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Expression of aggression attenuates stress-induced increases in rat brain noradrenaline turnover
Brain Research, 474 ( 19~8~ 174-180 Elscvicr
174 BRE 14072
Expression of aggression attenuates stress-induced increases in rat brain noradrenaline turnover Akira Tsuda, Masatoshi Tanaka, Yoshishige Ida, Ishou Shirao, Yuhji Gondoh, Masanobi Oguchi and Masami Yoshida Departmentof Pharmacology, Kurume UniversitySchool of Medicine, Kurume (Japan) (Accepted 31 May 1988)
Key words: Supine restraint stress; Noradrenaline turnover; 3-Methoxy-4-hydroxyphenylethyleneglycol sulfate; Rat brain region; Corticosterone; Expression of aggression
This experiment determined whether or not an aggressive biting response could attenuate stress-induced increases in brain noradrenaline (NA) turnover, by measuring contents of NA and its major metabolite, 3-methoxy-4-hydroxyphenylethylermglycol sulfate (MHPG-SO4), in discrete brain regions of male Wistar rats. Rats were exposed to a I0 rain supine restraint stress with or without being allowed to bite a wooden stick. In each group, the animals were sacrificed by decapitation either 0 rain or 50 rain after release from stress. NA and MHPG-SO 4 levels were unaffected in both biting and non-biting groups immediately after stress, as compared to controis. Fifty min after release from stress, increases in plasma corticosterone levels induced by stress recovered in the biting group but remained high in the non-biting group. MHPG-SO4 levels significantly increased in the hypothalamus, amygdala, thalamus, midbrain. basal ganglia, hippocampus and cerebral cortex in both stressed groups, however the increases in the non-biting group were significantly higher than those in the biting group in the first 5 of these regions. These findings clearly show that giving rats an opportunity to express aggression during stress exposure results in a significant attenuation of stress-induced increases in NA turnover in specific brain regions, such as the hypothalamus and limbic areas. The present experiment provides a possible neurochemical basis for clinical studies showing that suppression of anger in a stressful, frustrating situation leads to pathological consequences in humans. INTRODUCTION Several previous e x p e r i m e n t a l reports have indicated that, in comparison with animals exposed individually to inescapable shock, rats exposed to the same shock in pairs and thus being able to fight in response to shock, showed less elevation of p l a s m a corticosterone and a d r e n o c o r t i c o t r o p i n ( A C T H ) 3°, less increase in blood pressure 33 and fewer gastric mucosal lesions 9"31. In addition, Guile and McCutcheon s and Vincent et al. 29 r e p o r t e d that rats e x p o s e d to restraint stress and which were able to bite a brush or chew a block of w o o d , d e v e l o p e d less acute gastric mucosal lesions than did rats which were unable to display aggressive biting or gnawing responses. It would a p p e a r from these studies that the expression of stress-induced e m o t i o n a l states (anger) is beneficial in protecting the organism from the physiological
consequences of stress. H o w e v e r , there is very limited evidence concerning the stress-reducing effect of manifestation of an aggressive response on neurochemical activity in the brain 2°. Therefore, the present e x p e r i m e n t was conducted to d e t e r m i n e whether or not an aggressive biting response could a t t e n u a t e increases in brain noradrenaline ( N A ) turnover p r o d u c e d by supine restraint stress, by measuring levels of N A and its m a j o r metabolite 14, 3-methoxy-4-hydroxyphenylethyleneglycol sulfate ( M H P G - S O 4 ) , in discrete brain regions of male Wistar rats. It is well established that noradrenergic neurons in many brain regions in rats are activated by a variety of stressful stimuli 2'6. Moreover. our recent studies revealed that stress-induced increases in N A t u r n o v e r in certain brain regions (the h y p o t h a l a m u s , amygdala, thalamus etc.) might be closely related to the provocation of negative emo-
Correspondence: A. Tsuda, Department of Pharmacology, Kurume University School of Medicine, Kurume 830, Japan. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
175 tion (anxiety or fear) observed in animals during stress exposure 10-12'21'26'27. MATERIALS AND METHODS
Animals Forty-four male Wistar rats, weighing 180-200 g, were used as subjects. Rats were housed in groups of 4 in standard polypropylene cages (26.5 x 42.5 x 15.0 cm) containing wood shavings in an air-conditioned room (temperature, 24 + 1 °C; humidity, 50 + 10%) and were kept on a 12 h (light on 07.00-19.00 h) light-dark cycle. Food and water were provided ad lib.
plate by the method of Gispen et al.5: the hypothalamus, amygdala, thalamus, hippocampus, midbrain, cerebral cortex and basal ganglia. The locus coeruleus (LC) was also dissected out by the method of Reis and Ross 19. Brain tissues and separated plasma were stored at -45 °C until assayed. NA and MHPGSO4 levels in the brain regions were determined simultaneously by our fluorometric method 13. Plasma corticosterone levels were determined fluorometrically by the method of van der Vies 2s. All experimental procedures were performed between 10.00 and 15.00 h.
Statistical analysis Procedure By balancing body weights, animals were allocated to an untreated control group (n = 8) and 4 stressed groups. The stressed groups, consisting of two biting groups of 10 rats each and two non-biting groups of 8 rats each, were exposed to a 10 min restraint stress by immobilizing the rat in the supine position on a 13 x 22 cm wooden board 7. Each leg was extended at a 45 °C angle from the body midline and secured with a loop of adhesive tape. During the stress, the animals in the biting groups were visually and tactually stimulated by an 18 cm long x 0.5 cm diameter piece of wooden stick which was extended toward the mouth of the rat by the experimenter. Therefore, the rats were given an opportunity to bite the wooden stick. At the same time, the experimenter recorded the duration of any biting responses using a standard cumulative timer. Animals in the non-biting groups were restrained but were not confronted with the wooden stick. Half of the biting group and the non-biting group were sacrificed by decapitation immediately following the restraint period. The remaining animals in each of these groups were returned to home cages without food and water, and decapitated 50 min after release from stress. Two rats in the biting groups were discarded due to procedural error and due to spending less time exhibiting biting (less than 7 of 10 min), respectively.
