The Concept of Stress and Its Relevance for Animal Behavior

ADVANCES IN THE STUDY OF BEHAVIOR. VOL 21

The Concept of Stress and Its Relevance for Animal Behavior DIETRICH VON HOLST DEPARTMENT OF ANIMAL PHYSIOLOGY UNIVERSITY OF BAYREUTH

95440 BAYREUTH,

GERMANY

1. INTRODUCTION

Mammals live in social systems, which differ from species to species but are relatively constant for any species, although some variation as a function of the ecological situation is possible. These social systems are maintained by constant contact between the animals, which not only affects the behavior of the individuals, but may also positively or negatively influence their fertility and health. The negative consequences of social interactions are usually explained by the stress concept as shown in a particularly impressive way in the Australian dasyurid marsupials of the genus Antechinus. This genus is widely distributed in Australia and feeds mainly on insects and small vertebrates. All species examined so far exhibit an extremely synchronous life cycle: At the end of September-during the Australian spring-the females give birth to their young, which are weaned in January, but continue to live in harmony with their mothers for a few more months. At the end of May, the young leave their birthplace and spread out within their habitat. The short reproductive season commences in August, during the Australian winter. During the search for females, the males roam their territory and are continually involved in vehement fights with other males. Following the 2- to 3-week reproductive season and before the end of the first year of their life, virtually all the males “die off.” The females survive and after a 1-month gestation period they give birth to their young. A new cycle ensues (Woolley, 1966). The death of the males is due to typical stress reactions characterized by a tenfold increase in the plasma levels of free glucocorticosteroids and a simultaneous breakdown of the immune and inflammatory responses. As a consequence, gastrointestinal hemorrhaging associated with gastroduodenal ulcers, bacterially induced hepatic necrosis, heavy parasitic diseases, and other infections cause the death of all males 1

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within a short period of time (Barnett, 1964; Bradley et al., 1980; McDonald et nl., 1981, 1986). The physiological changes causing death are based mainly on the increased levels of aggression between the males. Accordingly, if males are captured before the breeding seasons and housed singly, they may live to about 2 years of age as do females under natural conditions. This means that the males die of stress mainly due to their enhanced aggression and persistent sexual activity. In this chapter, the significance of the stress concept in gaining a better understanding of social mechanisms in nonhuman mammals will be examined. In the second section the development of this concept during the last 50 years and the resulting current understanding of different stress reactions are described. The triggers of stress reactions are mainly psychical processes resulting from the assessment of a situation by an individual. Dependent on its coping behavior, these processes lead to different physiological response patterns, which can result in a number of pathophysiological effects. In the third section the most important currently applied methods in assessing stress levels in animals are introduced. Particular attention is paid to methodological problems as well as to the limits of interpretation. Focal points are the sympathetico-adrenomedullary and pituitary-adrenocortical systems, the pituitary-gonadal axis, and the immune system. In the fourth section an overview is provided of the relationships between social situations and stress responses, in which I concentrate mainly on our research on the monogamous and territorial tree shrews and the polygamous and territorial European wild rabbits. In these cases the social rank of an individual, as well as its sociopositive interactions with conspecifics, and the stability of the social system are determinants in the effects of a social situation on the individual’s vitality and fertility.

11. THECONCEPT OF STRESS A. INTRODUCTION

Few biomedical terms are as popular as stress. However, its definition is as inconsistent as the research strategies of the scientists from a variety of disciplines (biomedicine, psychology, or sociology) working on stressrelated topics (Lazarus and Folkman, 1984;Levine and Ursin, 1991;Weiner, 1991). Although it is probably impossible to find a definition that the majority of researchers will agree upon, and some authors even suggest that the concept is meaningless (e.g., Engel, 1985), the concept of stress has a long history that goes back to the ancient Greeks. As early as the

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year 400 B.c., Hippocrates ascribed the causes of disease to disturbing forces of nature and referred to the adaptive responses of the body as the “healing power of nature.” One hundred years later psychological stress was mentioned by Epicurus, who suggested that coping with emotional challenges is a way of improving the quality of life (cited from Chrousos et al., 1988). All recent stress concepts deal with the daily social and nonsocial stimuli that are challenging or threatening to the survival, health, and reproductive success of animals and that are, therefore, an essential part of natural selection. B. DEVELOPMENT OF THE STRESS CONCEPT Modern biomedical stress research is based in particular on the work of the American physiologist Walter B. Cannon and his colleagues, and the work of the Canadian physician Hans Selye. 1.

Cannon’s Fight or Flight Syndrome

In 1929, Cannon published an important monograph entitled “Bodily Changes in Pain, Hunger, Fear and Rage,” in which he summarized the results of decades of research into the effects of emotional challenges on physiological processes. Cannon did not regard emotions as purely subjective sensations, but as all-encompassing phenomena that also embrace objective physiological and ethological components and could, therefore, be analyzed scientifically. This opinion is still held today (Buck, 1988a). Cannon found a multifarious mosaic of changes in bodily functions in both animals and humans in emotionally stimulating situations: a reduction in gastric and intestinal function; an increase in heart rate, blood pressure, and breathing rate as well as in the number of red blood cells and the sugar content in the blood; and accelerated blood clotting. All these effects were attributed by Cannon to the increased activity of the sympathetic nervous system (Fig. 1). Cannon not only concentrated on the explanation of these causal controlling mechanisms, but also questioned the adaptive value of this variety of reactions. His conclusion was the following: All these effects increase the capability of an individual to react actively to critical situations in its environment-to prepare it for fight or flight. However, Cannon also realized that not every emotional process results in the activation of the organism. A difficult situation that cannot be changed by action can trigger apathetic, inactive behavior and lead to, among other things, a reduction in pulse rate and blood pressure. An early and detailed description of this reaction is given by Charles Darwin in 1872 in his book

Release of

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Decreased clotting time

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1

ventilation increased

Increased blood flow to brain, heart, and skeletal muscles

Lipolysis

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Glycolysis

Increased plasma levels of

FIG. 1. Cannon’s fight or flight response: Activation of the sympathetic nervous system and release of the adrenomedullary hormones epinephrine and norepinephrine. Their effector organs and the effects on the whole organism are shown (see also Fig. 11).

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“The Expression of the Emotions in Man and Animals,” in which he spoke of the feeling of despair or grief.

