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Catecholamines and their enzymes in discrete brain areas of rats after space flight on biosatellites Cosmos
Acta Astronautica Vol. 10, No. 5-6, pp. 295-300, 19e3 Printed in Great Britain.
0094-5765/83 $3.00+.00 Pergamon Press Ltd.
CATECHOLAMINES AND THEIR ENZYMES IN DISCRETE BRAIN AREAS OF RATS AFTER SPACE FLIGHT ON BIOSATELLITES COSMOS R. KVE~ANSK~', J. (~ULMAN,L. V. SEROVA?,R. A. TIGRANJANt,T. TORDAand L. MACHO Institute of Experimental Endocrinology, Centre of Physiological Sciences, Slovak Academy of Sciences, 83306 Bratislava, Vlfirska 3, Czechoslovakia (Received 6 January 1983) Abstrn?t--The activity of the catecholaminergic system was measured in the hypothalamus of rats which had experienced an 18.5-19.5-day-longstay in the state of weightlessnessduring space flightson board Soviet biosatellites of the type Cosmos. In the first two experiments, Cosmos 782 and 936, the concentration of norepinephrine and the activities of synthesizing enzymes tyrosine hydroxylase and dopamine-~8-hydroxylase and of the degrading enzyme monoamine oxidase were measured in the total hypothalamus. None of the given parameters was changed after space flight. In the light of the changes of these parameters recorded after exposure to acute stress on Earth, this findingindicates that long-termstate of weightlessnessdoes not represent an intensive stressogenic stimulus for the system studied. In the space experimentCosmos 1129,the concentration of norepinephrine,epinephrine,and dopaminewas studied in isolated nuclei of the hypothalamusof rats within 6-10 hr followingreturn from space. Norepinephrinewas found to be significantlyreduced in the arcuate nucleus, median eminence and periventricular nucleus, epinephrine in the median eminence, periventricular and suprachiasmatic nuclei, whereas dopamine was not significantlychanged after space flight. The decreased catecholamine levels found in some hypothalamic nuclei of rats which had undergone space flight indicate that no chronic intensive stressor could have acted during the flight, otherwise the catecholamine concentration would have been increased in the nuclei. The decreased levels must have been induced by the effect of a stressogenic factor acting for a short time only, and that either during the landing maneuver or immediately after landing. Thus long-term exposure of the organism to the state of weightlessness does not represent a stressogenic stimulus for the catecholaminergic system in the hypothalamus, which is one of the regulators of the activation of neuroendocrine reactions under stress.
1. INTRODUCTION
The effect of the state of weightlessness on the living organism during long-term space flights is one of the basic problems that the space medicine has still to solve. The state of weightlessness is an extremely non-physiologic situation, which the living organism has never encountered on Earth, and thus it may act as a stressor. Activation of the sympathetic-adrenomedullary system (SAS), resulting in the secretion of catecholamines (CA), and of the pituitary-adrenocortical system, resulting in the secretion of glucocorticoids, are classical indicators of stress reaction. The activity of these systems was studied in rats which participated in long-term space flights on biosatellites of the type Cosmos[l-4]. Activation of the given systems is the final effect of complex neuroendocrine reactions, in which brain catecholamines are also involved[5]. The brain adrSnergic system, particularly in the hypothalamus has been found to be activated under stress. Several authors have demonstrated a drop of norepinephrine (NE) and its increased turnover rate in the hypothalamus [6--9] as well as changes in the concentration of dopamine (DA) [6, 7, 10]. A few studies have also reported an increased activity of the epinephrinergic system in the brain under stress[ll-13]. ?Institute of Biomedical Problems, Moscow, U.S.S.R. 295
The aim of this study was to evaluate the influence of stressful factors and of the state of weightlessness during long-term space flight on the activity of the catecholaminergic systems in the rat brain, mainly in the hypothalamus.
2. MATERIALSANDMETHODS The question whether the state of weightlessness has a stressful influence on the activity of the hypothalamic adrenergic system was studied in rats which experienced space flight on the biosatellites Cosmos. On board the biosatellite Cosmos 782 all rats spent 19.5 days in space in the state of weightlessness. On board the biosatellite Cosmos 936, some rats were caged in a centrifuge (lg) to distinguish the effect of weightlessness from other stressful factors connected with space flight. The experiment of Cosmos 1129 was built on our previous results obtained in rats exposed to single or repeated immobilization stress on Earth[7, 14, 15]. On the basis of our experience in the field of stress we argued that if rats were chronically exposed to intensive stressors during space flight, after their return to Earth, they should react to immobilization stress as chronically stressed rats. If however, space flight is not connected with prolonged intensive stressful stimulation of the SAS and brain adrenergic system, then the rats should react to
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immobilization stress as animals acutely exposed to stress. The experiments were performed on male rats (SPF colony, Bratislava) weighting 200-250g. The rats from Cosmos 782 and 936 were decapitated 6--10 hr or 25-26 days after a 19.5-day-long space flight. Aboard the biosatellites, the rats were kept in individual cages in the state of weightlessness (F), and on board Cosmos 936 also in a centrifuge in which an artificial gravitational force of 1 g acted almost during the whole flight. During the touchdown maneuver the centrifuge was switched off because of technical reasons. The obtained results were compared with the control group (C) and with a synchronous group (S) which was exposed on Earth to similar conditions as group F in space except the state of weightlessness. The rats from Cosmos 1129 had very similar conditions to those from the previous ships, but there was no centrifuge on board and the biosatellite spent in space only 18.5 days. The animals were decapitated immediately after landing (7-10 hr) and 6 days after landing. The third flying group was after landing exposed to immobilization stress 5 times, daily for 150 rain[15], and the animals were killed immediately after the last exposure. The first immobilization was performed directly at the place of landing but the following day (transport of rats to Moscow) this procedure was omitted. The control and synchronous groups were also exposed to repeated immobilization stress. In our immobilization experiments on Earth, the rats were stressed either acutely or repeatedly (40 times, daily for 150min). In addition to these we had also a group of so-called adapted controls, which were decapitated 24 hours after the 39th immobilization. Immediately after decapitation the brain was removed and the hypothalamus was isolated. In the experiment Cosmos 1129, the whole brain was frozen and its individual nuclei were isolated by a special technique described by Palkovits[16]. We isolated a total of 16 microregions of the rat brain, in this paper, however, only the results concerning six hypothalmic nuclei will be reported, i.e. the arcuate nucl. (NARC), ventromedial nucl. (NVM), dorsomedial nucl. (NDM), periventricular nucl. (NPE), median eminence (EM), and the suprachiasmatic nucl. (NSC). The given hypothalamic regions are crucial with respect to the control of neuroendocrine reactions under stress. Catecholamines in the homogenate of the entire hypothalamus were determined by the radioenzymatic method according to Coyle and Henry[17], and in the isolated hypothalamic nuclei by the method of Peuler and Johnson[18] in our modification. Hypothalamic tyrosine hydroxylase activity (TH) was measured according to Saavedra et al.[19], dopamine - /3 - hydroxylase (DBH) according to Molinoff et al.[20], and monoamine oxidase (MAO) by the method of Wurtman and Axelrod[21]. The results are expressed as means -+ SEM of 5-7 rats and statistical significance was calculated by Student's t-test. 3. RESULTS
