Catecholamines and Stress: Proceedings of the International Symposium on Catecholamines and Stress, Held in Bratislava, Czechoslovakia, July 27–30, 1975

Wistar-Kyoto

PARTICIPANTS

There were 116 participants at the International Symposium on Catecholamines and Stress, representing 15 countries: Canada, Czechoslovakia, France, Great Britain, Holland, Hungary, India, Japan, Poland, Sweden, Switzerland, United States, U.S.S.R. and Yugoslavia.

I. Ahlers,     Košice, Czechoslovakia

E. Ahlersová,     Košice, Czechoslovakia

E. Aisene,     Paris, France

I. Albrecht,     Kr , Czechoslovakia

F. Babusikova,     Bratislava, Czechoslovakia

V. Baláz ,     Bratislava, Czechoslovakia

I. Balaz ovjech,     Bratislava, Czechoslovakia

Z. Bargiel,     Torun, Poland

E. Barta,     Bratislava, Czechoslovakia

I. Benedeczky,     Budapest, Hungary

P. Blaz í ek,     Bratislava, Czechoslovakia

R.T. Borchardt,     Lawrence, Kansas

J. Bruthans,     Prague, Czechoslovakia

B. Bucher,     Paris, France

P. Bulscak,     Bratislava, Czechoslovakia

A. Dlaba ,     Prague, Czechslovakia

M. Dobrakovová,     Bratislava, Czechoslovakia

M. Fatranská,     Bratislava, Czechoslovakia

M. Fekete,     Budapest, Hungary

C. Gagnon,     Basel, Switzerland

W.F. Ganong,     San Francisco, California

J. Gero,     Bratislava, Czechoslovakia

M. Gerova,     Bratislava, Czechoslovakia

F. Godefroy,     Paris, France

J.P. Henry,     Los Angeles, California

H. Hidaka,     Aichi, Japan

M. Holzbauer-Sharman,     Cambridge, England

J. Hrnciar,     Bratislava, Czechoslovakia

Z. Huszti,     Budapest, Hungary

H. Illnerová,     Kr , Czechoslovakia

F. Inczinger,     Bratislava, Czechoslovakia

D. Jacobowitz,     Bethesda, Maryland

E. Jahnová,     Bratislava, Czechoslovakia

L. Janský,     Prague, Czechoslovakia

J. Jedlicka,     Bratislava, Czechoslovakia

M. Juráni,     Bratislava, Czechoslovakia

J. Jur ovi ová,     Bratislava, Czechoslovakia

H. Kaciuba-Uscilko,     Warsaw, Poland

E. Kellerová,     Bratislava, Czechoslovakia

E. Kellerová,     Bratislava, Czechoslovakia

A. Kenessey,     Budapest, Hungary

D.C. Klein,     Bethesda, Maryland

Z. Klima,     Bratislava, Czechoslovakia

J. Knopp,     Bratislava, Czechoslovakia

R.M. Kobayashi,     San Diego, California

I.J. Kopin,     Bethesda, Maryland

J. Korf,     Groningen, Holland

M. Kouřilová,     Bratislava, Czechoslovakia

P. Kovacs,     Bratislava, Czechoslovakia

S. Kozlowski,     Warsaw, Poland

D. Krieger,     New York, N. Y.

V. Kujalová,     Prague, Czechoslovakia

L. Kuz ela,     Bratislava, Czechoslovakia

R. Kvetňanský,     Bratislava, Czechoslovakia

P. Langer,     Bratislava, Czechoslovakia

J. Lángoš,     Bratislava, Czechoslovakia

J. LeBlanc,     Québec City, Canada

B. Lichardus,     Bratislava, Czechoslovakia

L. Macho,     Bratislava, Czechoslovakia

Z. Malátová,     Košice, Czechoslovakia

E. Matlina,     Sh. Moscow, U.S.S.R.

J. Mejsnar,     Prague, Czechoslovakia

Z. Mikeš,     Bratislava, Czechoslovakia

L. Mikulaj,     Bratislava, Czechoslovakia

A. Mitro,     Bratislava, Czechoslovakia

K. Modigh,     Göteborg, Sweden

P.B. Molinoff,     Denver, Colorado

J. Moravec,     Prague, Czechoslovakia

P. Mráz,     Bratislava, Czechoslovakia

R.A. Mueller,     Chapel Hill, North Carolina

K. Murgaš,     Bratislava, Czechoslovakia

N.E. Naftchi,     New York, N. Y.

T. Nagatsu,     Nagoya, Japan

M.A.A. Namboodiri,     Bangalore, India

K. Nazar,     Warsaw, Poland

S. Németh,     Bratislava, Czechoslovakia

R. Nosál,     Bratislava, Czechoslovakia

J. Novotný,     Bratislava, Czechoslovakia

H. Nowica,     Torun, Poland

C. Nyakas,     Budapest, Hungary

U. Otten,     Basel, Switzerland

M. Palkovi ,     Bratislava, Czechoslovakia

M. Palkovits,     Budapest, Hungary

E. Paulíková,     Košice, Czechoslovakia

V. Petrovi ,     Belgrad, Yugoslavia

W.D. Pfeifer,     Norwich, Connecticut

J. Podoba,     Bratislava, Czechoslovakia

J. Poggioli,     Orsay, France

D.J. Reis,     New York, N. Y.

D. Rep eková,     Bratislava, Czechoslovakia

H. Saito,     Basel, Switzerland

N. Saleh,     Bratislava, Czechoslovakia

J. Sedlák,     Bratislava, Czechoslovakia

D.F. Sharman,     Cambridge, Great Britain

D.B. Stephens,     Cambridge, Great Britain

J. Samudovský,     Bardejov, Czechoslovakia

V. Strbák,     Bratislava, Czechoslovakia

G. Telegdy,     Pécs, Hungary

T. Torda,     Bratislava, Czechoslovakia

E. Tordova,     Bratislava, Czechoslovakia

M. Trabuchii,     Milan, Italy

V. Tr ka,     Prague, Czechoslovakia

K.N. Udupa,     Varanasi, India

E. Usdin,     Rockville, Maryland

M. Valchář,     Prague, Czechoslovakia

G.R. Van Loon,     Toronto, Canada

M. Vigaš,     Bratislava, Czechoslovakia

I. Vozár,     Ivanka pri Dunaji, Czechoslovakia

E. Wasilewska,     Torun, Poland

J. Weil-Fugazza,     Paris, France

N. Weiner,     Denver, Colorado

R. Weinshilboum,     Rochester, Minnesota

A. Ziegelhöffer,     Bratislava, Czechoslovakia

R.E. Zigmond,     Cambridge, Great Britain

I.