Tissue preparation and biochemical determination When animals were decapitated, their brains were rapidly removed and blood from the cervical wound was collected into heparinized tubes. The brain was dissected into the following brain regions on an ice
Factorial analysis of variance (ANOVA) followed by a post-hoc Tukey's HSD test, where significant main effects were detected with significance considered at the 0.05 level, were used for statistical analysis. RESULTS
Biting responses Most animals (19/20 rats) assigned to the biting groups exhibited a head thrust and biting toward the wooden stick which was extended near the mouth during stress exposure. There was no significant difference in duration of biting between biting groups sacrificed 0 min (mean + S.E.M. = 524.0 + 12.1 s) or 50 min (506 +_ 21.9 s) after release from stress.
Plasma corticosterone levels ANOVA of plasma corticosterone levels revealed highly significant main effects of biting (/71,30 = 7.6, P < 0.01) and post-stress delay (Fi.30 = 9.0, P < 0.01), as well as a significant interaction between these factors (F1,30 = 12.8, P < 0.01) (Table I). Tukey's post-hoc HSD test (P < 0.05) indicated that biting and non-biting groups examined 0 min after release from stress showed significantly higher levels of corticosterone as compared with the control group, and that these groups did not differ from each other. Fifty rain after release from stress, the biting group exhibited significantly lower levels of corticosterone than the biting group examined 0 min after release from stress, while the non-biting group maintained higher levels of corticosterone as compared with the control group.
176 significant main effect of post-stress delay in the amygdala (FI.30 = 6.4, P < 0.05). In general, NA levels were not markedly affected by our experimental treatments. Tukey's HSD tests indicated that NA levels for the non-biting group examined 50 min after release from stress were significantly decreased in the amygdala as compared with the non-biting group examined 0 min after release from stress. Although ANOVAs of MHPG-SO 4 levels revealed significant main effects of post-stress delay in the hypothalamus (F1,30 = 5.4, P < 0.05), amygdala (F1.30 = 11.6, P < 0.01) and thalamus (Fl.~0 = 4.8, P < 0.05), there were no significant main effects of biting in any brain region. ANOVAs also revealed significant interactions between biting and post-stress delay in the hypothalamus (F1,30 = 6.8, P < 0.05) and amydala (El,31 = 7.5, P < 0.05). Tukey's post-hoc HSD comparisons indicated that, as compared to control group, biting and nonbiting groups examined 0 min after release from stress showed no elevations of MHPG-SO4 levels in
TABLE I
Mean (+ S.E.M. ) levels o f plasma corticosterone in rats exposed to 10 rain supine restraint with or without being allowed to bite a wooden stick Treatment
Number of subjects
Plasma corticosterone (in tzg/dl)
Control 0 min after release from stress Biting group Non-biting group 50 rain after release from stress Biting group Non-biting group
8
12.2 + 3.3
9 8
33.7 + 1.6 a 31.5 + 1.9"
9 8
16.4 + 2.2 c 33.0 + 4.2 a'l'
Differs significantly (at least P < 0.05) from acontrol group, b the respective biting group, Cthe respective condition of 0 min after release from stress.
Regional brain NA and MHPG-SO~ levels Fig. t depicts levels of NA and MHPG-SO4 in the hypothalamus, amygdala, thalamus and midbrain for the 5 groups. ANOVAs of NA levels revealed only a
AMYGDALA
HYPOTHALAMUS
2,0
NA
0.4 __
MHPG-SO4
NA
0.5
0.4 - -
MHPG-SO4
*#§ .1_.
fl/l/ 0: m i
1.o
o
0
so
TIME
ul >
0
SO
AFTER
RELEASE
s0
0
s0
FROM STRESS (mln)
MIDBRAIN THALAMUS 0.6 F
NA
0.4
MHPG-SO4
* #§
NA
0-6
0.3
MHPG-$O4
i
*o §
0.3,
.
0.3
o
o so
0
TIME
80
AFTER
0
RELEASE
FROM STRESS
6o
o
so
train)
Fig. 1. Levels of NA and MHPG-SO 4 in brain regions of rats (the hypothalamus, amygdala, thalamus and midbrain) exposed a 10 Bin supine restraint stress with or without being allowed to bite a wooden stick. Each value indicates the mean (+ S.E.M.) of 8-9 rats. [2, control group; I111,biting group; Ill, non-biting group. * Differs significantly (at least P < 0.05) from control group, § the respective biting group, # the respective group of 0 min after release from stress. NA. noradrenalinc; MHPG-SO 4. 3-methoxy-4-hydroxyphenylethyleneglycol sulfate.