3.1 COSMOS 782 Norepinephrine in the total hypothalamus. NE concentrations in the hypothalamus of rats killed im-
mediately, i.e. 6-10 hr after the cosmic flight, were unchanged compared to both the control and synchronous groups (Fig. 1). Catecholamine-synthesizing enzymes in the hypothalamus. The results presented on Fig. 1 demonstrate that the activity of TH in the hypothalamus of flight group rats did not significantly change compared to the control and synchronous groups. The activity of DBH was also comparable with that of the control group, yet it was significantly higher compared to the synchronous group (Fig. 1). Catecholamine-degrading enzymes in the hypothalamus. MAO activity was measured in the hypothalamus of rats after long-term space flight. As seen in Fig. 1, no significant MAO changes occurred compared to either the control or synchronous group. 3.2 COSMOS 936 In the rats participating in a long-term flight aboard this biosatellite either under conditions of weightlessness or in a centrifuge, which provided conditions of earth gravitation, concentrations of CA and TH, DBH, and MAO activities were measured in the hypothalamus. Similarly as in the experiment on board the biosatellite Cosmos 782, none of these indicators was significantly changed compared to the control groups. The values recorded in the animals which had experienced the flight in the centrifuge were comparable with those obtained from rats which had lived in the state of weightlessness. Since the results are virtually the same as those obtained in the
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Fig. 1. Concentration of norepinephrine and activity of tyrosine hydroxylase (TH), dopamine-,8-hydroxylase (DBH) and monoamineoxidase (MAO)in the hypothalamusof rats after space flight aboard the biosatellite Cosmos 782. The animals were decapitated 6-10hr after landing (n = 5); x <0.01 compared to synchronous group.
Catecholamines and their enzymes in discrete brain areas of rats after space flight on biosatellites Cosmos experiment of Cosmos 782, they are not presented in this paper.
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Norepinephrine and tyrosine hydroxylase in the total hypothalamus in repeatedly immobilized rats. NE concentration decreased significantly after the first immobilization for 150 min, whereas in rats exposed to this stressor 35 times, no changes were observed (Fig. 2). TH activity, on the other hand, was unchanged after the first exposure, yet was found to be significantly increased after repeated immobilization (Fig. 2), suggesting an enhanced CA synthesis in the hypotalamus which accounts for the unchanged NE concentration in repeatedly stressed animals.
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CA changes in hypothalamic nuclei immediately alter landing. The concentrations of NE, DA, and epinephrine (EPI) in some hypothalamic nuclei of rats decapitated immediately after landing are given in Fig. 3. In the flight group, a significant NE decrease was recorded only in the NARC, NPE and EM, yet in all the regions studied NE tended to decrease. The decrease of NE in the NARC was significant compared to both control groups. EPI concentration was significantly reduced in the NPE, EM and NSC. The EPI decrease in the NE was significant compared to both control groups (Fig. 3). The concentration of DA displayed no significant changes in the regions studied (Fig. 3).
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Fig. 3. Catecholamines in some isolated hypothalamic nuclei in rats after space flight aboard the biosatellite Cosmos 1129.The animals were decapitated within 6-10hr after landing (n =6-7). Abbreviations of the nuclei are explained in methods. *<0.05, *** <0.01 compared to control group; x < 0.05, xx < 0.02 compared to synchronous group.
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Fig. 4. Effect of acuteimmobilizationon catecholamineconcentration in some hypothalamic nuclei of rats (n=4--11). Abbreviations of the nuclei are in Methods; NSO-supraoptic nucl.., NPV-paraventricular nucl. * < 0.05, ** < 0.02, *** < 0.001 compared to control group.
Effect of repeated immobilization on NE and DA in isolated hypothalamic nuclei. Repeated exposure to immobilization stress resulted in all the hypothalamic nuclei studied in a significant increase in NE concentration (Fig. 5). DA concentration was also significantly increased in the majority of hypothalamic nuclei in repeatedly stressed rats (Fig. 5).
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Fig. 5. Effect of repeated immobilization on catecholamine concentration in some hypothalamic nuclei of rats (n = 7-12). Abbreviations of the nuclei are in Methods. *<0.05, **<0.02, *** < 0.001 compared to control group, xx < 0.02, xxx < 0.01 compared to group 39-times immobilized + 24 hr rest. 4. DISCUSSION The main aim of this study was to investigate the effect of long-term space flight on the activity of catecholaminergic systems in the hypothalamus of rats and thus to answer the question whether a long-term state of weightlessness is stressogenic or not. Comparisons of the results obtained in "cosmic" and in "terrestiar' rats exposed to stress were expected to yield the answer. Acute immobilization of rats in experiments on Earth resulted in a decrease of NE in the hypothalamus (Fig. 2), whereas in repeatedly stressed animals NE concentration was not reduced (Fig. 2). Similar results were reported also by other authors using different models of stress [22-24]. In repeatedly immobilized animals, in which the NE concentration in the hypothalamus did no longer differ from unstressed control values, the significantly increased TH activity found suggested an increased CA synthesis in the hypothalamus of these rats (Fig. 2). Increased hypothalamic TH activity in repeatedly stressed rats, was reported also by other authors [25-27]. The activity of DBH was also found to increase in the hypothalamus of rats exposed to repeated stress[6, 28]. In the space experiments performed on board the biosatellites Cosmos 782 (Fig. l) and Cosmos 936, neither the concentration of NE nor TH and DBH activities in the hypothalamus were changed [29, 30]. The unchanged concentrations of NE in the hypothalamus of rats after space flight aboard Cosmos 782 and 936 can be interpreted in two ways: space flight, and particularly long-term state of weightlessness, does either not represent an intensive stressor, or the animals
al.