2. Selye’s General Adaptation Syndrome Cannon and associates were concerned mainly with the acute responses of an individual to potentially dangerous stimuli, while recognizing that repeated exposure to such stimuli results in adaptive changes in the organism that make it more resistent to challenge. The adaptation of an organism to chronic challenges, on the other hand, was the main interest in the research by Hans Selye, who also introduced the term stress into biomedical research (Selye, 1950). Contrary to the use of this term in everyday language and in other scientific disciplines, he designated stress as the response of an organism to any strong and potentially damaging stimulus, while for the damaging stimulus he introduced the term stressor. a. General Adaptation Syndrome. In 1936 Selye published a short paper “A Syndrome Produced by Diverse Nocuous Agents,” describing for the first time a pattern of physiological reactions in response to various damaging agents or critical situations, such as injuries, cold, infections, intoxications, burns, o r strong muscular exercise. An organism responds to these different stressors with stimulus-specific responses, such as with immunological responses to infections or with increased erythrocyte numbers to oxygen deficits. However, no matter how variable the nature of these stressors, according to Selye, they always elicit the same pattern of physiological responses, which seem to represent a generalized effort of the organism to adapt itself to the new situation. The response of the organism to stressors is accomplished in three stages, which Selye called the general adaptation or stress syndrome. 1. Alarm Reaction. The initial responses to physiological changes induced by a stressor are thymolymphatic involution, gastrointestinal ulceration, and loss of cortical lipids and medullary chromaffin substances from the adrenals, indicating an activation of the sympathetico-adrenomedullary and pituitary-adrenocortical systems. If the stressor is too strong (severe burns, extreme temperatures), death may result within a few hours. However, if the stressor is not too strong and has only a brief effect, then it usually has no further consequences for the organism, which quickly regains its original state. 2. Stage of Resistance. If the challenge persists, the body adapts itself to tolerable stressors, such as very low temperatures or unavoidable physical exertion, by changing its entire physiological state. According to Selye, the increased activity of the adrenal cortex during this stage of defense or

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resistance is of particular importance. The adrenal cortex adapts to the increased production and secretion of its hormones by markedly increasing its size. Concomitant with this, those functions unnecessary to coping with the stressor, such as growth, gonadal activity, and immunological resistance are suppressed.

3. Stage of Exhaustion. If the stressor is sufficiently severe and prolonged, the adaptation mechanisms will finally fail and lead to the death of the individual. Long-term, tolerable stress therefore impairs fertility and vitality in animals. Simultaneously, the initial advantageous physical adaptive reactions (particularly the increased production of adrenocortical hormones) were thought by Selye to lead to a number of diseases (referred to as “diseases of adaptation”), ranging from high blood pressure and gastric ulcers to diabetes and cancer (Selye, 1950, 1976, 1981). This concept of stress had a lasting effect on research. Ever since the 1950s, hundreds of scientific publications with the term stress in their titles have been published each year. Due to the central role of the adrenocortical system in the Selyean concept, research on stress centered to a large extent around the adrenal cortex and its hormones, while other endocrine responses or systems such as the gastrointestinal or the adrenomedullary system were largely neglected, even though changes in these systems were clearly recognized. As a consequence, it became common practice to equate stress with adrenocortical activity: Increased serum levels or excretion rates of glucocorticosteroids, such as cortisol and/or corticosterone, or other indications of heightened adrenocortical activity were used as an index of the adaptation of an organism to a stressful situation or to the intensity of a stressor. Although, even by today’s standards, this approach may appear attractive methodologically, it is important not to equate stress with adrenocortical function, as the responses of an organism to new and sudden demands comprise almost all physiological systems. Heightened adrenocortical activity constitutes only one part of this response pattern and is in no way sufficient to characterize the stress state of an animal, especially because adaptive responses to stressful situations may occur without any heightened adrenocortical activity (discussed later). b. Physical versus Psychical Stress. While originally the adrenocortical activity was assessed by changes in adrenal gland weight and morphology, developments in the late 1950s yielded the first biochemical methods enabling determination of adrenocortical activity by measurement of their hormone levels in plasma or urine. This led to a growing interest of psychologists and physiologists in emotionally induced adrenocortical activation. By 1956, Mason and Brady had demonstrated for the first time increased

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17-hydroxycorticosteroid plasma levels in rhesus monkeys in an emotionally distressing situation (“conditioned anxiety”), and at the same time previously impossible studies on humans began. In the following years, countless studies on mammals (including humans) demonstrated strong adrenocortical activation not only during acute emotional arousal, but also in longlasting emotionally disturbing situations. Nowadays, emotional “loads” are cited as the most common reasons for stress in humans and, as pointed out by Ursin and Olff in a recent review (1993), emotional processes are also the most commonly used stressors in animal research. These emotional processes must be considered even when the experimenter assumes he or she is dealing with physical stressors. It is necessary to bear in mind, however, that an activation of the adrenocortical system can also be induced without any concomitant emotional arousal (such as during surgery under deep anesthesia or during infections and the resulting release of mediators by the immune system). A most important contribution to the modern stress concept is the work of Mason and associates on the effects of psychological influences on the general endocrine response pattern (Mason, 1968a,c). His own work, as well as the results of the relevant literature, led Mason to the conclusion that situations of novelty, uncertainty, or unpredictability are especially potent in inducing heightened adrenocortical activity. Today it is generally accepted that unpredictability is most effective in stimulating adrenocortical activity in a variety of situations. Correspondingly, if an individual is given information about the occurrence of an adverse stimulus, its predictability leads to a reduction of the adrenocortical response. One illustration of this is the study by Dess and associates (1983) on dogs that were subjected to a series of either predictable or unpredictable electric shocks. In the predictable condition the animals were presented with a tone prior to the onset of shock, while in the unpredictable condition, no tone was presented. Dogs that did not have the signal preceding the shock showed an adrenocortical response two to three times that observed in animals with the predictable shock experience. Furthermore, as shown by Mason, even subtle everyday changes in the environment, usually not considered as stressful, such as presence or absence of familiar persons in a room in which monkeys were kept in cages, can result in measurable changes of adrenocortical activity. These results suggest “that the central nervous system exerts a constant ‘tonicity’ upon this endocrine system, in much the same fashion as has been previously demonstrated for the autonomic and skeletal muscular effector systems” (Mason, 1968b). c. UnspeciJicity of the Stress Response. Mason also questioned the basic premise of the Selyean stress concept of a nonspecific response by an