CATECHOLAMINES AND STRESS

Outline

Chapter 1: INTRODUCTION TO CATECHOLAMINES AND STRESS

INTRODUCTION TO CATECHOLAMINES AND STRESS

Irwin J. Kopin,     National Institute of Mental Health, Laboratory of Clinical Science, Bethesda, Maryland 20014

I am indeed privileged and honored to be chairman of this Symposium on Catecholamines and Stress. First I wish to express, on behalf of us all, our gratitude to the Institute of Experimental Endocrinology of the Slovak Academy of Sciences for the wonderful opportunity to visit this friendly and beautiful country. Dr. Vigas and Dr. Kvetnansky and the local organizing committee are to be congratulated for the attractive arrangements and stimulating program they have prepared for us.

The purpose of an introduction is to provide a suitable base and to create a receptive mood for the presentations and discussions to follow. It is appropriate as well as instructive to acknowledge our debt to the progenitors of many of the concepts which we will be discussing and to touch briefly on the milestones in the evolution of our understanding of the role of catecholamines in stress.

Nearly one hundred years ago Claude Bernard (Ref. 1) first defined precisely the concept that as organisms become more independent of their surroundings they develop more complex ways of stabilizing their internal environment, in spite of shifts in outer circumstances. Changes in the surroundings excite reactions to limit internal disturbances in the organism. It was the nature and mechanisms of these reactions which then became the subject of study.

The description by Oliver and Shaffer (Ref. 2) in 1895 of the remarkable activity of adrenal extracts led to the subsequent isolation and characterization of epinephrine (or adrenaline) by Abel (Ref. 3). The effects of sympathetic nerve stimulation, which had been extensively studied by Langley (Ref. 4), were strikingly similar to the effects of the catecholamine. This, of course, led Elliott (Ref. 5), then a graduate student in Langley’s laboratory, to make the ingenious suggestion that an adrenaline-like substance might be released from sympathetic nerve endings.

To Walter Cannon, however, must go the credit for the demonstration of the central role of the sympatho-adrenal medullary system in orchestrating the elaborate complex of adjustments involved in preserving the internal environment. About fifty years ago, Cannon coined the term homeostasis to describe the complex physiological reactions which maintain the steady states of the body. As early as 1911, Cannon and de la Paz (Ref. 6), had demonstrated that when a cat is frightened or angered by a barking dog, adrenaline is released from the adrenal gland into the blood. Generalization of the concept that such immediate responses to emergencies involve activation of the sympatho-adrenal system was further developed and firmly established during the following twenty years. Furthermore, physical as well as emotional disturbances triggered the onset of the same compensatory responses. By 1935 it was clearly recognized that there were limits in the ability to compensate and that critical stresses existed - either in terms of magnitude or duration - beyond which there is a failure of the homeostatic mechanisms (Ref. 7). The critical stress point and the ability to preserve the internal environment could vary under different general conditions and during the normal and pathologic ups and downs of existence in an ordinary life cycle. (Ref. 7).

Up to this time, physiologists had focused attention on the measurable responses of the organism after exposure to agents or circumstances which could lead to adverse changes in the internal environment. In 1936, Selye introduced a new dimension to such studies when in a brief report (Ref. 8) he described the sequence of pathological changes which occurred when an animal was exposed to a variety of nocuous agents and which he named the General Adaptation Syndrome.

The first stage, which Selye called the Alarm Reaction, consisted of the triad of lymphothymic involution, gastrointestinal ulceration, and loss of cortical lipoid and medullary chromaffin substance from the adrenals. If the animal survives, in the second stage, that of resistance, the adrenals are greatly enlarged but regain their lipoid, while the medullary cells show vacuolization. If the stress is sufficiently severe and prolonged there follows a stage of exhaustion in which the animal succumbs with symptoms similar to those seen in the first stage.

During the next thirty years, Selye led the development and popularization of the concept of Stress as a non-specific physiological response within the organism elicited by a wide variety of stressors (Ref. 9). Emotional stimuli were shown to be at least as potent as any physical damage in eliciting the stress response. The pathological changes found in experimental animals exposed to stressors were taken tobe models of human disease states which could be attributed to stress. Selye has called these diseases of adaptation. They include emotional illnesses as well as the so-called psychosomatic diseases. The particular disorder which surfaces is thought to depend upon a combination of internal conditioning factors (genetic, nutritional, etc.) and the specific effects of the stressor. Stress also attends non-stress-induced diseases and may modify their course and outcome.

Biochemical methods developed in the late 1940’s provided confirmation that adrenal steroid secretion was elevated during stress and was responsible for many of the pathologic changes. The hypothalamic-pituitary-adrenal cortical system received the most attention in studies of the endocrine response to stress. Although changes in the adrenal medulla were clearly recognized, except for some studies on epinephrine excretion, the role of catecholamines in the stress response was neglected and research on catecholamines progressed largely independently of research on stress.