might have become adapted to the effects of space flight and responded similarly as repeatedly stressed animals, so that CA were no longer reduced. However, the unchanged TH and DBH activities suggest that CA synthesis could not have been increased for a longer period of time during space flight. The unchanged CA levels in the hypothalamus of rats found after space flight would then not be the result of enhanced CA synthesis but rather the result of the unchanged activity of the noradrenergic system in the hypothalamus. The concentration of CA in the hypothalamus of rats may be affected also by changes in their degradation. MAO is one of the enzymes responsible for CA degradation. Our results[31], as well as literary data[25,32] showed decreased MAO activity in the hypothalamus of repeatedly stressed animals. In the hypothalamus of "cosmic" rats, MAO activity was not significantly changed, suggesting that CA degradation by oxidative deamination would not have changed either during space flight. The findings of unchanged activities of enzymes, which participate in the synthesis and degradation of CA, along with the unchanged CA concentrations in the hypothalamus indicate that the long-term effect of the state of weightlessness does not represent a stressogenic factor with respect to the catecholaminergic system studied in homogenates of the rat hypothalamus. The hypothalamus, however, is a very heterogeneous structure, so that CA determinations in homogenates of the whole hypothalamus may not reveal an altered function of this system occurring in some small specific areas, in which activities might have changed during long-term space flight. In rats exposed to intensive stress on Earth, substantial changes in CA concentrations were recorded in some hypothalamic nuclei. Acute immobilization induced decreases both in NE (Fig. 4) and EPI[12], in the majority of the hypothalamic nuclei studies, whereas repeated exposure to this stressor resulted in highly significant NE increases in all the hypothalamic nuclei studied, and in increased DA concentrations in the majority of them (Fig. 5). Decreased NE concentrations in some nuclei of the hypothalamus of rats exposed to acute stress were reported also by other authors[10, 33]. The concentration of NE tends to decrease in the majority of hypothalamic nuclei in "cosmic" rats (Fig. 3), which is a typical manifestation of acute stress (Fig. 4). The significant decrease of NE concentration in the NARC compared to both control and synchronous groups (Fig. 3), indicates an acute stress situation induced presumably by the landing maneuver. A similar NE decrease was demonstrated in several models of acute stress[33]. Chronic exposure to a stressor results in increased NE concentration in the NARC (Fig. 5). The decrease of both NE and EPI in the EM in acute stress presents an interesting, so far not reported, finding (Fig. 3). This change of NE in the EM appears to be a phenomenon specific for cosmic flight and suggests varied speculations on the potential mechanisms involved. Concerning the aim of our investigation, however, it is relevant that both the NE concentration (Fig. 5) and the EPI concentration (Kvetfansk~, et al.,
Catecholamines and their enzymes in discrete brain areas of rats after space flight on biosatellites Cosmos unpublished results) in the EM of repeatedly stressed rats is considerably increased, indicating, also for this region of the hypothalamus, that the rats which had undergone cosmic flight were exposed to the effect of an acute stressor. The concentration of DA did not display any significant changes (Fig. 3). After the long-term effect of an intensive stressor also this parameter would be expected to show increased values in some hypothalamic nuclei (Fig. 5). The EPI concentration in hypothalamic nuclei of rats after space flight was significantly decreased in the NPE, EM and NSC (Fig. 3). The diminished EPI concentration in N P E is a typical finding invariably demonstrated after acute stress in this region[12]. This is again an unequivocal manifestation of acute stress, since long-term exposure has been shown to increase the EPI level significantly in the N P E (KvetnanskS, et al., unpublished results). The presented result demonstrate that only some hypothalamic regions respond to cosmic flight by decreased catecholamine concentration, which accounts for the finding of unchanged levels of analyzing homogenates of the total hypothalamus. The most likely interpretation of the catecholamine changes determined in some hypothalmic nuclei of rats after space flights appears to be the effect of an acute stressor acting presumably during the landing maneuver.
REFERENCES
1. R. Kvetfiansk~', R. A. Tigranjan, T. Torda, D. Rep/~ekov~, E. Jahnov~ and K. Murgagr, Effect of a prolonged space flight on the responses of adrenal cortical and medullary layers. Kosmic. Biol. Aviakosmic. Med. 14(1), 24-27 (1980) (in Russian). 2. R. Kvetfiansk~,, R. A. Tigranjan, J. (~ulman, P. Bl~ff.ek, A. M. Genin and L. Macho, Effect of space flight in biosatellites Cosmos on sympathetic adrenomedullary activity of rats. In Advances in Physiologic Sciences, Vol. 19. Gravitational Physiology (Edited by J. Hideg and O. Gazenko), pp. 225228. Akad~miai Kiadb and Pergamon Press, Budapest (1981). 3. R. Kvtfiansky, T. Torda, L. Macho, R. A. Tigranjan, L. V. Serova and A. M. Genin, Effect of weightlessness on sympathetic-adrenomedullary activity of rats. Acta Astronautica 8,469-481 (1981). 4. R. Kvetfiansk~', M. Vigag, R. A. Tigranjan, S. N/~meth and L. Macho, Activity of the sympathetic-adrenomedullary system in rats after space flight on the Cosmos biosatellites. Adv. Space Res. 1, 187-192 (1981). 5. W. F. Ganong, The role of catecholamines and acetylcholine in the regulation of endocrine function. Life Sci. 15, 14011414 (1974). 6. R. Kvetfiansk~/, A. Mitro, M. Palkovits, M. Brownstein, T. Torda, M. Vigag and L. Mikulaj, Catecholamines in individual hypothalamic nuclei in stressed rats. In: Catecholamines and Stress (Edited E. Usdin, R. Kvetfiansk~, and I. J. Kopin) pp. 39-50. Pergamon Press, Oxford (1976). 7. R. Kvetfiansk~,, M. Palkovits, A. Mitro, T. Torda and L. Mikulaj, Catecholamines in individual hypothalamic nuclei of acutely and repeatedly stressed rats. Neuroendocrinology 23, 257-267 (1977). 8. R. Nakagawa, M. Tanaka, Y. Kohno, Y. Noda, N. Nagasaki, Regional responses of rat brain noradrenergic neurones to acute intense stress. Pharmac. Biochem. Behav. 14, 729732 (1981). 9. E. Stone, Accumulation and metabolism of norepinephrine in
