes of catecholamine levels in rat brain during swimming stress AYAKO SUDO Department of Industrial Physiology, National Institute of lndustrial Health, Nagao 6-chome, Tama-ku, Kawasaki 214 (Japan)
(Accepted June 7th, 1983) Key words': adrenaline - - noradrenaline -- dopamine -- stress -- rat brain
The time course of the changes in the levels of adrenaline, noradrenaline and dopamine was investigated in rat hypothalamus, ponsmedulla and midbrain during 4-h swimmingat 35 °C. The adrenaline levels showed gradual decrease whereas the noradrenaline and dopamine levels were kept in almost steady-state during the swimming. These findingssuggest that brainstem adrenaline levels are not so fast adaptive as the noradrenaline and dopamine levels under the stress. Many workers have reported that acute stress changes the levels of noradrenaline and dopamine in the rat brainl. Kvetnansky et al. 3 observed marked decrease of adrenaline contents in some hypothalamic nuclei of rats under immobilization stress. Electric foot-shock and cold swimming were also shown to produce the decrease of adrenaline in hypothalamus and pons-medulla of rats 5-~. Some 5.~ of these studies described that the degree of the stress-induced decrease in the adrenaline contents is greater than that of noradrenaline. However, little is known about the time course of the changes in brain adrenaline, noradrenaline and dopamine levels during stress. In this study, the levels of the 3 catecholamines were measured in the brains of rats exposed to swimming stress for various periods. Seven-week-old SD male rats, kept in 12-h lightdark cycle with light on at 10.30 h, were put in water individually for 0.25, 0.5, 1, 2 and 4 h. The experiments were performed during the last 5 h of the dark periods to minimize the effects of sleep deprivation during swimming periods, and the temperature of the water was maintained within the range of 34 and 37 °C. Six or 7 rats were used for each swimming period. Immediately after the swimming stress, the rectal temperature of the rats was measured by a thermister thermometer, and the animals were killed by decapitation. The brain was quickly removed and dissected into 7 parts on ice according to the method of Glowinski and lversen2. Dissected brain samples 0006-8993/83/$03.00 © 1983Elsevier Science Publishers B.V.
were weighed, homogenized in 10 vols. of 0.1 N HCIO4-5 mM EDTA.2 Na with a Polytron homogenizer and centrifuged at 4 °C and 20,000 g. After isolation on alumina columns 10, adrenaline and noradrenaline concentrations in hypothalamus, pons-medulla and midbrain, and dopamine concentrations in striatum and midbrain were determined by means of a high-performance liquid chromatograph connected with an autoanalyzer and a fluorometer using the THI method 1°. Dopamine in hypothalamus and pons-medulla could not be detected by the method used. The amine contents were expressed as nmol or pmol per g wet tissue. The results of the present work are summarized in Table 1. Noradrenaline levels in hypothalamus, pons-medulla and midbrain slightly decreased at the beginning of swimming, and then remained almost unchanged. Dopamine in midbrain and striatum did not show any significant change throughout the period of stress. In contrast, the level of adrenaline in hypothalamus showed a gradual decrease under the swimming stress and after 4 h of swimming had declined to 60% of the level of control rats. Similar changes were observed in ports-medulla and midbrain. The degree of the stress-induced decrease of adrenaline in pons-medulla (25%) was less than that in hypothalamus (40%), which is possibly attributable to higher activity of phenylethanolamine-Nmethyltransferase (PNMT), an adrenaline-forming enzyme, in pons-medulla than in hypothalamus 7.
373 TABLE I
Changes of brain catecholamine levels in rats under the stress of swimming Each value represents the mean and S.E. of 6--7 rats.
Control group
Duration of swimming (h) 0.25
0.5
230 + 12 35.1 + 1.7 16.6 + 0.5
195 + 15 30.8 + 2.0 14.7 + 0.9
Adrenaline (pmol/g wet tissue) Hypothalamus Pons-meduUa Midbrain
234 + 11 38.8 + 2.9 18.4 + 1.5
167 + 10"* 31.8 + 2.6 13.8 + 1.6
200 _+ 15 33.8 + 2.1 15.5 +_0.5
136 _+ 8** 28.6 + 1.3" 11.8 + 0.5**
Noradrenaline (nmol/g wet tissue) Hypothalamus Pons-medulla Midbrain
12.61 + 0.16 3.71 + 0.10 2.92 + 0.06
11.27 + 0.43* 10.83 + 0.54** 11.63 + 0.42* 3.20 + 0.09** 3.55 + 0.12 3.46 + 0.09 2.55 + 0.04** 2.84 + 0.09 2.69 +_0.29*
10.90 + 0.50** 11.31 + 0.22** 3.34 + 0.08* 3.36 + 0.07* 2.70 + 0.04* 2.86 + 0.07
Dopamine (nmol/g wet tissue) Striatum Midbrain
58.3 + 2.0 1.62 + 0.15
59.6 + 2.1 1.59 + 0.04
55.0 + 1.8 1.33 + 0.05
59.0 _+ 1.3 1.41 + 0.04
58.2 + 1.7 1.52 + 0.24
57.8 + 1.8 1.39 + 0.03
Difference from the value of control group was tested by the Student's t-test: * P < 0.05; ** P < 0.01.