In 1948, von Euler (Ref. 10) definitively demonstrated that norepinephrine was the neurotransmitter released from sympathetic nerve endings. On the basis of the regional localization and pharmacological alterations of norepinephrine in the hypothalamus and mesencephalon, Marthe Vogt (Ref. 11) suggested that norepinephrine and epinephrine might be neurotransmitters in the central nervous system. The availability of new techniques - radioactive compounds and spectrophotofluorimetry - triggered an explosion in catecholamine research. The investigations of Axelrod and his co-workers defined the metabolism and disposition of catecholamines (Ref. 12), demonstrated the importance of reuptake of norepinephrine as a mode of terminating its action (Ref. 13), and showed that a wide variety of drugs acted by altering the fate of the catecholamines (Ref. 14). Carlsson (Ref. 15), showed that dopamine, which had been regarded as a precursor of norepinephrine, was probably a neurotransmitter in the brain. Methods were developed for assay of the catecholamines and their metabolites, the enzymes involved in their biosynthesis were discovered, and as a result of the fluorescent-histological methods developed by Hillarp, Falck, and their co-workers (Ref. 16), catecholaminergic neuronal pathways in the central nervous system were defined. There became available new drugs which inhibited enzymes concerned with biosynthesis or metabolism of the catecholamines or interfered with their storage, inactivation, or action on receptors.

This basic research was productive of valuable new advances in therapy. The discovery that Parkinson’s Disease was associated with decreased levels of dopamine in brain (Ref. 17) resulted in the spectacular development of L-DOPA as a treatment for this movement disorder (Ref. 18). In short, there was born a new era rich in approaches to the study of aminergic neurones and fruitful in providing new concepts of the biological basis for understanding and treating behavioral and neurological disturbances.

It was inevitable that research on stress and on catecholamines would again converge. New information about control of catecholamines as neurotransmitters in brain and implication of these compounds in mediating secretion of hypothalamic releasing factors which control pituitary function has merged neuroendocrinology with catecholamine research. The application of such studies to stress was apparent at the Symposium on Hormones, Metabolism, and Stress (Ref. 19) held under the auspices of the Institute of Experimental Endocrinology nearly three years ago, when the first three papers presented were concerned with catecholamines in brain and the response to stress. Proliferation of research in this area has continued and it is appropriate that this symposium on Catecholamines and Stress be held at this time.

The aim of this symposium should not only be to provide a concise view of the state-of-the-art, but to stimulate and indicate the direction of further fruitful research. In addition, to the participants it offers through personal contacts the opportunity of exchanging ideas, concepts, and approaches. We should feel that the knowledge to be derived from such studies will provide keys to understanding how an intangible event outside the body is translated into physical or mental damage within and how these adverse effects of the inevitable stressors of life may be ameliorated. I hope that such a statement of these high aims does not seem overly ambitious and will succeed in creating the right atmosphere in which to begin this symposium.

REFERENCES

1. Bernard, Claude: Les Phénomènes de la Vie. Paris (1878).

2. Oliver, G., Shäffer, E. A. The physiological effects of extracts from the suprarenal capsules. J. Physiol. (Lond). 1895; 18:230.

3. Abel, J. J. Ueber den blutdruckerregenden Bestandtheil der Nebenniere, das Epinephrin. Hoppe-Selyer’s z. Physiol. Chem.. 1899; 28:318.

4. Langley, J. N. The autonomic nervous system. Brain. 1903; 26:1.

5. Elliott, T. R. The action of adrenalin. J. Physiol. (Lond). 1905; 32:401.

6. Cannon, W. B., de la Paz, D. Emotional stimulation of adrenal secretion. Am. J. of Physiol.. 1911; 28:64.

7. Cannon, W. B., Stresses and strains of homeostasis. Am. J. Med. Sci. 1935; 189:1

8. Selye, H. A syndrome produced by diverse nocuous agents. Nature (Lond). 1936; 138:32.

9. Selye, H. The evolution of the stress concept. Amer. Scientist. 1973; 61:692.

10. von Euler, U. S. Identification of the sympathomimetic ergone in adrenergic nerves of cattle (sympathin H) with laeva-noradrenaline. Acta Physiol. Scand.. 1948; 16:63.

11. Vogt, M. Norepinephrine and epinephrine in the central nervous system. Pharm. Rev.. 1954; 6:31.

12. Axelrod, J. Metabolism of epinephrine and other sympathomimetic amines. Physiol. Rev.. 1950; 39:751.

13. Hertting, G., Axelrod, J. Fate of tritiated noradrenaline at sympathetic nerve endings. Nature (Lond). 1961; 192:172.

14. Axelrod, J. The metabolism, storage, and release of catecholamines. Recent Progress Hormone Res.. 1965; 21:597.

15. Carlsson, A. The occurrence, distribution, and physiological role of catecholamines in the central nervous system. Pharmacol. Rev.. 1959; 11:490.

16. Falck, B., Hillarp, N. O., Thieme, G., Torp, A. Fluorescence of catecholamines and related compounds condensed with formaldehyde. J. Histochem. Cytochem.. 1962; 10:348.

17. Ehringer, H., Hornykiewicz, O. Verteilung von Noradrenalin und Dopamin (3-Hydroxytramin) im Gehirn des Menschen und IHR Verhalten bei Erkrankungen des extrapyramidalen Systems. Klin. Wschr. 1960; 38:1236.

18. Cotzias, G. C., Papasuasiliou, P. S., Gellene, R. Modification of parkinsonism – chronic treatment with L-DOPA. New Eng. J. Med.. 1969; 280:337.

19. Nemeth, S. ed.: Hormones, metabolism and stress. Recent progress and perspectives. Proceedings of International Symposium, Smolenice, Sept. 17–20, 1972. Publ. House of Slovak Academy of Sciences, Bratislava, 1973.

II.