299
rat hypothalamus after exhaustive stress. J. Neurochem. 21, 589-601 (1973). 10. M. I. K. Fekete, E. Stark, B. Kanyicska, J. P. Herman and M. Palkovits, Inhibition of stress induced hormonal changes by chronic ACTH treatment; possible relationship with changes of neuroaminergic transmission in the central nervous system. In: Catecholamines "and Stress: Recent Advances (Edited by E. Usdin, R. Kvetfiansk~, and I. J. Kopin), pp. 149-154. Elsevier, New York (1980). 11. M. Goldstein, A. Sauter, K. Ueta and K. Fuxe, Effect of stress on central catecholamine levels. In: Catecholamines and Stress: Recent Advances (Edited by E. Usdin, R. Kvet~ansk~ and I. J. Kopin), pp. 47-52. Elsevier, New York (1980). 12. R. Kvethansk~,, I. J. Kopin and J. M. Saavedra, Changes in epinephrine in individual hypothalamic nuclei after immobilization stress. Brain Res. 155, 387-390 (1978). 13. J. M. Saavedra, R. Kvet~ansk$', I. J. Kopin, Adrenaline, noradrenaline and dopamine levels in specific brain stem areas of acutely immobilized rats. Brain Res. 160, 271-280 (1979). 14. R. Kvetfiansk$' and L. Mikulaj, Adrenal and urinary catecholamines in rats during adaptation to repeated immobilization stress. Endocrinology 87, 738-743 (1970). 15. R. Kvethansk~,, V. K. Weise and I. J. Kopin, Elevation of adrenal tyrosine hydroxylase and phenylethanolamine - N methyltransferase by repeated immobilization of rats. Endocrinology 87,744-749 (1970). 16. M. Palkovits, Isolated removal of hypothalamic or other brain nuclei of the rat. Brain Res. 59, 449--450 (1973). 17. J. T. Coyle and D. T. Henry, Catecholamines in fetal and new born rat brain. J. Neurochem. 21, 61-67 (1973). 18. J. D. Peuler, G. A. Johnson, Simultaneous single isotope radioenzymatic assay of plasma norepinephrine, epinephrine and dopamine. Life Sci. 21, 625-636 (1977). 19. J. M. Saavedra, M. Brownstein, M. Palkovits, S. Kizer and J. Axelrod, Tyrosine hydroxylase and dopamine - /3 hydroxylase: distribution in the individual rat hypothalamic nuclei. J. Neurochem. 23, 869-871 (1974). 20. P. B. Molinoff, R. Weinshilboum, J. Axelrod, A sensitive enzymatic assay for dopamine -/3 - hydroxylase. J. Pharmacol. Exp. Ther. 178,425-431 (1971). 21. R. J. Wurtman and J. Axelrod, A sensitive and specific assay for the estimation of monoamine oxidase. Biochem. Pharmacol. 12, 1439-1440 (1963). 22. O. M. Avakian and E. A. Shirinjan, Compulsion of rats to muscular activity in the antiorthostatic position: Role of the sympatho-adrenal system. In: Catecholamines and Stress: Recent Advances (Edited by E. Usdin, R. Kvethansk~/and I. J. Kopin), pp. 431-435. Elsevier, New York (1980). 23. I. C)stman-Smith, Adaptive changes in the sympathetic nervous system and some effector organs of the rat following long-term exercise or cold acclimation and the role of cardiac sympathetic nerves in the genesis of compensatory cardiac hyperthrophy. Acta Physiol. Scand. Suppl. 477, 1-118 (1979). 24. A. M. Thierry, G. Blanc, J. Glowinski, Effect of stress on the disposition of catecholamines localised in various intraneuronal storage forms in the bralnstem of the rat. J. Neurochem. 18, 449-461 (1971). 25. Z. Huszti and A. Kenessey, 3H-tyrosine incorporation into proteins and catecholamines in immobilized rats. In: Catecholamines and Stress (Edited by E. Usdin, R. Kvetfiansk~, and I. J. Kopin), pp. 377-385. Pergamon Press, Oxford (1976). 26. F. Lamprecht, B. Eichelman, N. B. Thoa, R. B. Williams and I. J. Kopin, Rat fighting behaviour: serum dopamine - /3 hydroxylase and hypothalamic tyrosine hydroxylase. Science 177, 1214-1215 (1972). 27. E. A. Stone, L. S. Freedman, L. E. Morgano, Brian and adrenal tyrosine hydroxylase activity after chronic footshock stress. Pharmacol. Biochem. Behav. 9, 551-553 (1978). 28. G. R. Van Loon, Brain dopamine-~8-hydroxylase activity: response to stress, tyrosine hydroxylase inhibition, hypophysectomy and ACTH administration. In: Catecholamines
300
R. KVETI~ANSK~et al.
and Stress (Edited by E. Usdin, R. Kvetfiansk~, and 1. J. Kopin), pp. 77-85. Pergamon Press, Oxford (1976). 29. R. Kvethansk¢/, R. A. Tigranjan, T. Torda, D. BabugikovL E. Jahnov~., N. F. Kalita and M. Viga~, Catecholamines and enzymes involved in their metabolism in the hypothalamus of rats after space flight on biosatellite Cosmos-782. Kosmic. Biol. 13(3), 24-27 (1979) (in Russian). 30. T. Torda, R. Kvet~anskCA R. A. Tigranjan, J. Culman, A. M. Genin, Noradrenaline and enzymes involved in its synthesis and degradation in the hypothalamus of the rats flown on
Cosmos-936, Kosmic. Biol Auiakosmic. Med. 15 (6), 46-48 (1981) (in Russian). 31. R. Kvefftansk~,, F. Daher, S. Sev~:ik, J. (~ulman, Z. Opr~alov~, and A. Kiss, Degradation of catecholamines in hypothalamic nuclei of rats under stress. Abstracts, Integrative Neurohumoral Mechanisms, p. 128. Hungarian Academy of Sciences, Budapest (1982). 32. G. Maura, A. Vaccari, Relationships between age of submission to environmental stress and monoamine oxidase activity in rats. Experientia 31, 191-192 (1975).
0094-5765/83 $3.00+.00 Pergamon Press Ltd.