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organism to many different stimuli or agents (stressors). Instead, he considered the Selyean stress response to be a specific physiological response to its corresponding psychological reaction, which is probably induced by the different Selyean stressors. Whether an animal is immobilized, subjected to unavoidable electric shocks or extreme temperatures, or whether it is forced to swim to exhaustion, it is always in a hopeless situation that is out of its control and that may be responsible for the adrenocortical activation (Mason, 1968b). The same opinion was held by Bush (1962, p. 321), who stated in a review: It is probable that very severe burns, and large doses of certain agents such as bacterial pyrogens, histamine, and peptones. cause a brisk release of ACTH that is independent of any emotional concomitants; but . . . severe exercise, cold, and fasting produce little or no effect on the secretion and metabolism of cortisol in man unless they are part of a situation that provokes emotion. (1962, p. 321)

d. Predictability and Control. As mentioned above, the typical Selyean stress response occurs in those situations that are characterized by uncertainty or unpredictability. Prolonged stress responses can incur a high biological cost, leading to a number of immunological, gastrointestinal, and cardiovascular changes that may reduce the vitality of the animals. Therefore, mechanisms have evolved whereby the animals can reduce excessive adrenocortical activation. The most important factor involved in reducing hormonal responses to adverse stimuli is control. Control can be defined as the capacity of an animal to produce active responses during the presence of an adverse stimulus. These responses may allow the animal to avoid or escape from the stimulus, but they may also provide the animal with the opportunity to change from one set of stimulus conditions to another, rather than to escape the adverse situation entirely. In both cases control reduces an animal’s physiological stress response. Particularly impressive support for this is provided by the research conducted by Jay M. Weiss on the development of gastric ulceration in laboratory rats (Weiss, 1972). In one of the earliest experiments, two rats were restrained in an apparatus for 21 hours with identical electrodes on their tails attached to the same shock-delivering device. Every minute a tone was presented to the rats for 10 s, which was followed by a light electric shock. One of the animals (“avoidance-escape rat”) was given the possibility of avoiding the shock by touching a panel with its nose during the presentation of the signal, or of terminating it after the beginning of the shock; the other rat had no possibility of influencing the shock outcome (“yoked rat”). Every time the avoidance-escape rat received a shock the helpless yoked rat was given exactly the same shock. Thus, the two animals received exactly the same physical stressor, but they differed in their control over

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the situation. The two most important results of this study were (1) Simply receiving shock itself is not in particular responsible for the production of ulcers, but rather whether or not the animal is able to control the shock. The helpless yoked rats developed much more ulceration than did their partners; and (2) The more often an animal terminated the signal andlor the shock by its behavior, the less ulceration developed. That is, animals that can exercise control over a stressful situation do receive the relevant feedback when they respond by getting the information that they are “doing the right thing.” This is never the case in the helpless yoked animals. These results led Weiss to the conclusion that the most important aspect of an animal coping in a stressful situation is whether or not it can predict the consequences of its behavior. This conclusion was elegantly confirmed by experiments in which avoidance-escape animals with control over shock were given a brief electric shock every time they performed the previously correct response. Thus, each avoidance-escape response now produced precisely the wrong kind of feedback stimulus, a shock. In this “negative” feedback situation, the animals developed even more ulceration than did their helpless yoked partners (Fig. 2). The results of this research have been confirmed many times over by experiments designed to embrace endocrinological parameters and carried out on other species: Animals that are allowed to control the stimulus or

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Signal

p < .05

Signal + punishment

FIG.2. Length of gastric lesions (medians) of nonshock, avoidance-escape, and yoked groups of rats exposed to shock pulses that were preceded by a warning signal (lefi) and of groups that perceived a shock pulse whenever they performed an avoidance-escape response (right). Significant differences between the two shock groups and nonshock groups are indicated: *p < .05; **p < .01; ***p < .001. For details see text. Adapted from Weiss (1971). with kind permission from American Psychological Association, Washington, D.C.

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situation show less (and in some cases no) physiological stress responses (e.g., glucocorticosteroid levels not different from those of undisturbed controls), whereas their yoked counterparts exhibit extremely high levels of glucocorticosteroids and other signs of stress (e.g., Davis et al., 1977; Hanson et al., 1976; Seligman, 1975; Weiss, 1984). Hence, current opinion links Selye’s stress response or the activation of the pituitary-adrenocortical system to psychological processes, resulting from uncertainty to loss of control and helplessness.

3. Active and Passive Stress Responses An important modification of the original Selyean stress concept was made in 1977 by James P. Henry and Patricia M. Stephens. In their monograph “Stress, Health, and the Social Environment” they summarized the results of zoological, psychological, sociological, and medical research into stress and concluded that two independent chronic stress reactions needed to be distinguished from each other: active and passive stress. The central theme of this concept of two different stress responses is the relationship between styles of coping; limbic (emotional) processes and neuroendocrine stress responses. Every threatening stimulus or challenge to control immediately induces Cannon’s fight or flight response, followed within a few minutes by adrenocortical activation as the animal makes a behavioral effort to ensure that control over a conspecific or a situation is retained. If control is not possible, different types of coping are seen in nonhuman animals and humans alike, which clearly differ behaviorally and physiologically (Henry, 1986, 1992). a. Active Chronic Stress. If an animal reacts with a style of coping characterized by active attempts to control the situation, for example, by fighting to maintain or defend a social position or a territory or by fleeing to avoid the situation, Cannon’s sympathetico-adrenomedullary system is chronically activated; the activity of the adrenocortical axis can, but may not necessarily, be increased in this response. According to this concept, this active stress response is characterized by subjective feelings of anger or fear, depending on the context. Chronic active stress or the constantly heightened sympathetico-adrenomedullary activity may lead to arteriosclerosis and cardiovascular diseases. Recent studies have even shown a distinct response pattern, activated by the brain in differing emotional states within the sympathetico-adrenomedullary system. The neurosympathetic outflow of norepinephrine, the “fight hormone,” can be independently activated by the “flight hormone,” epinephrine, that is released from the adrenal medulla (Hucklebridge et al., 1981; de Boer et al., 1990). b. Passive Chronic Stress. When active coping (e.g., by flight) is not feasible, a state of helplessness emerges, characterized mostly by immobility

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and symptoms indicative of depression. This passive stress response is characterized by greatly enhanced activity of the pituitary-adrenocortical system, while the activity of the sympathetico-adrenomedullary system remains more or less unchanged (Fig. 3).