177 each of the brain regions depicted in Fig. 1, with the exception of the amygdala of the biting group. However, these groups exhibited significant increases of MHPG-SO4 levels 50 rain after release from stress. Of greater importance are the data showing that the non-biting group revealed marked increases of MHPG-SO 4 levels in these regions compared to the biting group. MHPG-SO4 levels for the non-biting group examined 50 min after release from stress significantly increased in the brain regions shown in Fig. 1, as compared with the respective group examined 0 min after release from stress, while those of the biting group revealed no changes. Fig. 2 depicts levels of NA and MHPG-SO4 in the basal ganglia, hippocampus, cerebral cortex and LC region for the five groups. ANOVAs of NA levels revealed only a significant main effect of post-stress delay in the basal ganglia (F1,30 = 5.1, P < 0.05). Tukey's HSD tests indicated that NA levels for the nonbiting group examined 50 min after release from
stress significantly increased in the basal ganglia as compared with each of the non-biting groups examined 0 rain after release from stress, and with their respective biting groups. ANOVAs of MHPG-SO 4 levels revealed significant main effects of post-stress delay in the basal ganglia (F1,29 = 5.5, P < 0.05), hippocampus (F1,3o = 15.7, P < 0.01), cerebral cortex (F1,30 = 13.9, P < 0.01) and LC region (FL30 = 5.0, P < 0.05), as well as a significant main effect of biting in the basal ganglia (F1,29 = 4.4, P < 0.05). ANOVAs also revealed significant interactions between biting and post-stress delay in the hippocampus (F1.30 = 6.2, P < 0.05) and cerebral cortex (F1,30 = 6.4, P < 0.05). Tukey's post-hoc comparisons generally revealed that, as compared to the control group, biting and non-biting groups examined 0 min after release from stress showed no elevations in MHPG-SO4 levels in each of the brain regions depicted in Fig. 2. However, these groups exhibited significant increases in MHPG-SO4 levels in the hippocampus and cerebral
BASAL GANGLIA NA
0.3--
0,18
# §
0.2
-11/.1°:1 so
o
HIPPOCAMPUS MHPG-SO4
D
0.4
SO
•
0.3
NA
0.2
o.,I
0"4/[-
gO
MHPO-804 *
•
* •
go
TIME AFTER RELEASE FROM STRESS (mini
CEREBRAL CORTEX
ul .i
NA
LC REGION MHPG-$O4
o.,
0.4
.
MHPG-IO4
o
o
0
SO
O
80
0
SO
TIME AFTER RELEASE FROM ETREa8 (rain)
Fig. 2. Levels of NA and MHPG-SO4in brain regions of rats (the basal ganglia, hippocampus, cerebral cortex and LC region) exposed a 10 rain supine restraint stress with or without being allowed to bite a wooden stick. Each value indicates the mean (-+S.E.M.) of 7-9 rats. Q, Control group; Ill, biting group; I , non-bitinggroup. Differs significantly(at least P < 0.05) from * control group, § the respective biting group, # the respective group of 0 min after release from stress. NA, noradrenaline; MHPG-SO4, 3-methoxy-4-hydroxyphenylethyleneglycolsulfate; LC, locus coeruleus.
178 cortex 50 min after release from stress. The non-biting group revealed marked increases in MHPG-SO 4 levels in the basal ganglia compared to the biting group. MHPG-SO4 levels for the non-biting group examined 50 min after release from stress significantly increased in the brain regions shown in Fig. 2, as compared with the respective group examined 0 min after release from stress, while those of the biting group revealed no changes. DISCUSSION This study clearly revealed that allowing rats an opportunity to engage in an aggressive biting response subsequently attenuated the enhancement of brain NA turnover induced by supine restraint stress. The data from this experiment indicated that rats which could bite a wooden stick extended toward the mouth under conditions of restraint stress produced less elevations of MHPG-SO 4 levels in the hypothalamus, amygdala, thalamus, midbrain and basal ganglia than seen in rats which were exposed to the same stress situation but without the opportunity to engage in biting. These attenuating effects of expression of aggressive biting responses on enhanced NA turnover occurred at 50 min after release from stress rather than immediately after stress. The present results are in agreement with previous literature reporting the effects of fighting and biting responses in reducing the severity of gastric lesions s'9'29'31 and elevations in plasma corticosterone and ACTH 3° and blood pressure 33. These findings suggest that the expression of aggression is related not only to reduced peripheral physiological-endocrine activation, but also to attenuated brain noradrenergic neuronal activity. This study is also comparable with the results of Stolk et al. 2° who demonstrated that allowing rats to fight determined the effects of inescapable footshock stress on central noradrenergic neuronal function. When rats were exposed to shock stress, [3H]NA in the brainstem decreased rapidly. On the other hand, when allowed to fight, the change in [aH]NA was negligible. The effect of an aggressive response on attenuating the magnitude of stress-induced brain NA turnover was not different depending upon the method used to determine this behavior. In previous studies reporting the protective effects of aggression on
pathophysiological processes 20,30"31,33, animals