Brain Catecholamines and Stress

Outline

Chapter 2: INTRODUCTION TO BRAIN CATECHOLAMINES AND STRESS

Chapter 3: BRAIN CATECHOLAMINES AND HOMEOSTATIC BEHAVIOUR

Chapter 4: INFLUENCE OF SOCIAL STRESS ON BRAIN CATECHOLAMINE MECHANISMS

Chapter 5: SELECTIVE ALTERATIONS OF CATECHOLAMINES AND TYROSINE HYDROXYLASE ACTIVITY IN THE HYPOTHALAMUS FOLLOWING ACUTE AND CHRONIC STRESS

Chapter 6: CATECHOLAMINES IN INDIVIDUAL HYPOTHALAMIC NUCLEI IN STRESSED RATS

Chapter 7: EFFECT OF STRESS ON SEROTONIN AND TRYPTOPHAN HYDROXYLASE ACTIVITY OF BRAIN NUCLEI

Chapter 8: THE EFFECT OF PROLONGED ISOLATION ON THE CATECHOLAMINE AND SEROTONIN CONCENTRATION OF DISCRETE AREAS OF THE RAT BRAIN

Chapter 9: ACTH INDUCED CHANGES IN THE TRANSMITTERAMINE CONCENTRATION OF INDIVIDUAL BRAIN NUCLEI OF THE RAT

Chapter 10: BRAIN DOPAMINE BETA HYDROXYLASE ACTIVITY: RESPONSE TO STRESS, TYROSINE HYDROXYLASE INHIBITION, HYPOPHYSECTOMY AND ACTH ADMINISTRATION

Chapter 11: CATECHOLAMINES IN THE REGIONS OF CIRCUMVENTRICULAR ORGANS OF RATS EXPOSED TO STRESS

Chapter 12: COMPARISON OF THE EFFECTS OF VARIOUS STRESSES ON BIOGENIC AMINES IN THE CENTRAL NERVOUS SYSTEM AND ANIMAL SYMPTOMS

Chapter 13: LOCUS COERULEUS, NORADRENALINE METABOLISM AND STRESS

Chapter 14: THE EFFECT OF HIND-LIMB ISCHAEMIA ON THE CONCENTRATION OF 4-HYDROXY-3 METHOXYPHENYLETHYLENE GLYCOL SULPHATE (MOPEG-SO4) IN THE HYPOTHALAMUS AND HIND-BRAIN OF THE RAT

Chapter 15: A PROTECTIVE ROLE OF NERVE ENDINGS IN THE STRESS-STIMULATED INCREASE IN PINEAL N-ACETYLTRANSFERASE ACTIVITY

Chapter 16: THE EFFECT OF IMMOBILISATION ON THE ACTIVITY OF SEROTONIN N-ACETYLTRANSFERASE IN THE RAT EPIPHYSIS

INTRODUCTION TO BRAIN CATECHOLAMINES AND STRESS

BRAIN CATECHOLAMINES AND HOMEOSTATIC BEHAVIOUR

E. Endroczi and C. Nyakas,     Research Division, Postgraduate Medical School, 1389 Budapest, Hungary

Involvement of catecholaminergic neurons in controlling the innate and learned behavioural reactions had been studied extensively in past decades. It is generally accepted that chemical destruction of catecholaminergic neurons by 6-hydroxy-dopamine (6-OHDA) is followed by a marked deficit of the different behavioural parameters (Lenard et al., 1972; Stern et al., 1972; Taylor and Laverty, 1972; Breese et al., 1973). In recent studies it was found that intraventricular injections of 6-OHDA to newborn rats resulted in a permanent alteration of both exploratory activity and avoidance behaviour as well as in their electrophysiological correlates (Nyakas et al., 1973; Nyakas and van Delft, 1975). Interpretation of noradrenaline (NA) and dopamine (DA) content of central nervous system has been reduced by the treatment.The present investigations were aimed to study the effects of 6-OHDA on elementary behavioural reactions, the correlations between exploratory activity and brain NA content and the influence of stress as well as ACTH on brain NA metabolism in rats.

Effects of 6-OHDA treatment on exploratory activity and avoidance behaviour

Intraventricular injections of total doses of 100 or 200 μg 6-OHDA-HCl to new-born rats produced permanent changes in behavioural reactions and a significant decrease of the brain NA and DA content. The animals were sacrificed at 2 to 3 months of age and the brain NA and DA content was reduced to 10 to 20 per cent of control littermates. No apparent change was found in the general motor activity of animals which had been tested by an Animex activity meter. In contrast to this finding the exploratory activity was significantly less in 6-OHDA treated rats than in control animals (Table 1).

TABLE 1

Effect of neonatal intraventricular injections of 6-OHDA on exploratory activity and shuttle box avoidance acquisition (Nyakas et al., 1973)

Values are means ± S.E.M. /Student’s t-test

xp < 0.05

xxp < 0.01

In the present experiments the exploratory activity was tested in a twelve-compartment experimental cage and the behaviour was scored according to the crossings of compartments and the rearing activity. The intracisternal injections of 6-OHDA (3 × 200 μg of the HCl salt) led to a marked decrease of the exploration in adult age and this effect seemed to be unchanged during the one-month observation period (Table 2). These observations confirmed the earlier data of Jalfre and Haefely (1971) who found that intraventricular administration of 6-OHDA produced a suppression of the exploratory activity in adult rats.

TABLE 2

Effect of intracisternal injections of 6-OHDA

Fig. 2 c – extinction of CAR by non-reinforced trials following 6-OHDA treatment.

Active avoidance behaviour was studied in a shuttle box in rats treated with 6-OHDA either in newborn or adult age. Sound stimuli were associated with electrical foot-shocks and 20 associations were presented each day. Intracisternal injections of 6-OHDA-HCl (3 × 200 μg) produced a marked decrease of the acquisition of avoidance response and the intertrial activity was also suppressed (Fig. 1). In further studies the animals were trained for acquisition of a two-way active avoidance conditioned response up to 90 per cent criterion level and then 6-OHDA was injected intracisternally in same doses as the previous experiment. The extinction of avoidance response was tested 20 days after injections by administration of nonreinforced trials. It was found that 6-OHDA led to a 70-60% depletion in NA and around 60% depletion in DA content of the brain. The behavioural observations here are partly in accordance with the earlier findings of Cooper et al (1972) who found a decrease of the acquisition of avoidance response but an increased intertrial activity following 6-OHDA treatment.