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FIG. 3. Schematic diagram contrasting the active and passive stress responses. The sympathetico-adrenomedullary system is divided into two branches: one of fight, anger, and norepinephrine; another of flight, fear, and epinephrine. Adapted from Henry et al. (1995). with kind permission from Lippincott-Raven Publishers, Philadelphia.

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c. Coping Behavior and Appraisal. These two styles of coping depend on the appraisal of the situation by the animal and on the quality and/or duration of the stressor. They thus may represent alternative strategies to the solution of a problem. For example, to avoid the constant attacks of a dominant rival, active escape may in the short term have the same result as hiding quietly in the corner of a cage, especially if escape is impossible. Furthermore, van Oortmerssen and associates (1985) demonstrated in a study of wild house mice (Mus rnusculus) that the two coping patterns may play different roles depending on the social structures and dynamics of populations: Aggressive mice do better in settled stable demes, whereas nonaggressive mice fare better in growing colonies. Appraisal of a stimulus or situation as well as the resulting coping behavior are basically psychological processes. There are, therefore, no clear relationships between stimuli imposed on individuals and their physiological responses. It is the behavioral, psychological, and thus the physiological responses of individuals to stimuli that differ depending on their genetics, prenatal influences, and especially postnatal learning processes (e.g., Fokkema et al., 1988; Henry et al., 1993). A striking example for the significance of social experiences on stress responses is provided by the work of Sachser and associates on guinea pigs (Cavia aperea f porcellus). Male and female guinea pigs can be kept in large groups without any behavioral or physiological signs of stress. When two adult males from different colonies are confronted in an experimental arena in the presence of an unfamiliar female, they arrange themselves in a dominance order within a short time in the absence of any serious fighting (Sachser, 1986; Sachser and Lick, 1991). However, if two males, each reared with only a single female, are confronted in the same way, both display continuously very high levels of aggressive behavior and extreme stress responses and die within a few days unless separated (Sachser and Lick, 1989). The ability to come to a peaceful arrangement with conspecifics is dependent on social experiences with male conspecifics around and shortly after puberty (Sachser, 1993). A few low-key confrontations with an unfamiliar male, introduced into their enclosure 5 times for 10 min between the age of 90 and 138days, were sufficient to reduce the fights with unknown males in later chronic confrontations to the same low levels of animals raised in colonies. That is, only 50 min of aggressive experience around puberty is required to enable adult male guinea pigs to come to a stressfree arrangement with conspecifics (Sachser et al., 1994). The crucial role of social experiences for behavior and stress responses was confirmed in a further approach (Sachser and Renninger, 1993). Colony- and individually reared males were singly introduced into unfamiliar colonies of conspecifics for a period of 30 days. Colony-reared males

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easily adjusted to the new social situation: On the first day they only explored the new environment but did not court any female, thereby avoiding attacks from the male residents. In the course of the following days they gradually integrated into the social network of the established colonies. Changes could not be determined in either their body weights or their plasma concentrations of cortisol, androgens, or catecholamines. In contrast, individually reared males were involved from the beginning in courtship behavior, threat displays, and fighting. As a consequence, they responded to the new situation with substantial body weight loss as well as with extreme increases in plasma cortisol levels (Fig. 4). These data from guinea pigs clearly demonstrate the causal relationship between social experience around puberty, behavior of the individuals as adults, and the degree of their stress in unfamiliar social situations. d. General Physiological Response Pattern, Physiological studies of recent decades have revealed that many, if not all, neuroendocrine systems respond to stressors. In his comprehensive treatise on motivation and emotions, Buck (1988b) differentiates between behavior involved in selfpreservation (offensive and defensive behavior) and behavior concerned with reproduction. These are accompanied by the arousal of different parts of the limbic system and the hypothalamus as well as by patterns of neuroendocrine response, each peculiar to the particular emotion involved. Apart from modifications in the sympathetic nervous system and the adrenal

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Days before and after introduction into an unfamiliar colony FIG. 4. Plasma cortisol levels ( M 2 SEM) of 6 colony- and 6 individually reared male guinea pigs before and after transfer into an unfamiliar colony. Significant differences between the two groups: **p < .01; ***p < ,001. Adapted from Sachser and Renninger (1993), with kind permission from II Sedicesimo, Florence, Italy.

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glands, examples are to be found in the modification of neuroendocrine systems that are involved in the regulation of reproduction (e.g., folliclestimulating hormone [FSH], luteinizing hormone [LH], testosterone, estrogen, prolactin), in metabolism (e.g., growth hormone, thyroid-stimulating hormone [TSH], thyroxine, insulin), in osmoregulation and regulation of blood pressure (e.g., aldosterone, vasopressin, renin), and in immune response. In accordance with the Selyean hypothesis, it appears that gonadal activity is always inhibited by passive stress, whereas, dependent on the context, active stress can have an inhibiting or activating effect (see Section 111,BJ). However, the immune system appears, at least in the long term, to be more or less inhibited by all stress reactions. Divergent hormonal patterns probably have differential effects on the function of the immune system. Current knowledge is insufficient regarding the other systems and prevents any general statements on their participation in a given stress reaction or on their long-term effects. Future findings will most certainly lead to further differentiation of the Henry-Stephens concept, particularly regarding the participation of the immune system in stress reactions. However, this concept of two independent stress axes has proved durable in zoology as well as in medicine and psychology over the past 20 years (e.g., Bohus et al., 1987; Henry and Meehan, 1981; von Holst, 1986a,b; Lemaire et al., 1993; Lundberg and Frankenhaeuser, 1980; Mormkde et al., 1990). 4. Stress-A