displayed aggression attack toward each other in response to shock. On the other hand, rats in our experiment had access to a biting attack against a wooden target. The results from this experiment suggest that the release of any aggressive response sufficiently reduces stress-enhanced brain NA turnover, and are consistent with previous studies, in which rats were given the opportunity to express the aggression against an inanimate object such as a nylon brush 29or a wooden block 8. It is unlikely that the present data showing that changes in brain NA turnover occurred 50 min after, but not immediately after release from stress, are simply reflections of a 'time-lag' in that the released NA is metabolized to MHPG-SO4. It is believed that the NA released into the synaptic cleft is rapidly converted to MHPG-SO 4 (refs. 1, 15). NA turnover in most brain regions examined was enhanced in both the biting and non-biting groups 50 min after release from stress due to the aversive nature of the restraint stress treatment. However, the non-biting group exhibited considerably higher increases in NA turnover. This difference was particularly striking in the hypothalamus, amygdala, thalamus, midbrain and basal ganglia, and probably reflects the 'emotional' component of the inability to express aggression. In our previous pharmacological studies, it was shown that enhanced NA turnover in these brain areas was related to the appearance of 'fear' and 'anxiety' in rats exposed to the stress 11'23. We have previously shown that changes in NA turnover in the hypothalamus and limbic structures were indicators of psychological dimensions including the ability to cope with the stressor 12'24'25. The present findings not only support our notion of regional brain characteristics related to susceptibility to stress 22, but also add more precise evidence that noradrenergic neuronal activity in the hypothalamus and limbic areas is apparently involved in provoking unpleasant emotions in stressed rats. In accordance with our view, Redmond and Huang Is and Weiss et al. 32 pointed out the importance of increased NA neuronal activity in the LC and distal regions of the ventral system innervated by the LC. Moreover, it was suggested that brain noradrenergic function in the LC and amygdala is involved in the elaboration of certain types of aggressive behaviors 16, although aggression is a corn-
179 plicated behavior, which has many forms including territorial, predatory and defensive aspects. What is it about aggressive behavior which produces attenuating effects against stress-enhanced NA turnover in these brain regions, as well as increased endocrine activity in the adrenal cortex? There are some interpretations which may help to explain these results. The first hypothesis is that aggressive biting behavior might distract the biting rat's attention from the discomfort of the restraint stress situation and thereby to reduce the effects of that stressor, suggested by Weiss et a i r . Secondly, because aggressive biting is associated with self-protection under threatening situations, such responses may facilitate the termination of sympathetic arousal; that is, the expression of relevant negative emotional responses is beneficial to the animal 4"17. Thus, we speculate that access to an aggressive response has a protective effect in situations which result in tension and possible organic disease conditions (e.g. gastric ulcers). Engaging in aggressive response may serve to reduce the input of stressors which trigger NA neuronal transmission within specific brain circuits. It is, however, difficult to identify from this experiment when NA metabolism for the biting and nonbiting rats reached their maximal responses nor returned to the steady levels. It might be argued that the biting rats had their responses spread over a longer period of time than the non-biting rats. In any
event, the present experiment clearly demonstrates that among rats which could not express aggression, the rate of NA utilization increased markedly during the 50 min post stress period, as evidenced by higher levels of MHPG-SO4. We are currently investigating the time course of changes in brain NA turnover for both groups of rats at various phases after release from the stress. In conclusion, the present study not only seems to support clinical studies suggesting that suppression of anger or hostility leads to psychopathological consequences 3, but also seems to provide a possible neurochemical basis for these conditions.
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1 Ad~r, J.-P., Muskiet, F.A.J., Jeuring, H.J. and Korf, J., On the origin of vanillylmandelicacid and 3-methoxy-4-hydroxy-phenylglycol in the rat brain, J. Neurochem., 30 (1978) 1213-1216. 2 Anisman, H. and Zacharko, R.M., Behavioral and neurochemical consequences associated with stressors, Ann. N. Y. Acad. Sci., 467 (1986) 205-225. 3 Freud, S., Inhibition, Symptoms and Anxiety, 1926, Cited from H. Gleitman, Psychology, Norton, New York, 1981, 464 pp. 4 Gellhorn, E., The consequences of the suppression of overt movements in emotional stress: a neurophysiological interpretation, Confin. Neurol., 31 (1969) 289-299. 5 Gispen, W.H., Schotman, P. and De Kloet, E.R., Brain RNA and hypophysectomy: a topographical study, Neuroendocrinol., 9 (1972) 285-296. 6 Glavin, G.B., Stress and brain noradrenaline: a review, Neurosci. Biobehav. Rev., 9 (1985) 233-243. 7 Glavin, G.B., Tanaka, M., Tsuda, A., Kohno, Y., Hoaki, Y. and Nagasaki, N., Regional rat brain noradrenaline turnover in response to restraint stress, Pharmacol. Bio-
ACKNOWLEDGEMENTS This research was supported, in part, by a Grant-in Aid for Encouragement of Young Scientists (No. 61710096 and No. 62710095) to A.T. from the Ministry of Education, Science and Culture of Japan, and by a grant to M.T. from Japan Foundation for Health Sciences. We are grateful to Dr. S. Koga for his technical assistance and to Dr. Gary B. Glavin from the Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Canada for his kind reviewing of an earlier version of this manuscript. Gratitude is also due to Nippon Roche K.K. for the generous supply of MHPG-SO4.