Cold swimming is often used as a stress 1. In the in-
study indicates that after the beginning of stress the
terpretation of the results about a stress of such type,
biosynthesis rates were quickly readjusted to main-
possible reduction of the amine synthesis rate caused
tain the normal catecholamine levels in these re-
by hy p o t h erm i a 9 must be considered. In the present
gions. In contrast, the adrenaline levels were not so
study, however, the t e m p e r a t u r e of the water in the
fast adaptive under the stress, suggesting insufficient
swimming bath was controlled at 35 °C, and the rec-
increase in P N M T activities or enhanced release of
tal t e m p e r a t u r e of the rats did not fall below 36 °C af-
this amine from the nerve terminals, c o m p a r e d with
ter swimming. W e can exclude, therefore, the effects
the cases of noradrenaline and dopamine.
of hypothermia as a cause of the catecholamine de-
Stress usually accompanies emotional responses
crease. Brain tyrosine hydroxylase 4 and P N M T 7 activities
and enhanced cardiovascular activities. A d r e n a l i n e neurons in hypothalamus and pons-medulla are as-
were found to increase under acute stress. Increased
sumed to be involved in central stress responses
amounts of m a j o r metabolites of noradrenaline and
probably relating to endocrinoiogical and autonomic
d o p a m i n e were observed in the brain areas under the
functionsT. Increase of these central activities under
stress1, 5. E l e v a t e d levels of 3,4-dihydroxyphenylgly-
the stress might have been a cause of the dramatic de-
col, a m a j o r metabolite of noradrenaline, were found
crease of adrenaline levels in these areas.
in the rat hypothalamus and pons-medulla after 4 h swimming at 35 °C (unpublished results). These find-
The author is grateful to Prof. M. Otsuka, To k y o
ings suggest increased turnover rates of brain nor-
Medical and D e n t a l University, and Dr. S. Y a m a m o -
adrenaline and d o p a m i n e u n d e r the stress. T h e time
to, National Institute of Industrial Health, for their
course of the two amines observed in the present
kind advice.
1 Anisman, H., Neurochemical changes elicited by stress: behavioral correlates. In H. Anisman and G. Bignami (Eds.),
Psychopharmacology of Aversively Motivated Behavior, Plenum Press, New York, 1978, pp. 119-172. 2 Glowinski, J. and Iversen, L. L., Regional studies of catecholamines in the rat brain. I. The distribution of H3-norephinephrine, H3-dopamine and H3-DOPA in various regions of the brain, J. Neurochem., 13 (1966) 655-669.
3 Kvetnansky, R., Kopin, I. L. and Saavedra, J. M., Changes in epinephrine in individual hypothalamic nuclei after immobilization stress, Brain Research, 155 (1978) 387-390. 4 Masserano, J. M., Takimoto, G. S. and Weiner, N., Electroconvulsive shock increases tyrosine hydroxylase activity in the brain and adrenal gland of the rat, Science, 214 (1981) 662-665. 5 Roth, K. A., Mefford, I. M. and Barchas, J. D., Epineph-
374 rine, norepinephrine, dopamine and serotonin: differential effects of acute and chronic stress on regional brain amines, Brain Research, 239 (1982) 417-424. 6 Saavedra, J. M., Kvetnansky, R. and Kopin, I. J., Adrenaline, noradrenaline and dopamine levels in specific brain stem areas of acutely immobilized rats, Brain Research, 160 (1979) 271-280. 7 Saavedra, J. M., Brain and pineal adrenaline: its level and possible function in stress. In E. Usdin, R. Kevemansky and I. k. Kopin (Eds.), Catecholamines and Stress: Recent Advances, Elsevier/North-Holland, Amsterdam, 1980, pp.
37-45. 8 Sauter, A. M., Baba, Y., Stone, E. A. and Goldstein, M., Effects of stress and of phenylethanolamine-N-methyltransferase inhibition on central norepinephrine and epinephrine levels, Brain Research, 144 (1978) 415-419. 9 Stone, E. A., Behavioral and neurochemical effects of acute swim stress are due to hypothermia, Life Sci., 9 (1970) 877-888. 10 Sudo, A., Measurement of adrenaline in the rat brain by high-performance liquid chromatography with fluorometric detection, Ind. Health, 20 (1982) 151-156.