Useful Concept for Behavioral Research

In a very general form, Selye (1952) defined stress as “a non-specific response of the body to any demand made on it.” It is only in this sense that the term stress can be employed usefully today. In contrast to the original Selyean assumption, “nonspecific” must be interpreted as those reactions triggered within the body that are not a result of peripheral changes (e.g., a drop in blood sugar content or blood pressure) and, therefore, represent correction mechanisms of homeostatic processes. These are neuroendocrine response patterns induced by the central nervous system, which change the organism’s physiological state and thereby generally lead to its activation. These neuroendocrine stress reactions differ depending on the situation as well as the behavior of the animals and concomitant emotional processes. This definition of stress in no way implicates definitive reaction patterns or the participation of specific endocrine systems. However, it does assume physiological reactions that could be detrimental to the individual if they reached sufficient intensity or were of long duration. Social stress or psychosocial stress describes the state of an animal, in which interactions with

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conspecifics trigger central nervous processes, which themselves induce physiological reactions that can lead to detrimental effects on the animal’s vitality in the long term. Reduced fertility is not automatically also a consequence of these stress reactions. As the underlying factor in stress reactions is usually to be found in emotional processes, a given situation or stimulus can vary in its effect from one individual to the next by acting as an extremely damaging stressor, a harmless influence, or even a positive trigger. Hence, the recording of physiological stress reactions is-independent of the relevance of these reactions to diseases-a methodological basis used to rate the appraisal of a given situation by an individual and, therefore, to evaluate the biological significance of social interactions and situations as well as that of nonsocial influences (such as climatic factors or housing conditions).

C. ASSESSMENT OF STRESS

1. General Methodological Considerations Basically the effects of stressors can be measured on two levels, which, of course, are not exclusive. a. Epidemiological Approach. This approach is often taken in medicosociologically oriented research; the actual aim of research is to clarify the role of stressors in the development of malfunctions and diseases, from cardiovascular, gastrointestinal, or renal diseases to tumor growth and infertility. These mainly epidemiological studies usually focus on a few variables relevant for the respective organ or system, without paying much attention to the underlying physiological (regulatory) mechanisms (e.g., Ader et al., 1991; Adler et al., 1986; Dohrenwend and Dohrenwend, 1974; Friedman and Rosenman, 1974; Levi, 1971; Price, 1982). b. Physiological Approach. This approach mainly investigates those structures of the central nervous system involved in stress reactions and their effects on peripheral neuroendocrine and immunological parameters. It is not the aim of this chapter to discuss those central nervous structures involved in controlling neuroendocrine processes, but rather to focus on changes in peripheral parameters. These are of particular importance, as they not only indicate the presence of a stressful situation, but also allow limited statements on possible pathophysiological consequences of the situation for the individual. Hence, this is the preferred approach in research into stress (for details, see Section 111,B). A multitude of very different physiological parameters must be assessed, as individuals can react to the same stimuli in very different ways. However, even today this is not the case in most studies. Measures are usually selected on the basis of methodological constraints rather than based on present

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knowledge, which makes interpretation of apparently contradictory results often difficult or impossible. Although the determination of many different hormones, immunological mediators, and other clinically relevant parameters present few problems today, the interpretation of data is complicated by numerous possibilities for methodological errors. These are described briefly in the following section (for details, see textbooks of endocrinology). 2. Methodological Problems a. Animal Housing and Handling. Every organism responds to challenges with arousal responses of varying intensity (acute stress response). Moving an animal that is accustomed to a specific laboratory environment into a new cage or an unfamiliar room is sufficient disruption to act as a strong stressor for hours (e.g.. laboratory rats: Schuurman, 1981) or even days depending on the species. This can result in a total masking of actually interesting social situations or interactions. Thus, in tree shrews, transfer into a new cage within an experimental room results in increased serum levels of glucocorticosteroids and catecholamines for about 1week. Interestingly, serum levels of testosterone follow a biphasic pattern; after an initial decrease over a few days, the testosterone levels increase significantly above initial levels as the animals become habituated to the new situation (Fig. 5). This same biphasic testosterone response was found already in 1968 by Mason and co-workers (1968a) in rhesus monkeys during and after a 3day avoidance session. All handling of animals (e.g., weighing or the taking of blood samples) also functions as a stressor that acts on the corresponding variables within

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FIG. 5. Some endocrine responses of male tree shrews after a transfer into an unfamiliar room. Significant differences to initial values: * p < .05.

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seconds (catecholamines), minutes (glucocorticosteroids, thyroid and gonadal hormones), or a few hours (some immunological parameters); the repeated taking of blood samples on consecutive days can therefore lead to extensive stress reactions. After withdrawal of larger quantities of blood, a physiological stimulus is added to the psychological one, leading among other things to a sustained stimulation of the sympatheticoadrenomedullary system of variable duration. Finally, even in laboratory conditions, changes in climatic conditions or in food and introduced (nonvirulent) germs are capable of influencing many different endocrine as well as other parameters that indicate the presence or absence of stress. These problems can largely be avoided by standardized laboratory conditions. b. Diurnal and Other Variations of Physiological Parameters. There are a number of other problems associated with the measurement of hormone levels. In the majority of hormones secretion is not continuous, but occurs in a pulsatile fashion. The pulse amplitude is usually highest at the beginning of the activity period and lowest at the end, resulting in a marked diurnal rhythmicity of the hormone output (Fig. 6). Due to this pulsatile secretion, the hormone concentrations in the blood can change by a factor of 10 or more within minutes. Hence, baseline values exhibit much intra- and interindividual variation, even if great care is taken to exclude all potential interfering factors. This prevents interpretation on the level of the individual of most endocrine parameters based on single blood samples. Although it is possible to obtain blood samples from larger laboratory animals, over several hours and up to a few days, by insertion of cannulas into the blood vessels, this method is generally stressful to the animals, inhibits freedom of movement, and is therefore only of limited application in laboratory experiments (Fagin et al., 1983; Schuurman, 1981). Furthermore, many physiological parameters also show annual rhythms or other periodicities, which may influence hormone values. It is possible to circumvent the general influence of such rhythms in controlled laboratory conditions by, for example, always taking blood samples for endocrinological investigations at the same time of day. Nevertheless, because of the pulsatile secretory pattern of hormone release, marked individual variations will remain. Furthermore, no information is available on the influence of changing day length and other naturally occurring factors on such daily rhythms and, therefore, how comparable such values are even if they are collected at the same time each day. It is also more or less unknown whether stressful situations lead always to the same changes in the levels of different parameters during the day or not. A case in point are our investigations carried out on tree shrews with the aid of telemetry, which reveal a particular increase in heart rate during the night (sleep