180 Nagasaki, N., Simu~aneous determination of noradrenaline and 3-methoxy-4-hydroxyphenylethyleneglycol sulfate in discrete brain regions of the rat, Anal. Biochem., 97 (1979) 352-358. 14 Kohno, Y., Tanaka, M., Nakagawa, R., Toshima, N. and Nagasaki, N., Regional distribution and production rate of 3-methoxy-4-hydroxyphenylethyleneglycol sulfate (MHPG-SO4) in rat brain, J. Neurochem., 36 (1981) 286-289. 15 Korf, .I., Aghajanian, G.K. and Roth, R., Stimulation and destruction of the locus coeruleus: opposite effects on 3methoxy-4-hydroxyphenylethyleneglycol sulfate levels in the rat cerebral cortex, Eur. J. Pharmacol., 21 (1973) 305-310. 16 Liou, S.Y., Shibata, S. and Ueki, S., Differential effects of electroconvulsive shock on four models of experimentallyinduced aggression in rats, Jpn. J. Pharmacol., 37 (1985) 167-172. 17 Par6, W.P. and Vincent, G.P., Stomach acid secretion in submissive and aggressive rats, Behav. Neurosci., 100 (1986) 381-389. 18 Redmond, Jr., D.E. and Huang, Y.H., Current concepts: II. New evidence for a locus coeruleus-norepinephrine connection with anxiety, Life Sci., 25 (1979) 2149-2162. 19 Reis, D.J. and Ross, R.A., Dynamic changes in brain dopamine-fl-hydroxylase activity during anterograde and retrograde reactions to injury of central noradrenergic axons, Brain Research, 57 (1973) 307-326. 20 Stolk, J.M., Conner, R.L., Levine, S. and Barchas, J.D., Brain norepinephrine metabolism and shock-induced fighting behavior in rats: differential effects of shock and fighting on the neurochemical response to a common footshock stimulus, J. Pharmacol. Exp. Ther., 190 (1974) 193-209. 21 Tanaka, M., Kohno, Y., Nakagawa, R., Ida, Y., Iimori, K., Hoaki, Y., Tsuda, A. and Nagasaki, N., Naloxone enhances stress-increases in noradrenaline turnover in specific brain regions in rats, Life Sci., 30 (1982) 1663-1669. 22 Tanaka, M., Kohno, Y., Nakagawa, R., Ida, Y., Takeda, S., Nagasaki, N. and Noda, Y., Regional characteristics of stress-induced increases in brain noradrenaline release in rats, Pharmacol. Biochem. Behav., 19 (1983) 543-547. 23 Tanaka, M., Kohno, Y., Tsuda, A., Nakagawa, R., Ida,
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Y., Iimori, K., Hoaki, Y. and Nagasaki, N., Differential cffects of morphine on noradrenaline release in brain regions of stressed and non-stressed rats, Brain Research, 275 (1983) 105-115. Tsuda, A. and Tanaka, M., Differential changes in noradrenaline turnover in specific regions of rats brain produced by controllable and uncontrollable shocks, Behav. Neurosci., 99 (1985) 802-817. Tsuda, A., Tanaka, M., Ida, Y., Tsujimaru, S. and Nagasaki, N., Effects of shock controllability on rat brain noradrenaline turnover under FR-1 and FR-3 Sidman avoidance schedules, Physiol. Behav., 37 (1986) 945-950. Tsuda, A., Tanaka, M., Ida, Y., Tsujimaru, S., Ushijima, I. and Nagasaki, N., Effect of preshock experience on enhancement of rat brain noradrenaline turnover induced by psychological stress, Pharrnacol. Biochem. Behav., 24 (1986) 115-119. Tsuda, A., Tanaka, M., Kohno, Y., Ida, Y., Hoaki, Y., limori, K., Nakagawa, R., Nishikawa, T. and Nagasaki, N., Daily increase in noradrenaline turnover in brain regions of activity-stressed rats, Pharmacol. Biochem. Behav., 19 (1983) 393-396. Van der Vies, J., Individual determination of cortisol and corticosterone in a single small sample of peripheral blood, Acta Endocrinol., 38 (1961) 399-406. Vincent, G.P., Pare, W.P., Prenatt, J.D. and Glavin, G.B., Aggression, body temperature, and stress ulcer, Physiol. Behav., 32 (1984) 265-268. Weinberg, J., Erskine, M. and Levine, S., Shock-induced fighting attenuates the effects of prior shock experience in rats, Physiol. Behav., 25 (1980) 9-16. Weiss, J.M., Pohorecky, L.A., Satman, S. and Gruenthal, M., Attenuation of gastric lesions by psychological aspects of aggression in rats, J. Cornp. Physiol. Psychol., 90 (1976) 252-259. Weiss, J.M., Simson, P.G., Hoffman, L.J., Ambrose, M.J., Cooper, S. and Webster, A., Infusion of adrenergic receptor agonists and antagonists into the locus coeruleus and ventricular system of the brain, Neuropharmacology, 25 (1986) 367-384. Williams, R.B. and Eichelman, B., Social setting: influence on the physiological response to electric shock in the rat, Science, 174 (1971) 613-614.
174 BRE 14072
Expression of aggression attenuates stress-induced increases in rat brain noradrenaline turnover Akira Tsuda, Masatoshi Tanaka, Yoshishige Ida, Ishou Shirao, Yuhji Gondoh, Masanobi Oguchi and Masami Yoshida Departmentof Pharmacology, Kurume UniversitySchool of Medicine, Kurume (Japan) (Accepted 31 May 1988)
Key words: Supine restraint stress; Noradrenaline turnover; 3-Methoxy-4-hydroxyphenylethyleneglycol sulfate; Rat brain region; Corticosterone; Expression of aggression
This experiment determined whether or not an aggressive biting response could attenuate stress-induced increases in brain noradrenaline (NA) turnover, by measuring contents of NA and its major metabolite, 3-methoxy-4-hydroxyphenylethylermglycol sulfate (MHPG-SO4), in discrete brain regions of male Wistar rats. Rats were exposed to a I0 rain supine restraint stress with or without being allowed to bite a wooden stick. In each group, the animals were sacrificed by decapitation either 0 rain or 50 rain after release from stress. NA and MHPG-SO 4 levels were unaffected in both biting and non-biting groups immediately after stress, as compared to controis. Fifty min after release from stress, increases in plasma corticosterone levels induced by stress recovered in the biting group but remained high in the non-biting group. MHPG-SO4 levels significantly increased in the hypothalamus, amygdala, thalamus, midbrain. basal ganglia, hippocampus and cerebral cortex in both stressed groups, however the increases in the non-biting group were significantly higher than those in the biting group in the first 5 of these regions. These findings clearly show that giving rats an opportunity to express aggression during stress exposure results in a significant attenuation of stress-induced increases in NA turnover in specific brain regions, such as the hypothalamus and limbic areas. The present experiment provides a possible neurochemical basis for clinical studies showing that suppression of anger in a stressful, frustrating situation leads to pathological consequences in humans. INTRODUCTION Several previous e x p e r i m e n t a l reports have indicated that, in comparison with animals exposed individually to inescapable shock, rats exposed to the same shock in pairs and thus being able to fight in response to shock, showed less elevation of p l a s m a corticosterone and a d r e n o c o r t i c o t r o p i n ( A C T H ) 3°, less increase in blood pressure 33 and fewer gastric mucosal lesions 9"31. In addition, Guile and McCutcheon s and Vincent et al. 29 r e p o r t e d that rats e x p o s e d to restraint stress and which were able to bite a brush or chew a block of w o o d , d e v e l o p e d less acute gastric mucosal lesions than did rats which were unable to display aggressive biting or gnawing responses. It would a p p e a r from these studies that the expression of stress-induced e m o t i o n a l states (anger) is beneficial in protecting the organism from the physiological