Fig. 1 a – acquisition of conditioned avoidance responses (CAR), b – inter-trial activity (IA). + p < 0.05, ++ p < 0.01

In the effects of 6-OHDA on active avoidance responding, the predominant role of brain DA has been emphasized more recently (Cooper et al., 1973; Fibiger et al., 1974). Stereotaxic injection of 6-OHDA into the substantia nigra resulted in a heavy deficit of the learning behaviour which was associated with a 90% loss of DA content in the telencephalon; however, the hypothalamic NA content was also markedly reduced in these studies (Fibiger et al. 1974). The authors claimed the specificity of dopaminergic neurons in these behavioural processes in that a lesion of NA bundle caudal to the substantia nigra resulted in a marked decrease of the hypothalamic NA content but did not simulate the effect of 6-OHDA injection into the dopaminergic nuclei. On the other hand, we must be aware that lesioning the substantia nigra (both electrolytically or chemically) is followed by an impairment of learning in rats (Stokes and Thompson, 1970; Mitcham and Thomas, 1972).

Participation of NA neurons in the activation of forebrain structures has been confirmed in both electrophysiological and behavioural studies (see Kety, 1970; Jouvet, 1972; Stein and Wise, 1971, among others). Intraventricular injection of NA led to an increased behavioural arousal in rat (Segal and Mandell, 1970) and in cat (Cordeau et al., 1971).

In the present investigations we have studied the correlation between the exploratory activity of 3-month-old rats and the noradrenaline content of the rostral brainstem and the forebrain. The exploratory activity was tested in a 12-cell maze. A week following the behavioural test the animals were killed by decapitation, the brain was removed and the appropriate structures were dissected for determination of NA content by the method of Shellenberger and Gordon (1971). An inverse correlation of the exploratory activity to the NA content of the rostral brainstem (mesencephalon and posterior hypothalamus) could be observed (Fig. 3). There was no significant correlation between the exploratory activity and the NA content of the forebrain. These observations indicate that noradrenergic neurons of brainstem structures are involved in organization of exploratory behaviour and ascending NA neurons of forebrain areas may play only a secondary role in these events. Nevertheless, differences in NA turnover rates of rostral and caudal brain structures cannot be excluded.

Fig. 3 Correlation between exploratory activity and NA content of rostral brainstem

Stress induced changes in brain NA metabolism

Changes in the brain catecholamines to noxious stimuli have been observed by a number of investigators (Vogt, 1954; Barchas and Freedman, 1963; Maynert and Lewi, 1964; Moore and Lariviere, 1964). Moreover, it was found that electrical footshocks produced a marked acceleration of the decline of 3H-NA specific activity in various parts of the brain in rats (Thierry et al., 1968). It is generally accepted that labelled NA injected into the ventricle mixes with an endogenous catecholamine pool and can be used as a tracer to monitor uptake, storage and metabolism of brain catecholamines (Milhaud and Glowinski, 1962; Goldstein and Gerber, 1963; Glowinski et al., 1965; Carr and Moore, 1969, etc.). At least two stores have been postulated in the brain: one with a rapid turnover of 3-4 hours, and the other with a slower turnover, about 17-18 hours. Moreover, it was found that an inverse correlation exists between the NA content and its turnover (Iversen and Glowinski, 1966; Glowinski and Baldessarini, 1966). In studying the effect of stress on the brain NA metabolism the influence of electrical shocks was tested in the first stage of NA turnover and the observations were performed in rats with intact pituitary-adrenal function (Glowinski, 1970). In the present experiments intraventricular injection of 3H-NA was followed by a series of electrical footshocks 16 hours later and the animals were sacrificed in the 17th hour. The experiment was carried out in adrenalectomized rats. The administration of 10 painful stimuli within 5 minutes one hour prior to decapitation resulted in a slight decrease of NA pools in the hippocampus and neocortex and a more pronounced decrease in the hypothalamus (Fig. 4). These observations indicated that stress-induced changes in brain NA metabolism are not mediated through pituitary-adrenal axis and the corticosteroids are not involved in this event. Nevertheless, an increase of pituitary ACTH secretion to stress even after adrenalectomy is well-known, and its influence on NA metabolism cannot be excluded.

Fig. 4 Effect of electrical foot-shock on the disappearance of radioactivity following the intraventricular injection of 3H-NA. + p < 0.05, ++ p < 0.01

Influence of ACTH on exploratory activity in adrenalectomized rats

Numerous observations have been accumulated in the literature that ACTH peptides and its analogues may exert a direct influence on brain function (DeWied, 1966; Endroczi et al., 1969; Bohus and Koranyi, 1969; DeWied et al., 1974).

An increased exploratory activity and a greater resistance to extinction of avoidance response could be observed in different laboratories. Recently, we have studied the influence of ACTH 1-24 on different behavioural reactions, and it was found that the ACTH administration produced an increase of the exploratory activity found in a 12-cell naze on the one hand, and a decrease of the extinction of two-way active avoidance response on the other hand (Fig. 5). The mechanism of ACTH action on brain function is still obscure, although there are observations which revealed its direct influence on both RNA and protein synthesis (Gispen and Schotman, 1973). Moreover, it was found that ACTH peptides exert a longlasting influence on hehavioural reactions (for 12 to 24 hours) which cannot be attributed to membrane effects or vascular reactions as a result of the peptide actions.

Fig. 5 Effect of ACTH on exploratory activity in intact and adrenalectomized rats.

Influence of ACTH 1-24 and ACTH 4-10 on brain NA metabolism in adrenalectomized rats

Intraventricular injection of 5 and 10 μg ACTH 1-24 and ACTH 4-10 to adrenalectomized rats (3 to 5 days after adrenalectomy) simultaneously with intraventricular injection of 3H-NA on the opposite side, resulted in a significant decrease of labelled NA pool measured in the 12th and 18th hours after injections. The increased disappearance of labelled NA to ACTH treatment had been observed in both the hypothalamus and hippocampus as well as in the neocortex (Figs. 6 and 7).

Fig. 6 Effect of ACTH at different hours after intraventricular injection of 3H-NA. + p 0.05.

Fig. 7 Radioactivity in the 18th hour after ACTH 1-24

Since ACTH 4-10 proved to be effective, although the fragment has no steroidogenic property, it seems that a relatively short central part of the amino acid chain of ACTH 1-24 is responsible for the action.