18

DIETRICH VON HOLST

Body temperature ("C)

Heart rate (beatshin)

41

360

39

280

37

20 0

35

120

Triiodothyronine (nglrnl serum)

Glucose (mg/100 ml blood)

06

140

05

120

04

100

03

80

Cortisol (ng/ml serum)

Testosterone (ng/ml serum)

12

24

0

18

4

12

0

6 0

4

8

12

Time

16

20

24

0

4

8

12

16

20

24

Time

FIG. 6. Diurnal rhythms of some physiological parameters in male tree shrews. Night periods are characterized by dark color. Depending on the blood parameter, each point represents the mean (t SEM) of 20-80 males: heart rate and body temperature are hourly means ( 2 SEM) measured with implanted radio transmitters from 12 males and females.

periods) if the animals are in a stressful situation, even though they appear to be sleeping normally (e.g., Figs. 20 and 40). In summary, in order to gain relatively reliable data on endocrine and other physiological processes on the individual level, it is necessary to collect these data only on individuals that are kept in a controlled laboratory environment; in natural conditions in the field it is usually possible to detect only strong effects.

3. Physiological Markers of Stress In this section, I shall briefly discuss those methods that appear to me to be the most important or those that are most commonly employed in assessing the level of stress in an individual, as well as their application

STRESS A N D ITS RELEVANCE FOR ANIMAL BEHAVIOR

19

and power. To begin with, those systems will be briefly introduced that are necessary to understand the choice of variables that are measured. a. Pituitary-Adrenocortical System. Each of the paired adrenal glands of higher vertebrates is composed of two distinct and functionally different tissues-the adrenal cortex and the adrenal medulla. The cortex forms the outer part of the adrenal gland and consists of three zones: the outer glomerulosa; the zona fasciculata; and the inner zona reticularis, all of which produce large numbers of different steroid hormones. Glucocorticosteroids. For the present discussion, the hormones of the zona fasciculata are of special relevance, as they are released immediately in stressful situations. The most important and biologically relevant hormones are cortisol and corticosterone, the presence of which varies from species to species. For example, it is corticosterone almost exclusively that is found in the blood of rats and mice; in primates and guinea pigs cortisol is predominant; whereas other species (such as rabbits, hamsters, or tree shrews) exhibit both hormones, although they are liable to differential change during stress (e.g., rabbit: Kass et af., 1954; Krum and Glenn, 1965; hamster: Ottenweller etal, 1985). Because of their strong effects on carbohydrate and protein metabolism, all hormones of the zona fasciculata are grouped together as glucocorticosteroids: They increase the production of glucose from protein resources, and this is then stored in the liver as glycogen (a process referred to as gluconeogenesis), thus increasing the available glucose necessary for energetic processes during stress. Furthermore, they inhibit inflammatory processes and suppress many immunological responses by directly acting on receptors of the thymus and blood cells. Finally, glucocorticosteroids are required for the action of catecholamines such as for the induction of vasoconstriction by norepinephrine (e.g., Beato and Doenecke, 1980; Munck et af., 1984). Long-term increased glucocorticosteroid levels selectively reduce glucocorticosteroid receptors in the hippocampus (Brooke et af., 1994). Furthermore, high levels of glucocorticosteroids, such as are found in individuals suffering from chronic stress, are known to cause severe dendritic atrophy. This atrophy is particularly notable in hippocampal neurons and may contribute to the cognitive impairment found in persistently challenged individuals (e.g., Aus der Muhlen and Ockenfels, 1969; Magarinos et af., 1996; McEwen et al., 199.5; Uno et af., 1994; Sapolsky, 1991, 1992) (Table I). The synthesis and release of glucocorticosteroids are controlled by the pituitary hormone ACTH (adrenocorticotrophic hormone), which itself is controlled by the hypothalamic corticotrophin-releasing hormone (CRH). In emotionally induced stress reactions the release of corticosteroids appears to be largely controlled by CRH, whereas physical pressures can result in an increase in ACTH and, therefore, also in an increase in glucocor-

20

DIETRICH VON HOLST

TABLE I ACUTEA N D POTENTIAL LONG-TERM EFFECTS OF GLUCOCORTICOSTEROIDS Elevated levels of glucocorticosteroid hormones Acute effects Chronic effects Mobilization of energy (Gluconeogenesis) Lipolysis (synergistic with catecholamines) Raised muscle contractibility (permissive to catecholamines) Sodium retention and diuresis Release of calcium from bones Elevated release of hydrochloric acid and pepsinogen in stomach Antiinflammatory and immunosuppressive actions Suppression of gonadal activity

?

Neural responses, including altered cognition and sensory threshold

Loss of muscle mass, fatigue, steroid diabetes Arteriosclerosis Hypertension Hypertension Osteoporosis Ulcerat ion Decreased wound healing, increased disease susceptibility Decreased sexual behavior, sterility Dendritic atrophy (especially of hippocampal neurons) Psychoses and depression

ticosteroids through other mechanisms (such as through direct action of interleukin 1 during infections or through vasopressin during disturbances of the electrolyte balance: e.g., Aguilera et al., 1992; Berkenbosch et al., 1992; Brown, 1991; Dallman, 1991; Dempsher and Gann, 1983; Lilly et al., 1983; Rivier, 1991; Smelik and Vermes, 1980) (Fig. 7). While the release of glucocorticosteroids is usually controlled by ACTH, an additional possibility for the modification of the adrenocortical activity that has so far largely been ignored, is by its innervation. Henry and associates (1976) discussed the morphological and physiological evidence and presented their own data indicating that the activation of the adrenal cortex in dominant and aggressive fighting mice is due to direct sympathetic nervous stimulation, while in subordinate and repeatedly defeated mice the normal hormonal ACTH pathway is involved (see also Hucklebridge et al., 1981). Apart from its effect on the adrenal cortex, ACTH acts directly on the central nervous system. This indicates that ACTH may play an important role in the establishment and maintenance of social hierarchies, as was pointed out by Brain and Poole in 1974: Injection of ACTH suppresses defensive fighting behavior in mice pitted against trained fighters. In addition, acquisition of both actively and passively conditioned avoidance responses is enhanced by ACTH and the disappearance of these responses