consequences of stress. H o w e v e r , there is very limited evidence concerning the stress-reducing effect of manifestation of an aggressive response on neurochemical activity in the brain 2°. Therefore, the present e x p e r i m e n t was conducted to d e t e r m i n e whether or not an aggressive biting response could a t t e n u a t e increases in brain noradrenaline ( N A ) turnover p r o d u c e d by supine restraint stress, by measuring levels of N A and its m a j o r metabolite 14, 3-methoxy-4-hydroxyphenylethyleneglycol sulfate ( M H P G - S O 4 ) , in discrete brain regions of male Wistar rats. It is well established that noradrenergic neurons in many brain regions in rats are activated by a variety of stressful stimuli 2'6. Moreover. our recent studies revealed that stress-induced increases in N A t u r n o v e r in certain brain regions (the h y p o t h a l a m u s , amygdala, thalamus etc.) might be closely related to the provocation of negative emo-
Correspondence: A. Tsuda, Department of Pharmacology, Kurume University School of Medicine, Kurume 830, Japan. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
175 tion (anxiety or fear) observed in animals during stress exposure 10-12'21'26'27. MATERIALS AND METHODS
Animals Forty-four male Wistar rats, weighing 180-200 g, were used as subjects. Rats were housed in groups of 4 in standard polypropylene cages (26.5 x 42.5 x 15.0 cm) containing wood shavings in an air-conditioned room (temperature, 24 + 1 °C; humidity, 50 + 10%) and were kept on a 12 h (light on 07.00-19.00 h) light-dark cycle. Food and water were provided ad lib.
plate by the method of Gispen et al.5: the hypothalamus, amygdala, thalamus, hippocampus, midbrain, cerebral cortex and basal ganglia. The locus coeruleus (LC) was also dissected out by the method of Reis and Ross 19. Brain tissues and separated plasma were stored at -45 °C until assayed. NA and MHPGSO4 levels in the brain regions were determined simultaneously by our fluorometric method 13. Plasma corticosterone levels were determined fluorometrically by the method of van der Vies 2s. All experimental procedures were performed between 10.00 and 15.00 h.
Statistical analysis Procedure By balancing body weights, animals were allocated to an untreated control group (n = 8) and 4 stressed groups. The stressed groups, consisting of two biting groups of 10 rats each and two non-biting groups of 8 rats each, were exposed to a 10 min restraint stress by immobilizing the rat in the supine position on a 13 x 22 cm wooden board 7. Each leg was extended at a 45 °C angle from the body midline and secured with a loop of adhesive tape. During the stress, the animals in the biting groups were visually and tactually stimulated by an 18 cm long x 0.5 cm diameter piece of wooden stick which was extended toward the mouth of the rat by the experimenter. Therefore, the rats were given an opportunity to bite the wooden stick. At the same time, the experimenter recorded the duration of any biting responses using a standard cumulative timer. Animals in the non-biting groups were restrained but were not confronted with the wooden stick. Half of the biting group and the non-biting group were sacrificed by decapitation immediately following the restraint period. The remaining animals in each of these groups were returned to home cages without food and water, and decapitated 50 min after release from stress. Two rats in the biting groups were discarded due to procedural error and due to spending less time exhibiting biting (less than 7 of 10 min), respectively.
Tissue preparation and biochemical determination When animals were decapitated, their brains were rapidly removed and blood from the cervical wound was collected into heparinized tubes. The brain was dissected into the following brain regions on an ice
Factorial analysis of variance (ANOVA) followed by a post-hoc Tukey's HSD test, where significant main effects were detected with significance considered at the 0.05 level, were used for statistical analysis. RESULTS
Biting responses Most animals (19/20 rats) assigned to the biting groups exhibited a head thrust and biting toward the wooden stick which was extended near the mouth during stress exposure. There was no significant difference in duration of biting between biting groups sacrificed 0 min (mean + S.E.M. = 524.0 + 12.1 s) or 50 min (506 +_ 21.9 s) after release from stress.
Plasma corticosterone levels ANOVA of plasma corticosterone levels revealed highly significant main effects of biting (/71,30 = 7.6, P < 0.01) and post-stress delay (Fi.30 = 9.0, P < 0.01), as well as a significant interaction between these factors (F1,30 = 12.8, P < 0.01) (Table I). Tukey's post-hoc HSD test (P < 0.05) indicated that biting and non-biting groups examined 0 min after release from stress showed significantly higher levels of corticosterone as compared with the control group, and that these groups did not differ from each other. Fifty rain after release from stress, the biting group exhibited significantly lower levels of corticosterone than the biting group examined 0 min after release from stress, while the non-biting group maintained higher levels of corticosterone as compared with the control group.