CATECHOLAMINES AND THEIR ENZYMES IN DISCRETE BRAIN AREAS OF RATS AFTER SPACE FLIGHT ON BIOSATELLITES COSMOS R. KVE~ANSK~', J. (~ULMAN,L. V. SEROVA?,R. A. TIGRANJANt,T. TORDAand L. MACHO Institute of Experimental Endocrinology, Centre of Physiological Sciences, Slovak Academy of Sciences, 83306 Bratislava, Vlfirska 3, Czechoslovakia (Received 6 January 1983) Abstrn?t--The activity of the catecholaminergic system was measured in the hypothalamus of rats which had experienced an 18.5-19.5-day-longstay in the state of weightlessnessduring space flightson board Soviet biosatellites of the type Cosmos. In the first two experiments, Cosmos 782 and 936, the concentration of norepinephrine and the activities of synthesizing enzymes tyrosine hydroxylase and dopamine-~8-hydroxylase and of the degrading enzyme monoamine oxidase were measured in the total hypothalamus. None of the given parameters was changed after space flight. In the light of the changes of these parameters recorded after exposure to acute stress on Earth, this findingindicates that long-termstate of weightlessnessdoes not represent an intensive stressogenic stimulus for the system studied. In the space experimentCosmos 1129,the concentration of norepinephrine,epinephrine,and dopaminewas studied in isolated nuclei of the hypothalamusof rats within 6-10 hr followingreturn from space. Norepinephrinewas found to be significantlyreduced in the arcuate nucleus, median eminence and periventricular nucleus, epinephrine in the median eminence, periventricular and suprachiasmatic nuclei, whereas dopamine was not significantlychanged after space flight. The decreased catecholamine levels found in some hypothalamic nuclei of rats which had undergone space flight indicate that no chronic intensive stressor could have acted during the flight, otherwise the catecholamine concentration would have been increased in the nuclei. The decreased levels must have been induced by the effect of a stressogenic factor acting for a short time only, and that either during the landing maneuver or immediately after landing. Thus long-term exposure of the organism to the state of weightlessness does not represent a stressogenic stimulus for the catecholaminergic system in the hypothalamus, which is one of the regulators of the activation of neuroendocrine reactions under stress.
1. INTRODUCTION
The effect of the state of weightlessness on the living organism during long-term space flights is one of the basic problems that the space medicine has still to solve. The state of weightlessness is an extremely non-physiologic situation, which the living organism has never encountered on Earth, and thus it may act as a stressor. Activation of the sympathetic-adrenomedullary system (SAS), resulting in the secretion of catecholamines (CA), and of the pituitary-adrenocortical system, resulting in the secretion of glucocorticoids, are classical indicators of stress reaction. The activity of these systems was studied in rats which participated in long-term space flights on biosatellites of the type Cosmos[l-4]. Activation of the given systems is the final effect of complex neuroendocrine reactions, in which brain catecholamines are also involved[5]. The brain adrSnergic system, particularly in the hypothalamus has been found to be activated under stress. Several authors have demonstrated a drop of norepinephrine (NE) and its increased turnover rate in the hypothalamus [6--9] as well as changes in the concentration of dopamine (DA) [6, 7, 10]. A few studies have also reported an increased activity of the epinephrinergic system in the brain under stress[ll-13]. ?Institute of Biomedical Problems, Moscow, U.S.S.R. 295
The aim of this study was to evaluate the influence of stressful factors and of the state of weightlessness during long-term space flight on the activity of the catecholaminergic systems in the rat brain, mainly in the hypothalamus.
2. MATERIALSANDMETHODS The question whether the state of weightlessness has a stressful influence on the activity of the hypothalamic adrenergic system was studied in rats which experienced space flight on the biosatellites Cosmos. On board the biosatellite Cosmos 782 all rats spent 19.5 days in space in the state of weightlessness. On board the biosatellite Cosmos 936, some rats were caged in a centrifuge (lg) to distinguish the effect of weightlessness from other stressful factors connected with space flight. The experiment of Cosmos 1129 was built on our previous results obtained in rats exposed to single or repeated immobilization stress on Earth[7, 14, 15]. On the basis of our experience in the field of stress we argued that if rats were chronically exposed to intensive stressors during space flight, after their return to Earth, they should react to immobilization stress as chronically stressed rats. If however, space flight is not connected with prolonged intensive stressful stimulation of the SAS and brain adrenergic system, then the rats should react to
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immobilization stress as animals acutely exposed to stress. The experiments were performed on male rats (SPF colony, Bratislava) weighting 200-250g. The rats from Cosmos 782 and 936 were decapitated 6--10 hr or 25-26 days after a 19.5-day-long space flight. Aboard the biosatellites, the rats were kept in individual cages in the state of weightlessness (F), and on board Cosmos 936 also in a centrifuge in which an artificial gravitational force of 1 g acted almost during the whole flight. During the touchdown maneuver the centrifuge was switched off because of technical reasons. The obtained results were compared with the control group (C) and with a synchronous group (S) which was exposed on Earth to similar conditions as group F in space except the state of weightlessness. The rats from Cosmos 1129 had very similar conditions to those from the previous ships, but there was no centrifuge on board and the biosatellite spent in space only 18.5 days. The animals were decapitated immediately after landing (7-10 hr) and 6 days after landing. The third flying group was after landing exposed to immobilization stress 5 times, daily for 150 rain[15], and the animals were killed immediately after the last exposure. The first immobilization was performed directly at the place of landing but the following day (transport of rats to Moscow) this procedure was omitted. The control and synchronous groups were also exposed to repeated immobilization stress. In our immobilization experiments on Earth, the rats were stressed either acutely or repeatedly (40 times, daily for 150min). In addition to these we had also a group of so-called adapted controls, which were decapitated 24 hours after the 39th immobilization. Immediately after decapitation the brain was removed and the hypothalamus was isolated. In the experiment Cosmos 1129, the whole brain was frozen and its individual nuclei were isolated by a special technique described by Palkovits[16]. We isolated a total of 16 microregions of the rat brain, in this paper, however, only the results concerning six hypothalmic nuclei will be reported, i.e. the arcuate nucl. (NARC), ventromedial nucl. (NVM), dorsomedial nucl. (NDM), periventricular nucl. (NPE), median eminence (EM), and the suprachiasmatic nucl. (NSC). The given hypothalamic regions are crucial with respect to the control of neuroendocrine reactions under stress. Catecholamines in the homogenate of the entire hypothalamus were determined by the radioenzymatic method according to Coyle and Henry[17], and in the isolated hypothalamic nuclei by the method of Peuler and Johnson[18] in our modification. Hypothalamic tyrosine hydroxylase activity (TH) was measured according to Saavedra et al.[19], dopamine - /3 - hydroxylase (DBH) according to Molinoff et al.[20], and monoamine oxidase (MAO) by the method of Wurtman and Axelrod[21]. The results are expressed as means -+ SEM of 5-7 rats and statistical significance was calculated by Student's t-test. 3. RESULTS