Administration of Pargyline to block monoamine oxidase activity, which led to an increase of labelled NA pool, did not prevent the ACTH-induced facilitation of disappearance of labelled pool (Fig. 8). On the other hand, the intraventricular injection of α-Methyl-m-tyrosine resulting in a marked decrease of labelled NA pool was also used to study the effect of ACTH on NA metabolism. It was found that simultaneous injection of α-methyl-m-tyrosine with ACTH produced an augmentation of the NA disappearance in the 18th hour following the labelled NA injection (Fig. 9).

Fig. 8 Effect of ACTH on the decreased disappearance of 3H-activity following Pargyline administration

Fig. 9 Action of ACTH given together with α-methyl-tyrosine intraventricularly to rats

In connection with the effect of ACTH it is of significance that intravenous injection of 5 U ACTH per 100 g body weight led to a significant increase of the disappearance of labelled NA pool of the hypothalamus. The ACTH administration was performed in the 16th hour following intraventricular injection of 3H-NA and the animals were decapitated one hour later. In contrast to the significant decrease of hypothalamic NA pool after ACTH administration, the hippocampus and neocortex showed no significant alterations. The reason that ACTH administration had no effect in the hippocampus and neocortex may be explained by a greater penetration of ACTH through blood-brain barrier in the tubero-infundibular region than in other parts of the brain.

Summary and conclusions

Present observations indicate that catecholaminergic neurons participate in the control of elementary behavioural reactions like exploratory activity and avoidance behaviour. A primary role of NA neurons seems to be supported by the findings of pharmacologists who have reported an increased exploration and an enhanced EEG arousal to amphetamine which could be blocked by a pretreatment with phentolamine and propranolol (Cahn and Herold, 1970). It is known that amphetamine affects the NA stores with either short and longlasting turnover and its administration in high doese depleted the 3H-NA in both storage compartments (Glowinski and Axelrod, 1965).

The intraventricular injections of 6-OHDA produced permanent destruction of both NA and DA neurons which was associated with significant alterations in both exploration and avoidance behaviour. There are many observations which suggest the specific role of dopaminergic neurons (nigro-neostriatal system) in homeostatic behavioural reactions, although experimental findings accumulated in the literature are contradictory and the participation of noradrenergic neurons in the behavioural deficits following destruction of dopaminergic system cannot be excluded (Fibiger et al., 1974). It seems to be confirmed that both NA and DA neurons are involved in self-stimulation (Rolls, 1971; Crow, 1972; Phillips and Fibiger, (1973). These observations suggest that catecholaminergic neurons participate in both nonspecific activation of brainstem-forebrain structures (exploration) and decoding of environmental signals in conditioned reflex situation (necessary for reinforcement.

Stress-induced changes in brain NA metabolism at least seem to be mediated through pituitary ACTH secretion. The intraventricular injection of ACTH to adrenalectomized rats suggested a direct effect of the ACTH and its fragment on brain NA metabolism. Studies using blocking agents strongly suggest that ACTH results in an increased release of NA from its storage compartment. Further studies are required to understand the biochemical bases of ACTH action and its role in such behavioural reactions as increased exploration observed after administration of both ACTH and noradrenaline or after an increase of NA release from storage by amphetamine.

References

Barchas, J. D., Freedman, D. Response to Psychological stress. Biochem. Pharmacol.. 1963; 12:1232.

Bohus, B., Koranyi, L. Hormonal conditioning of adaptive behavioural processes. In: Lissak K., ed. Recent developments of neurobiology in Hungary. Budapest: Akademia Kiado; 1969:50–76.

Breese, G. R., Smith, R. D., Cooper, B. R., Grant, L. D. Alteration in consummatory behaviour following intracisternal injection of 6-hydroxydopamine. Pharmac. Biochem. Behav.. 1973; 1:319.

Cahn, J., Herold, M. Effects of amphetamines in rats and rabbits injected with alpha and beta-adrenergic blocking agents. In: Costa E., Garattini S., eds. Amphetamines and related ccmpounds. N.Y.: Raven Press; 1970:493–510.

Carr, L. A., Moore, K. E. Distribution and metabolism of norepinephrine after its administration into the cerebroventricular system of the cat. Biochem. Pharmacol.. 1969; 18:1907.

Cooper, B. R., Breese, G. R., Howard, J. L., Grant, L. D. Effect of central catecholamine alteration by 6-hydroxydopamine on shuttle-box avoidance acquisition. Physiol. Behav.. 1972; 9:727.

Cooper, B. R., Breese, G. R., Grant, L. D., Howard, J. L. Effects of 6-hydroxydopamine treatment on active avoidance responding: Evidence for involvement of brain dopamine. J. Pharmac. ex. Ther.. 1973; 185:358.

Cordeau, J. P., DeChamplain, J., Jacks, B. Excitation and prolonged waking produced by catecholamines injected into the ventricular system of cats. Can. J. Physiol. Pharmacol.. 1971; 49:627.

Crow, T. J. A map of the rat mesencephalon for electrical self-stimulation. Brain Res.. 1972; 36:265.

DeWied, D. Influence of anterior pituitary on avoidance learning and escape behavior. Amer.J.Physiol.. 1966; 207:255.

DeWied, D., Bohus, B. and Wimersma-Greidanus, T.B. The hypothalamo-neurophyseal system and the preservation of conditioned avoidance behaviour in rats. In: Integrative hypothalamic activity (Swaab, D.F. and Schade eds.), Progress in Brain Research, 41, 417, 1974.

Endroczi, E., Lissak, K., Fekete, M., Effects of ACTH on EEG habituation in human subjects. Progress in Brain Research; 32. Elsevier, Amsterdam, 1969.:254.

Fibiger, H. C., Phillips, A. G., Zis, A. P. Deficits in instrumental responding after 6-hydroxydopamine lesions of the nigro-striatal dopaminergic projection. Pharmac. Biochem. Behav.. 1974; 2:87.

Gispen, W. H., Schotman, P., Drug effects on neuroendocrine regulation. Progress in Brain Research; 39. Elsevier, Amsterdam, 1973.:443.