176 significant main effect of post-stress delay in the amygdala (FI.30 = 6.4, P < 0.05). In general, NA levels were not markedly affected by our experimental treatments. Tukey's HSD tests indicated that NA levels for the non-biting group examined 50 min after release from stress were significantly decreased in the amygdala as compared with the non-biting group examined 0 min after release from stress. Although ANOVAs of MHPG-SO 4 levels revealed significant main effects of post-stress delay in the hypothalamus (F1,30 = 5.4, P < 0.05), amygdala (F1.30 = 11.6, P < 0.01) and thalamus (Fl.~0 = 4.8, P < 0.05), there were no significant main effects of biting in any brain region. ANOVAs also revealed significant interactions between biting and post-stress delay in the hypothalamus (F1,30 = 6.8, P < 0.05) and amydala (El,31 = 7.5, P < 0.05). Tukey's post-hoc HSD comparisons indicated that, as compared to control group, biting and nonbiting groups examined 0 min after release from stress showed no elevations of MHPG-SO4 levels in
TABLE I
Mean (+ S.E.M. ) levels o f plasma corticosterone in rats exposed to 10 rain supine restraint with or without being allowed to bite a wooden stick Treatment
Number of subjects
Plasma corticosterone (in tzg/dl)
Control 0 min after release from stress Biting group Non-biting group 50 rain after release from stress Biting group Non-biting group
8
12.2 + 3.3
9 8
33.7 + 1.6 a 31.5 + 1.9"
9 8
16.4 + 2.2 c 33.0 + 4.2 a'l'
Differs significantly (at least P < 0.05) from acontrol group, b the respective biting group, Cthe respective condition of 0 min after release from stress.
Regional brain NA and MHPG-SO~ levels Fig. t depicts levels of NA and MHPG-SO4 in the hypothalamus, amygdala, thalamus and midbrain for the 5 groups. ANOVAs of NA levels revealed only a
AMYGDALA
HYPOTHALAMUS
2,0
NA
0.4 __
MHPG-SO4
NA
0.5
0.4 - -
MHPG-SO4
*#§ .1_.
fl/l/ 0: m i
1.o
o
0
so
TIME
ul >
0
SO
AFTER
RELEASE
s0
0
s0
FROM STRESS (mln)
MIDBRAIN THALAMUS 0.6 F
NA
0.4
MHPG-SO4
* #§
NA
0-6
0.3
MHPG-$O4
i
*o §
0.3,
.
0.3
o
o so
0
TIME
80
AFTER
0
RELEASE
FROM STRESS
6o
o
so
train)
Fig. 1. Levels of NA and MHPG-SO 4 in brain regions of rats (the hypothalamus, amygdala, thalamus and midbrain) exposed a 10 Bin supine restraint stress with or without being allowed to bite a wooden stick. Each value indicates the mean (+ S.E.M.) of 8-9 rats. [2, control group; I111,biting group; Ill, non-biting group. * Differs significantly (at least P < 0.05) from control group, § the respective biting group, # the respective group of 0 min after release from stress. NA. noradrenalinc; MHPG-SO 4. 3-methoxy-4-hydroxyphenylethyleneglycol sulfate.
177 each of the brain regions depicted in Fig. 1, with the exception of the amygdala of the biting group. However, these groups exhibited significant increases of MHPG-SO4 levels 50 rain after release from stress. Of greater importance are the data showing that the non-biting group revealed marked increases of MHPG-SO 4 levels in these regions compared to the biting group. MHPG-SO4 levels for the non-biting group examined 50 min after release from stress significantly increased in the brain regions shown in Fig. 1, as compared with the respective group examined 0 min after release from stress, while those of the biting group revealed no changes. Fig. 2 depicts levels of NA and MHPG-SO4 in the basal ganglia, hippocampus, cerebral cortex and LC region for the five groups. ANOVAs of NA levels revealed only a significant main effect of post-stress delay in the basal ganglia (F1,30 = 5.1, P < 0.05). Tukey's HSD tests indicated that NA levels for the nonbiting group examined 50 min after release from
stress significantly increased in the basal ganglia as compared with each of the non-biting groups examined 0 rain after release from stress, and with their respective biting groups. ANOVAs of MHPG-SO 4 levels revealed significant main effects of post-stress delay in the basal ganglia (F1,29 = 5.5, P < 0.05), hippocampus (F1,3o = 15.7, P < 0.01), cerebral cortex (F1,30 = 13.9, P < 0.01) and LC region (FL30 = 5.0, P < 0.05), as well as a significant main effect of biting in the basal ganglia (F1,29 = 4.4, P < 0.05). ANOVAs also revealed significant interactions between biting and post-stress delay in the hippocampus (F1.30 = 6.2, P < 0.05) and cerebral cortex (F1,30 = 6.4, P < 0.05). Tukey's post-hoc comparisons generally revealed that, as compared to the control group, biting and non-biting groups examined 0 min after release from stress showed no elevations in MHPG-SO4 levels in each of the brain regions depicted in Fig. 2. However, these groups exhibited significant increases in MHPG-SO4 levels in the hippocampus and cerebral
BASAL GANGLIA NA
0.3--
0,18
# §
0.2
-11/.1°:1 so
o
HIPPOCAMPUS MHPG-SO4
D
0.4
SO
•
0.3
NA
0.2
o.,I
0"4/[-
gO
MHPO-804 *
•
* •
go
TIME AFTER RELEASE FROM STRESS (mini
CEREBRAL CORTEX
ul .i
NA
LC REGION MHPG-$O4
o.,
0.4
.
MHPG-IO4
o
o
0
SO
O
80
0
SO
TIME AFTER RELEASE FROM ETREa8 (rain)
Fig. 2. Levels of NA and MHPG-SO4in brain regions of rats (the basal ganglia, hippocampus, cerebral cortex and LC region) exposed a 10 rain supine restraint stress with or without being allowed to bite a wooden stick. Each value indicates the mean (-+S.E.M.) of 7-9 rats. Q, Control group; Ill, biting group; I , non-bitinggroup. Differs significantly(at least P < 0.05) from * control group, § the respective biting group, # the respective group of 0 min after release from stress. NA, noradrenaline; MHPG-SO4, 3-methoxy-4-hydroxyphenylethyleneglycolsulfate; LC, locus coeruleus.