3.1 COSMOS 782 Norepinephrine in the total hypothalamus. NE concentrations in the hypothalamus of rats killed im-
mediately, i.e. 6-10 hr after the cosmic flight, were unchanged compared to both the control and synchronous groups (Fig. 1). Catecholamine-synthesizing enzymes in the hypothalamus. The results presented on Fig. 1 demonstrate that the activity of TH in the hypothalamus of flight group rats did not significantly change compared to the control and synchronous groups. The activity of DBH was also comparable with that of the control group, yet it was significantly higher compared to the synchronous group (Fig. 1). Catecholamine-degrading enzymes in the hypothalamus. MAO activity was measured in the hypothalamus of rats after long-term space flight. As seen in Fig. 1, no significant MAO changes occurred compared to either the control or synchronous group. 3.2 COSMOS 936 In the rats participating in a long-term flight aboard this biosatellite either under conditions of weightlessness or in a centrifuge, which provided conditions of earth gravitation, concentrations of CA and TH, DBH, and MAO activities were measured in the hypothalamus. Similarly as in the experiment on board the biosatellite Cosmos 782, none of these indicators was significantly changed compared to the control groups. The values recorded in the animals which had experienced the flight in the centrifuge were comparable with those obtained from rats which had lived in the state of weightlessness. Since the results are virtually the same as those obtained in the
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Fig. 1. Concentration of norepinephrine and activity of tyrosine hydroxylase (TH), dopamine-,8-hydroxylase (DBH) and monoamineoxidase (MAO)in the hypothalamusof rats after space flight aboard the biosatellite Cosmos 782. The animals were decapitated 6-10hr after landing (n = 5); x <0.01 compared to synchronous group.
Catecholamines and their enzymes in discrete brain areas of rats after space flight on biosatellites Cosmos experiment of Cosmos 782, they are not presented in this paper.
297
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Norepinephrine and tyrosine hydroxylase in the total hypothalamus in repeatedly immobilized rats. NE concentration decreased significantly after the first immobilization for 150 min, whereas in rats exposed to this stressor 35 times, no changes were observed (Fig. 2). TH activity, on the other hand, was unchanged after the first exposure, yet was found to be significantly increased after repeated immobilization (Fig. 2), suggesting an enhanced CA synthesis in the hypotalamus which accounts for the unchanged NE concentration in repeatedly stressed animals.
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CA changes in hypothalamic nuclei immediately alter landing. The concentrations of NE, DA, and epinephrine (EPI) in some hypothalamic nuclei of rats decapitated immediately after landing are given in Fig. 3. In the flight group, a significant NE decrease was recorded only in the NARC, NPE and EM, yet in all the regions studied NE tended to decrease. The decrease of NE in the NARC was significant compared to both control groups. EPI concentration was significantly reduced in the NPE, EM and NSC. The EPI decrease in the NE was significant compared to both control groups (Fig. 3). The concentration of DA displayed no significant changes in the regions studied (Fig. 3).
MARC
NVM
NDM
NPE
EM
NSC
Fig. 3. Catecholamines in some isolated hypothalamic nuclei in rats after space flight aboard the biosatellite Cosmos 1129.The animals were decapitated within 6-10hr after landing (n =6-7). Abbreviations of the nuclei are explained in methods. *<0.05, *** <0.01 compared to control group; x < 0.05, xx < 0.02 compared to synchronous group.
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Effect of acute immobilization on NE and DA in isolated hypothalamic nuclei. After the first im-
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300
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mobilization, NE concentration tended to decrease in all the nuclei studied with the exception of the NDM where a significant increase of NE was seen (Fig. 4). The concentration of DA was not substantially changed in the majority of the nuclei studied, but in the NARC it was significantly decreased and in the NDM significantly increased.
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Fig. 2. Changes of hypothalamic norepinephrine concentration and tyrosine hydroxylase activity in rats exposed to acute and repeated immobilization, daily for 150 min (n = 7); x <0.001 compared to both groups.
NSO
NPV
~" EM NARC NVM NDH
Fig. 4. Effect of acuteimmobilizationon catecholamineconcentration in some hypothalamic nuclei of rats (n=4--11). Abbreviations of the nuclei are in Methods; NSO-supraoptic nucl.., NPV-paraventricular nucl. * < 0.05, ** < 0.02, *** < 0.001 compared to control group.
Effect of repeated immobilization on NE and DA in isolated hypothalamic nuclei. Repeated exposure to immobilization stress resulted in all the hypothalamic nuclei studied in a significant increase in NE concentration (Fig. 5). DA concentration was also significantly increased in the majority of hypothalamic nuclei in repeatedly stressed rats (Fig. 5).
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Fig. 5. Effect of repeated immobilization on catecholamine concentration in some hypothalamic nuclei of rats (n = 7-12). Abbreviations of the nuclei are in Methods. *<0.05, **<0.02, *** < 0.001 compared to control group, xx < 0.02, xxx < 0.01 compared to group 39-times immobilized + 24 hr rest. 4. DISCUSSION The main aim of this study was to investigate the effect of long-term space flight on the activity of catecholaminergic systems in the hypothalamus of rats and thus to answer the question whether a long-term state of weightlessness is stressogenic or not. Comparisons of the results obtained in "cosmic" and in "terrestiar' rats exposed to stress were expected to yield the answer. Acute immobilization of rats in experiments on Earth resulted in a decrease of NE in the hypothalamus (Fig. 2), whereas in repeatedly stressed animals NE concentration was not reduced (Fig. 2). Similar results were reported also by other authors using different models of stress [22-24]. In repeatedly immobilized animals, in which the NE concentration in the hypothalamus did no longer differ from unstressed control values, the significantly increased TH activity found suggested an increased CA synthesis in the hypothalamus of these rats (Fig. 2). Increased hypothalamic TH activity in repeatedly stressed rats, was reported also by other authors [25-27]. The activity of DBH was also found to increase in the hypothalamus of rats exposed to repeated stress[6, 28]. In the space experiments performed on board the biosatellites Cosmos 782 (Fig. l) and Cosmos 936, neither the concentration of NE nor TH and DBH activities in the hypothalamus were changed [29, 30]. The unchanged concentrations of NE in the hypothalamus of rats after space flight aboard Cosmos 782 and 936 can be interpreted in two ways: space flight, and particularly long-term state of weightlessness, does either not represent an intensive stressor, or the animals
al.