STRESS AND ITS RELEVANCE FOR ANIMAL BEHAVIOR

-u=-

21

Stressor

Interleukin 1

I

Epinephrine

]

Vasopressin

I

Adrenal cortex

I

I

I Effector cells I FIG. 7. Schematic diagram of the hypothalamo-pituitary-adrenocorticalaxis. Stimulating

(+) and inhibiting (-) influences are indicated.

is delayed. On the basis of these results, Brain and Poole proposed that subordination in a dominance hierarchy may be a form of conditioned avoidance response, which causes subordinates to avoid further attacks by dominants either by fleeing or by signaling subordination. Corticosterone treatment has no apparent effect on offensive, aggressive behavior, but increases submissiveness in mice. Evidence for this is found in the occurrence of “the rigid upright posture” and the failure to defend themselves when attacked by an opponent (Leshner, 1981; Leshner and Politch, 1979; Leshner et al., 1980; Politch and Leshner, 1977). The authors

22

DIETRICH VON HOLST

conclude that, whereas ACTH may be important in the regulation of aggression, corticosterone regulates submission. As already mentioned, the influence of stressors induces an increased production and release of glucocorticosteroids. Long-term stress can therefore lead to ACTH-induced hypertrophy and hyperplasia in cells, resulting in a substantial enlarging of the zona fasciculata and hence of the entire adrenal gland. Minerafocorticosteroids. The second group of adrenocortical hormones are produced in the zona glomerulosa and are called mineralocorticosteroids after their function. The only physiologically relevant hormone is aldosterone, which affects sodium reabsorption in the distal tubuli of the kidneys and is hence involved in water and electrolyte metabolism. Its secretion is regulated by several factors (mainly by the concentrations of potassium and/or angiotensin I1 in the serum). Although a participation of aldosterone in stress responses (“conditioned anxiety”) has been demonstrated in rhesus monkeys, the direction of the initial change varies between the animals (Mason et al., 1968b). Because such studies on aldosterone and stress are few and far between, it will not be considered here. Sex steroids. The third group of adrenocortical hormones are sex steroids, particularly androgens such as dehydroepiandrosterone and androstenedione, which are normally released in considerable amounts by ACTH. However, in certain states (e.g., puberty, aging, and stress) there is a divergence between the stimulation of cortisol release on one hand and adrenal androgens on the other, which indicates the additional release of adrenal androgens by other, probably pituitary, factors (for details, e.g., Labhart, 1986). Compared to the biologically relevant testicular androgen testosterone, the biological effectiveness of adrenal androgens is very weak and little is known about their physiological role under normal conditions. However, the possibility cannot be ruled out that female reproduction (inclusive of fetal development) may be impaired by increased androgen concentrations in stressful situations (for a recent review, see Collaer and Hines, 1995). In contrast to the mineralocorticoids, glucocorticosteroids and sex hormones in the blood are mainly bound to transport proteins (corticoidbinding protein (CBP) and albumin) and free and bound fractions are at equilibrium. Only the free fractions exhibit biological activity. Concentration of these transport proteins is variable (e.g., increase during pregnancy: decrease during starvation). Assessment of adrenocortical activity

In the simplest case, changes in the size and weight of the adrenals can be useful to infer changes in activity. Adrenal weight and histology were the first parameters used to asess the

A D R E N A L G L A N D WEIGHT A N D HISTOLOGY.

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23

extent of adrenocortical activity by Selye and other scientists up to the end of the 1950s. Its usefulness is restricted, as the animals have to be killed to gain access to the organs and it is not possible to follow responses of the adrenocortical system in animals on the individual level. Even so, weighing is still often the only way of assessing adrenal activity in smaller animals in the wild, particularly as the weight of the adrenals is not affected by the capture and killing of the animal. Adrenal weights do not, however, provide information either on current hormone levels or on short-term changes in adrenal activity, as changes in size require several days of continual stress. Therefore, weights are more indicative of the adaptive state of the adrenal cortex. In addition, they provide little more than semiquantitative indications of current hormone concentrations. In many cases of less recent laboratory and field research on small rodents no absolute adrenal weights were given, and only results of calculations relative to the body weights of the animals were supplied. These relative adrenal weights are aimed at highlighting developmental differences between individuals and should compensate for differences in the size of their organs. In my opinion, these values are not, however, satisfactory measures of the adrenal activity in individuals, as body weights are particularly prone to rapid change if animals are stressed. Although relative adrenal weights do not allow any conclusions as to the adrenocortical activities or serum glucocorticosteroid concentrations in animals, increased relative adrenal weights do indicate stress. Chemical, histological, or histochemical studies of adrenal glands, as used to determine adrenocortical function in the initial research into stress, have since lost all relevance because of the development of direct methods in the determination of hormone concentrations. HORMONE MEASUREMENTS. The direct measurement of glucocorticosteroid concentrations in blood samples (serum or plasma) by radioimmunoassays and other methods is quite simple. Since, however, glucocorticosteroid concentrations increase after only 3 min subsequent to the beginning of the blood sampling procedure, “true” baseline levels can usually be obtained only under laboratory conditions. In the controlled laboratory environment, this methodology is applicable to assessing the effects of social and other stimuli on adrenal activity in individuals, as the necessary blood sample size is so small ( 4 0 ~ 1 that, ) even in small animals with body weights far below 100 g, blood sampling at 1- to 2-day intervals over long time spans is possible without detrimental effects due to blood loss. As mentioned previously, though, depending on the species and its emotional reaction and resulting psychological processes, an insufficient time lapse between each blood sampling procedure may result in typical stress responses with heightened glucocorticosteroid levels