Glowinski, J., Axelrod, J. Effects of drugs on the uptake, release and metabolism of 3H-norepinephrine in the rat brain. J. Pharmac. exp. Ther.. 1965; 149:43.

Clowinski, J., Kopin, I. J., Axelrod, J. Metabolism of 3H-norepinephrine in the rat brain. J. Neurochem.. 1965; 12:25.

Glowinski, J., Baldessarini, R. Metabolism of norepinephrine in the central nervous system. Pharmacol. Rev.. 1966; 18:1201.

Glowinski, J., Effects of amphetamine on various aspects of catecholamine metabolism in the central nervous system of the ratCosta E., Garattini S., eds. Amphetamines and related compounds. Raven Press: New York, 1970:301–316

Goldstein, M., Gerber, H. Phenolic alcohols in the brain after administration of DOPA-C¹⁴ or dopamine-C⁴¹. Life Sci.. 1963; 2:97.

Iversen, L., Glowinski, J. Regional studies of catecholamines in the rat brain. II. Rate of turnover of catecholamines in various regions. J. Neurochem.. 1966; 13:671.

Jalfre, M., Haefely, W. Effects of some centrally acting agents in rats after intraventricular injections of 6-hydroxydopamine. In: Malmfors T., Thoenen H., eds. 6-hydroxydopamine and catecholamine neurons. Amsterdam: North-Holland; 1971:333–346.

Jouvet, M. Some monoaminergic mechanisms controlling sleep and waking. In: Karezmar A.G., Eccles J.C., eds. Brain and human behavior. Berlin: Springer; 1972:131–182.

Kety, S. S. The biogenic amines in the central nervous system, their possible roles in arousal, emotion, and learning. In: Schmitt F.O., ed. The neurosciences second study program. New York: Rockefeller University Press; 1970:324–336.

Lenard, L. G., Clody, D. E., Beer, B. Long-term conditioned avoidance deficits in rats after intraventricular injection of 6-hydroxydopamine: Restoration of performance with 1-norepinephrine. Proc. Am. Psychol. Assoc.. 1972.

Maynert, E. W., Lewi, R. Stress induced release of brain norepinephrine and its inhibition by drugs. J. Pharmacol. exp. Ther.. 1964; 143:90.

Milhaud, G., Glowinski, J. Metabolisme de la dopamine-C¹⁴ dans le cerveau du rat. Etude de monde d’administration. C.R. Acad. Sci.. 1962; 255:203.

Mitcham, J. C., Thomas, R. K. Effects of substantia nigra and caudate nucleus lesions on avoidance learning in rats. J. comp. physiol. Psychol.. 1972; 81:101.

Moore, K. E., Lariviere, E. W. Effects of stress and d-amphetamine on rat brain catecholamines. Biochem. Pharmacol.. 1964; 13:1098.

Nyakas, C., van Delft, A. M.L., Kaplanski, J., Smelik, P. G. Exploratory activity and conditioned avoidance acquisition after early postnatal 6-hydroxydopamine administration. J. Neural Transm.. 1973; 34:253.

Nyakas, C., van Delft, A. M.L. Behavioral and electrocortical activity in rats after neonatel intraventricular 6-hydroxydopamine administration. Pharmac. Biochem. Behav.. 1975; 3:271.

Phillips, A. G., Fibiger, H. C. Dopaminergic and noradrenergic substrates of positive reinforcement: Differential effects of d- and 1-amphetamine. Science. 1973; 179:575.

Rolls, E. T. Contrasting effects of hypothalamic and nucleus accumbens septi self-stimulation on brain stem unit activity and cortical arousal. Brain Res.. 1971; 31:275.

Shellenberger, M. K., Gordon, J. H. A rapid, simplified procedure for simultaneous assay of norepinephrine, dopamine, and 5-hydroxy-tryptamine from discrete brain areas. Anal. Biochem.. 1971; 39:356.

Segal, D. S., Mandell, A. J. Behavioral activation of rats during intraventricular infusion of norepinephrine. Proc. Nat. Acad. Sci.. 1970; 66:289.

Stein, L., Wise, C. D. Possible etiology of schizophrenia: progressive damage to the noradrenergic reward system of 6-hydroxydopamine. Science. 1971; 171:1032.

Stern, W. L., Hartman, E. E., Draskoczy, P. R., Schildkraut, J. J. Behavioral effects of centrally administered 6-hydroxydopamine. Psychol. Rep.. 1972; 30:815.

Stokes, L. D., Thompson, R. Combined damage to the medial cerebral penduncle and anterior hypothalamus and escape behavior in the rat. J. comp. physiol. Psychol.. 1970; 71:303.

Taylor, K. M., Laverty, R. The effects of drugs on the behavioral and biochemical actions of intraventricular 6-hydroxydopamine. Eur. J. Pharmac.. 1972; 17:16.

Thierry, A. M., Javoy, F., Glowinski, J., Kety, S. S. Effects of stress on the metabolism of norepinephrine, dopamine and serotonin in the central nervous system of the rat. I. Modifications of norepinephrine turnover. J. Pharmac. exp. Ther.. 1968; 163:163.

Vogt, M. Concentration of sympathin in different parts of the central nervous system under normal conditions and after the administration of drugs. J. Physiol.. 1954; 123:451.

DISCUSSION

Dr. Usdin inquired as to whether Dr. Endroczi had tested other peptides (vasopressin, scotophobin, etc.) but was told that this had not been done. Dr. van Loon showed some data indicating that after administration of ACTH there was a differential effect on DBH activity in various regions of the brain; this seemed to be at variance with the results of Dr. Endroczi – uniform effects throughout the brain. Dr. Endroczi replied that he had not yet investigated ACTH effects in the cerebellum. Dr. van Loon showed a slide demonstrating that in the hypophysectomized rat ACTH 1-10 increased hypothalamic DBH activity and decreased cerebellar DBH activity whereas ACTH 11-24 was without effect.