might have become adapted to the effects of space flight and responded similarly as repeatedly stressed animals, so that CA were no longer reduced. However, the unchanged TH and DBH activities suggest that CA synthesis could not have been increased for a longer period of time during space flight. The unchanged CA levels in the hypothalamus of rats found after space flight would then not be the result of enhanced CA synthesis but rather the result of the unchanged activity of the noradrenergic system in the hypothalamus. The concentration of CA in the hypothalamus of rats may be affected also by changes in their degradation. MAO is one of the enzymes responsible for CA degradation. Our results[31], as well as literary data[25,32] showed decreased MAO activity in the hypothalamus of repeatedly stressed animals. In the hypothalamus of "cosmic" rats, MAO activity was not significantly changed, suggesting that CA degradation by oxidative deamination would not have changed either during space flight. The findings of unchanged activities of enzymes, which participate in the synthesis and degradation of CA, along with the unchanged CA concentrations in the hypothalamus indicate that the long-term effect of the state of weightlessness does not represent a stressogenic factor with respect to the catecholaminergic system studied in homogenates of the rat hypothalamus. The hypothalamus, however, is a very heterogeneous structure, so that CA determinations in homogenates of the whole hypothalamus may not reveal an altered function of this system occurring in some small specific areas, in which activities might have changed during long-term space flight. In rats exposed to intensive stress on Earth, substantial changes in CA concentrations were recorded in some hypothalamic nuclei. Acute immobilization induced decreases both in NE (Fig. 4) and EPI[12], in the majority of the hypothalamic nuclei studies, whereas repeated exposure to this stressor resulted in highly significant NE increases in all the hypothalamic nuclei studied, and in increased DA concentrations in the majority of them (Fig. 5). Decreased NE concentrations in some nuclei of the hypothalamus of rats exposed to acute stress were reported also by other authors[10, 33]. The concentration of NE tends to decrease in the majority of hypothalamic nuclei in "cosmic" rats (Fig. 3), which is a typical manifestation of acute stress (Fig. 4). The significant decrease of NE concentration in the NARC compared to both control and synchronous groups (Fig. 3), indicates an acute stress situation induced presumably by the landing maneuver. A similar NE decrease was demonstrated in several models of acute stress[33]. Chronic exposure to a stressor results in increased NE concentration in the NARC (Fig. 5). The decrease of both NE and EPI in the EM in acute stress presents an interesting, so far not reported, finding (Fig. 3). This change of NE in the EM appears to be a phenomenon specific for cosmic flight and suggests varied speculations on the potential mechanisms involved. Concerning the aim of our investigation, however, it is relevant that both the NE concentration (Fig. 5) and the EPI concentration (Kvetfansk~, et al.,
Catecholamines and their enzymes in discrete brain areas of rats after space flight on biosatellites Cosmos unpublished results) in the EM of repeatedly stressed rats is considerably increased, indicating, also for this region of the hypothalamus, that the rats which had undergone cosmic flight were exposed to the effect of an acute stressor. The concentration of DA did not display any significant changes (Fig. 3). After the long-term effect of an intensive stressor also this parameter would be expected to show increased values in some hypothalamic nuclei (Fig. 5). The EPI concentration in hypothalamic nuclei of rats after space flight was significantly decreased in the NPE, EM and NSC (Fig. 3). The diminished EPI concentration in N P E is a typical finding invariably demonstrated after acute stress in this region[12]. This is again an unequivocal manifestation of acute stress, since long-term exposure has been shown to increase the EPI level significantly in the N P E (KvetnanskS, et al., unpublished results). The presented result demonstrate that only some hypothalamic regions respond to cosmic flight by decreased catecholamine concentration, which accounts for the finding of unchanged levels of analyzing homogenates of the total hypothalamus. The most likely interpretation of the catecholamine changes determined in some hypothalmic nuclei of rats after space flights appears to be the effect of an acute stressor acting presumably during the landing maneuver.
REFERENCES
1. R. Kvetfiansk~', R. A. Tigranjan, T. Torda, D. Rep/~ekov~, E. Jahnov~ and K. Murgagr, Effect of a prolonged space flight on the responses of adrenal cortical and medullary layers. Kosmic. Biol. Aviakosmic. Med. 14(1), 24-27 (1980) (in Russian). 2. R. Kvetfiansk~,, R. A. Tigranjan, J. (~ulman, P. Bl~ff.ek, A. M. Genin and L. Macho, Effect of space flight in biosatellites Cosmos on sympathetic adrenomedullary activity of rats. In Advances in Physiologic Sciences, Vol. 19. Gravitational Physiology (Edited by J. Hideg and O. Gazenko), pp. 225228. Akad~miai Kiadb and Pergamon Press, Budapest (1981). 3. R. Kvtfiansky, T. Torda, L. Macho, R. A. Tigranjan, L. V. Serova and A. M. Genin, Effect of weightlessness on sympathetic-adrenomedullary activity of rats. Acta Astronautica 8,469-481 (1981). 4. R. Kvetfiansk~', M. Vigag, R. A. Tigranjan, S. N/~meth and L. Macho, Activity of the sympathetic-adrenomedullary system in rats after space flight on the Cosmos biosatellites. Adv. Space Res. 1, 187-192 (1981). 5. W. F. Ganong, The role of catecholamines and acetylcholine in the regulation of endocrine function. Life Sci. 15, 14011414 (1974). 6. R. Kvetfiansk~/, A. Mitro, M. Palkovits, M. Brownstein, T. Torda, M. Vigag and L. Mikulaj, Catecholamines in individual hypothalamic nuclei in stressed rats. In: Catecholamines and Stress (Edited E. Usdin, R. Kvetfiansk~, and I. J. Kopin) pp. 39-50. Pergamon Press, Oxford (1976). 7. R. Kvetfiansk~,, M. Palkovits, A. Mitro, T. Torda and L. Mikulaj, Catecholamines in individual hypothalamic nuclei of acutely and repeatedly stressed rats. Neuroendocrinology 23, 257-267 (1977). 8. R. Nakagawa, M. Tanaka, Y. Kohno, Y. Noda, N. Nagasaki, Regional responses of rat brain noradrenergic neurones to acute intense stress. Pharmac. Biochem. Behav. 14, 729732 (1981). 9. E. Stone, Accumulation and metabolism of norepinephrine in