178 cortex 50 min after release from stress. The non-biting group revealed marked increases in MHPG-SO 4 levels in the basal ganglia compared to the biting group. MHPG-SO4 levels for the non-biting group examined 50 min after release from stress significantly increased in the brain regions shown in Fig. 2, as compared with the respective group examined 0 min after release from stress, while those of the biting group revealed no changes. DISCUSSION This study clearly revealed that allowing rats an opportunity to engage in an aggressive biting response subsequently attenuated the enhancement of brain NA turnover induced by supine restraint stress. The data from this experiment indicated that rats which could bite a wooden stick extended toward the mouth under conditions of restraint stress produced less elevations of MHPG-SO 4 levels in the hypothalamus, amygdala, thalamus, midbrain and basal ganglia than seen in rats which were exposed to the same stress situation but without the opportunity to engage in biting. These attenuating effects of expression of aggressive biting responses on enhanced NA turnover occurred at 50 min after release from stress rather than immediately after stress. The present results are in agreement with previous literature reporting the effects of fighting and biting responses in reducing the severity of gastric lesions s'9'29'31 and elevations in plasma corticosterone and ACTH 3° and blood pressure 33. These findings suggest that the expression of aggression is related not only to reduced peripheral physiological-endocrine activation, but also to attenuated brain noradrenergic neuronal activity. This study is also comparable with the results of Stolk et al. 2° who demonstrated that allowing rats to fight determined the effects of inescapable footshock stress on central noradrenergic neuronal function. When rats were exposed to shock stress, [3H]NA in the brainstem decreased rapidly. On the other hand, when allowed to fight, the change in [aH]NA was negligible. The effect of an aggressive response on attenuating the magnitude of stress-induced brain NA turnover was not different depending upon the method used to determine this behavior. In previous studies reporting the protective effects of aggression on
pathophysiological processes 20,30"31,33, animals
displayed aggression attack toward each other in response to shock. On the other hand, rats in our experiment had access to a biting attack against a wooden target. The results from this experiment suggest that the release of any aggressive response sufficiently reduces stress-enhanced brain NA turnover, and are consistent with previous studies, in which rats were given the opportunity to express the aggression against an inanimate object such as a nylon brush 29or a wooden block 8. It is unlikely that the present data showing that changes in brain NA turnover occurred 50 min after, but not immediately after release from stress, are simply reflections of a 'time-lag' in that the released NA is metabolized to MHPG-SO4. It is believed that the NA released into the synaptic cleft is rapidly converted to MHPG-SO 4 (refs. 1, 15). NA turnover in most brain regions examined was enhanced in both the biting and non-biting groups 50 min after release from stress due to the aversive nature of the restraint stress treatment. However, the non-biting group exhibited considerably higher increases in NA turnover. This difference was particularly striking in the hypothalamus, amygdala, thalamus, midbrain and basal ganglia, and probably reflects the 'emotional' component of the inability to express aggression. In our previous pharmacological studies, it was shown that enhanced NA turnover in these brain areas was related to the appearance of 'fear' and 'anxiety' in rats exposed to the stress 11'23. We have previously shown that changes in NA turnover in the hypothalamus and limbic structures were indicators of psychological dimensions including the ability to cope with the stressor 12'24'25. The present findings not only support our notion of regional brain characteristics related to susceptibility to stress 22, but also add more precise evidence that noradrenergic neuronal activity in the hypothalamus and limbic areas is apparently involved in provoking unpleasant emotions in stressed rats. In accordance with our view, Redmond and Huang Is and Weiss et al. 32 pointed out the importance of increased NA neuronal activity in the LC and distal regions of the ventral system innervated by the LC. Moreover, it was suggested that brain noradrenergic function in the LC and amygdala is involved in the elaboration of certain types of aggressive behaviors 16, although aggression is a corn-
179 plicated behavior, which has many forms including territorial, predatory and defensive aspects. What is it about aggressive behavior which produces attenuating effects against stress-enhanced NA turnover in these brain regions, as well as increased endocrine activity in the adrenal cortex? There are some interpretations which may help to explain these results. The first hypothesis is that aggressive biting behavior might distract the biting rat's attention from the discomfort of the restraint stress situation and thereby to reduce the effects of that stressor, suggested by Weiss et a i r . Secondly, because aggressive biting is associated with self-protection under threatening situations, such responses may facilitate the termination of sympathetic arousal; that is, the expression of relevant negative emotional responses is beneficial to the animal 4"17. Thus, we speculate that access to an aggressive response has a protective effect in situations which result in tension and possible organic disease conditions (e.g. gastric ulcers). Engaging in aggressive response may serve to reduce the input of stressors which trigger NA neuronal transmission within specific brain circuits. It is, however, difficult to identify from this experiment when NA metabolism for the biting and nonbiting rats reached their maximal responses nor returned to the steady levels. It might be argued that the biting rats had their responses spread over a longer period of time than the non-biting rats. In any
event, the present experiment clearly demonstrates that among rats which could not express aggression, the rate of NA utilization increased markedly during the 50 min post stress period, as evidenced by higher levels of MHPG-SO4. We are currently investigating the time course of changes in brain NA turnover for both groups of rats at various phases after release from the stress. In conclusion, the present study not only seems to support clinical studies suggesting that suppression of anger or hostility leads to psychopathological consequences 3, but also seems to provide a possible neurochemical basis for these conditions.
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ACKNOWLEDGEMENTS This research was supported, in part, by a Grant-in Aid for Encouragement of Young Scientists (No. 61710096 and No. 62710095) to A.T. from the Ministry of Education, Science and Culture of Japan, and by a grant to M.T. from Japan Foundation for Health Sciences. We are grateful to Dr. S. Koga for his technical assistance and to Dr. Gary B. Glavin from the Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Canada for his kind reviewing of an earlier version of this manuscript. Gratitude is also due to Nippon Roche K.K. for the generous supply of MHPG-SO4.
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