INFLUENCE OF SOCIAL STRESS ON BRAIN CATECHOLAMINE MECHANISMS

Kjell Modigh,     Department of Pharmacology, University of Goteborg, Goteborg, Sweden

Publisher Summary

Prolonged social isolation is a method to induce aggressiveness in male animals of various species. The phenomenon has been studied in mice that fight each other intensively when brought together after individual housing for about 4 weeks. The effect of isolation on the synthesis and turnover of brain monoamines has been the subject of several investigations. The turnover of noradrenaline (NA) and 5-hydroxytryptamine has been found to be lower in isolated than in grouped mice. Studies on the turnover of brain dopamine (DA) during isolation are contradictory. The rate of depletion of DA after tyrosine hydroxylase inhibition has been reported to be either decreased or increased. The catecholamine (CA) synthesis is retarded in isolated animals, when estimated in vivo as the accumulation of DA and NA after monoamine oxidase (MAO) inhibition. The tyrosine hydroxylase activity, measured in vitro in midbrain and striatum, has been reported to be increased. Fighting among previously isolated animals has been reported to be without significant effect on the synthesis and turnover of brain CA when estimated as their accumulation after MAO inhibition and depletion after tyrosine hydroxylase inhibition. These findings were taken as support for the hypothesis that stress induces a partial and reversible inhibition of mitochondrial MAO in the brain.

Introduction

Prolonged social isolation is a well known method to induce aggressiveness in male animals of various species. The phenomenon is usually studied in mice which generally fight each other intensively when brought together after individual housing for about 4 weeks. The effect of isolation on the synthesis and turnover of brain monoamines has been subject of several investigations. The turnover of noradrenaline (NA) and 5-hydroxytryptamine has repeatedly been found to be lower in isolated than in grouped mice (for review see ¹, ², ³, ⁴). Studies on the turnover of brain dopamine (DA) during isolation are however contradictory. Thus the rate of depletion of DA after tyrosine hydroxylase inhibition has been reported to be either decreased⁵ or increased⁶. Furthermore the catecholamine (CA) synthesis is retarded in isolated animals, when estimated in vivo as the accumulation of DA and NA after monoamine oxidase (MAO) inhibition⁷. The tyrosine hydroxylase activity, measured in vitro in midbrain and striatum, is however reported to be increased⁸.

Fighting among previously isolated animals is reported to be without significant effect on the synthesis and turnover of brain CA when estimated as their accumulation after MAO inhibition and depletion after tyrosine hydroxylase inhibition, respectively⁹, ¹⁰. These findings were taken as support for the hypothesis that stress induces a partial and reversible inhibition of mitochondrial MAO in the brain¹¹. The brain CA neurons are, according to this hypothesis, activated during various forms of stress, including fighting. The increase in CA release should thereby primarily be compensated for by a decreased catabolism of intraneuronal CA rather than by an increased CA synthesis.

Synthesis of brain CA in isolated, grouped and fighting mice

The hydroxylation of tyrosine to dihydroxyphenylalanine (DOPA) is under normal conditions considered to be the rate-limiting step in the CA synthesis. A new and simple method has recently been developed for estimating the rate of tyrosine hydroxylation in vivo¹². The method utilizes the fact that DOPA normally is rapidly decarboxylated and therefore not readily detectable in the brain. The concentration of DOPA increases, however, linearly after inhibition of DOPA decarboxylase (DC). The accumulation of DOPA after administration of a DC inhibitor therefore provides a measure of the rate of tyrosine hydroxylation. Previous experiments indicate that acute physiological variations in CA synthesis are detectable by this method (see e g¹³). Furthermore, animals’ gross behaviour is generally unaffected by the experimental procedure when NSD 1015 (3-hydroxybenzylhydrazine) is used as DC inhibitor. The availability of this method prompted a reinvestigation of the CA synthesis in isolated, grouped and fighting animals.

Male mice were housed one per cage (isolated) for several weeks. Animals of the same batch, serving as controls, were housed in groups of 25-30 per cage (grouped). Some of the animals were in the acute experiments brought together in groups of 25-30 for 30 min. These animals (fighters) started almost immediately to fight vigorously. The fighting involved all the animals. At the end of the 30 min periods most of the animals appeared exhausted and usually 2-4 mice per group had died, some of them with generalized convulsions. NSD 1015 was administered intraperitoneally in a dose of 100 mg/kg, 30 min before sacrifice. Biochemical analyses were performed on 3 parts of the brain; the corpus striatum including the olfactory tubercles (referred to as striatum), the rest of the cerebral hemispheres (referred to as hemispheres) and the rest of the brain (referred to as rest).

Administration of NSD 1015 had no apparent effects on the behaviour of neither isolated, grouped nor fighting animals. The NSD 1015 induced DOPA accumulation occurred in all the three parts of the brain, at a higher rate in grouped than in isolated animals, and was further accelerated in fighters (Fig. 1). The brain concentration of tyrosine was approximately the same in NSD 1015 treated isolated and grouped animals but was increased in fighters (Fig. 1). The concentration of homovanillic acid (HVA), the main metabolite of DA, was determined in whole brains of untreated animals. Isolated animals showed lower levels and fighters showed higher levels of HVA than grouped animals (Fig. 2).

Fig. 1 Effects on differential housing and of fighting on the concentrations of tyrosine and dihydroxyphenylalanine (DOPA) in different parts of the brains of male mice treated with NSD 1015. Each value represents the mean of 4 determinations (+ s e m) (from Modigh, 1973 ²⁸)

Fig. 2 Effects of differential housing and of fighting on the brain concentration of homovanillic acid (HVA) in male mice. Each value represents the mean of 4 determinations (+ s e m) (from Modigh, 1973 ²⁸)

The results indicate that in brains of male mice, the tyrosine hydroxylation is retarded during social isolation but rapidly accelerated when the animals are brought together and begin to fight. The differences in DOPA accumulation, elicited by the differential housing or by the fighting, were of the same magnitude in hemispheres, striatum and rest, whereas the relations between the concentrations of DA and NA are quite different in the three parts of the brain (Fig. 3). The changes in DOPA accumulation therefore probably reflect corresponding changes of the tyrosine hydroxylation in both DA and NA neurons. The differences in HVA concentration provide additional support for variations in turnover of