299
rat hypothalamus after exhaustive stress. J. Neurochem. 21, 589-601 (1973). 10. M. I. K. Fekete, E. Stark, B. Kanyicska, J. P. Herman and M. Palkovits, Inhibition of stress induced hormonal changes by chronic ACTH treatment; possible relationship with changes of neuroaminergic transmission in the central nervous system. In: Catecholamines "and Stress: Recent Advances (Edited by E. Usdin, R. Kvetfiansk~, and I. J. Kopin), pp. 149-154. Elsevier, New York (1980). 11. M. Goldstein, A. Sauter, K. Ueta and K. Fuxe, Effect of stress on central catecholamine levels. In: Catecholamines and Stress: Recent Advances (Edited by E. Usdin, R. Kvet~ansk~ and I. J. Kopin), pp. 47-52. Elsevier, New York (1980). 12. R. Kvethansk~,, I. J. Kopin and J. M. Saavedra, Changes in epinephrine in individual hypothalamic nuclei after immobilization stress. Brain Res. 155, 387-390 (1978). 13. J. M. Saavedra, R. Kvet~ansk$', I. J. Kopin, Adrenaline, noradrenaline and dopamine levels in specific brain stem areas of acutely immobilized rats. Brain Res. 160, 271-280 (1979). 14. R. Kvetfiansk$' and L. Mikulaj, Adrenal and urinary catecholamines in rats during adaptation to repeated immobilization stress. Endocrinology 87, 738-743 (1970). 15. R. Kvethansk~,, V. K. Weise and I. J. Kopin, Elevation of adrenal tyrosine hydroxylase and phenylethanolamine - N methyltransferase by repeated immobilization of rats. Endocrinology 87,744-749 (1970). 16. M. Palkovits, Isolated removal of hypothalamic or other brain nuclei of the rat. Brain Res. 59, 449--450 (1973). 17. J. T. Coyle and D. T. Henry, Catecholamines in fetal and new born rat brain. J. Neurochem. 21, 61-67 (1973). 18. J. D. Peuler, G. A. Johnson, Simultaneous single isotope radioenzymatic assay of plasma norepinephrine, epinephrine and dopamine. Life Sci. 21, 625-636 (1977). 19. J. M. Saavedra, M. Brownstein, M. Palkovits, S. Kizer and J. Axelrod, Tyrosine hydroxylase and dopamine - /3 hydroxylase: distribution in the individual rat hypothalamic nuclei. J. Neurochem. 23, 869-871 (1974). 20. P. B. Molinoff, R. Weinshilboum, J. Axelrod, A sensitive enzymatic assay for dopamine -/3 - hydroxylase. J. Pharmacol. Exp. Ther. 178,425-431 (1971). 21. R. J. Wurtman and J. Axelrod, A sensitive and specific assay for the estimation of monoamine oxidase. Biochem. Pharmacol. 12, 1439-1440 (1963). 22. O. M. Avakian and E. A. Shirinjan, Compulsion of rats to muscular activity in the antiorthostatic position: Role of the sympatho-adrenal system. In: Catecholamines and Stress: Recent Advances (Edited by E. Usdin, R. Kvethansk~/and I. J. Kopin), pp. 431-435. Elsevier, New York (1980). 23. I. C)stman-Smith, Adaptive changes in the sympathetic nervous system and some effector organs of the rat following long-term exercise or cold acclimation and the role of cardiac sympathetic nerves in the genesis of compensatory cardiac hyperthrophy. Acta Physiol. Scand. Suppl. 477, 1-118 (1979). 24. A. M. Thierry, G. Blanc, J. Glowinski, Effect of stress on the disposition of catecholamines localised in various intraneuronal storage forms in the bralnstem of the rat. J. Neurochem. 18, 449-461 (1971). 25. Z. Huszti and A. Kenessey, 3H-tyrosine incorporation into proteins and catecholamines in immobilized rats. In: Catecholamines and Stress (Edited by E. Usdin, R. Kvetfiansk~, and I. J. Kopin), pp. 377-385. Pergamon Press, Oxford (1976). 26. F. Lamprecht, B. Eichelman, N. B. Thoa, R. B. Williams and I. J. Kopin, Rat fighting behaviour: serum dopamine - /3 hydroxylase and hypothalamic tyrosine hydroxylase. Science 177, 1214-1215 (1972). 27. E. A. Stone, L. S. Freedman, L. E. Morgano, Brian and adrenal tyrosine hydroxylase activity after chronic footshock stress. Pharmacol. Biochem. Behav. 9, 551-553 (1978). 28. G. R. Van Loon, Brain dopamine-~8-hydroxylase activity: response to stress, tyrosine hydroxylase inhibition, hypophysectomy and ACTH administration. In: Catecholamines
300
R. KVETI~ANSK~et al.
and Stress (Edited by E. Usdin, R. Kvetfiansk~, and 1. J. Kopin), pp. 77-85. Pergamon Press, Oxford (1976). 29. R. Kvethansk¢/, R. A. Tigranjan, T. Torda, D. BabugikovL E. Jahnov~., N. F. Kalita and M. Viga~, Catecholamines and enzymes involved in their metabolism in the hypothalamus of rats after space flight on biosatellite Cosmos-782. Kosmic. Biol. 13(3), 24-27 (1979) (in Russian). 30. T. Torda, R. Kvet~anskCA R. A. Tigranjan, J. Culman, A. M. Genin, Noradrenaline and enzymes involved in its synthesis and degradation in the hypothalamus of the rats flown on
Cosmos-936, Kosmic. Biol Auiakosmic. Med. 15 (6), 46-48 (1981) (in Russian). 31. R. Kvefftansk~,, F. Daher, S. Sev~:ik, J. (~ulman, Z. Opr~alov~, and A. Kiss, Degradation of catecholamines in hypothalamic nuclei of rats under stress. Abstracts, Integrative Neurohumoral Mechanisms, p. 128. Hungarian Academy of Sciences, Budapest (1982). 32. G. Maura, A. Vaccari, Relationships between age of submission to environmental stress and monoamine oxidase activity in rats. Experientia 31, 191-192 (1975).