24

DIETRICH VON HOLST

in the serum. For example, regular blood sample collection at 7-day intervals over several months induces no quantifiable physiological changes in tree shrews, whereas sampling at 4-day intervals o r less induces clear stress reactions after only two to three blood sample collections. One largely neglected aspect that may be particularly relevant to stress research is the relationship between free and protein-bound hormone levels. In the majority of studies only the total amount of the hormones (bound and unbound) is determined. As mentioned earlier, the biologically active fraction of the glucocorticosteroids are the free hormones: They affect tissues and regulate the release of glucocorticosteroids from the adrenal glands by their negative feedback effects on hypothalamic and hypophyseal structures. The concentration in the blood of these proteins, that bind to and transport the hormones, is usually restricted and can be saturated by increased hormone concentrations. Dependent on the concentration of transport proteins in the blood, this means that a small increase in total hormone concentration can lead to a substantial increase in the concentration of biologically active free hormones, as shown in laboratory mice (Bronson and ElefthCriou, 1965a). However, there appear to be substantial interspecific differences: Serum concentrations of both cortisol and corticosterone in tree shrews in acute stressful situations can rise by factors of 4-5 within 30 min, without affecting the ratio of free to bound hormones (correlation between initial values and stress values of free and proteinbound glucocorticosteroids is always >.90). On the other hand, a decrease in concentration of transport proteins, as the consequence of a glucocorticosteroid-induced general protein mobilization, body weight loss during stress, and/or as the consequence of increased testosterone levels, can increase the free hormone levels, although the total concentration of hormones remains the same or even decreases (e.g., Blanchard et al., 1993; Bradley et af., 1980). In mammals, free (non-protein-bound) hormones and their metabolites are largely excreted with the urine. The determination of the excretion rates of glucocorticosteroids (as well as those of other hormones) should therefore be especially appropriate in making statements on hormonal changes in mammals in stressful situations. The main limitation associated with the measurement of hormone levels in the urine is the considerable time lag between the appearance of the hormones in the blood and their excretion with the urine. Furthermore, the concentration of hormones in the urine varies according to the amount of urine produced, and both urine production and the drinking behavior of animals are influenced by stressors. Already in 1859, Claude Bernard reported the occurrence of oliguria in association with pain or emotional reactions, and the antidiuretic effect of emotional stimuli has repeatedly been confirmed by numerous subsequent

STRESS A N D ITS RELEVANCE FOR ANIMAL BEHAVIOR

25

studies on mammals including human beings. There are, however, also reports of diuretic responses to psychological stimuli (Mason et al., 1968b). It is not yet known what situations or specific stress responses are associated with these divergent effects on urine excretion. Conclusions on the activity of the adrenocortical (or other) endocrine systems, drawn from the concentrations of specific hormones or their metabolites in individual urine samples (e.g., collected at the beginning of the activity period of an animal), are therefore not particularly reliable. This problem is aggravated by the fact that reliable internal standards correcting for changes in urine concentration and/or loss of urine are not available. The creatinine concentration in urine samples is often used as an internal standard, but is subject to substantial change under stress conditions, as are all other urinary parameters, thus making it unsuitable as an internal standard. This means that statements on the activity of endocrine systems are usually possible only if the urine is collected quantitatively, throughout the entire day, in either one or several samples. With the exception of humans, this is possible only in laboratory conditions and requires the isolation of the animals. Very often the animals have to be kept in specialized urine collection or metabolic cages (or, in psychological research on monkeys, often in restraint chairs), which results in extreme restriction of freedom of movement and hence usually in stress reactions or the requirement of long habituation periods of the animals to the situation. This usually precludes any research into the effects of social interactions or factors on endocrine processes. In many species that can be kept in cages on lattices, the influence of specific social stimuli (e.g., sight or smell of rivals or sex partners or separation of mother and its infant) can be investigated by collection of the urine in a basin beneath the cage. As steroid hormones (such as the glucocorticosteroids and sex hormones) are not destroyed by delayed collection or by drying out, the urine can be collected at fixed time intervals (e.g., 24 hours), dissolved in distilled water and the hormone concentrations can be determined (Fenske, 1989). In this manner acute changes in hormone secretion due to stressors, as well as chronic effects of social input, can be assessed (e.g., Fig. 8). In field studies, some groups have also used concentrations of steroid hormones in samples of feces as indications of adrenocortical activity (e.g., Miller et al., 1991). Although the quantitative collection of feces of individuals is sometimes possible, even under natural conditions usually only single samples are collected (e.g., morning feces). The interpretation of these fecal hormone concentrations is subject to the same methodological constraints as those of single urinary samples, even when sampled at predetermined times of the day. For the past few years salivary steroid hormone levels have been used to analyze the stressful effects of different social situations. The hormone

26

DIETRICH VON HOLST

m c .-c

-

15

e3

10

2

5

c 0 L

Male 475 dominant

Daily confrontations and visual contact

0) P

s o

.-

c

-6

-4

-2

C C C

2

4

6

Days before resp. after confrontation FIG. 8. Daily urinary cortisol excretion of two male tree shrews that lived in a cage separated by a nontransparent partition. After habituation to the new cage the partition between the animals was removed on 3 days daily for 10 min, which resulted in slight fights and the establishment of a dominance order (C). For the rest of the days both animals were separated by a wire mesh partition to allow visual contact between the rivals. As evident from the figure, cortisol excretion increased in the subordinate male during the period of visual contact with its rival and returned to initial values after separation by a nontransparent partition, while the opposite was found in the dominant individual. Horizontal dashed line: Mean cortisol excretion during the 6 days before the confrontation.

concentrations in the saliva correspond in most species to those in the blood and are independent of saliva production. This method has many advantages compared to blood sampling procedures, as it is noninvasive, very fast (about 1 ml of saliva is needed), and it measures only the biologically relevant free (non-protein-bound) fraction of the hormones (RiadFahmy et af., 1982; Vining and McGinley, 1986; Wade, 1991). For these reasons, this method is widely used in psychological studies on humans and in some animal welfare studies on larger mammals, such as dogs or pigs (e.g., Beerda et al., 1996; De Jonge et al., 1996; van Eck et al., 1996; Ekkel et al., 1996; Kirschbaum and Hellhammer, 1989); recently Fenske (1996) showed that this method is also applicable to small mammals. In studies with guinea pigs he found a good correlation between saliva and plasma concentrations of cortisol, but, in contrast to studies in humans, not of testosterone. The reason for this discrepancy cannot be explained so far. CHALLENGE TESTS. Due to the time required to catch and handle the animals for blood sampling, in many cases it is impossible, even in the laboratory,

STRESS AND ITS RELEVANCE FOR ANIMAL BEHAVIOR

27

to obtain “real” baseline hormonal levels; therefore, some authors have used challenge tests to gain information on the state of the adrenocortical system. The most common challenge used in studies on small rodents in the laboratory is the “open field test.” Animals transferred to an open field respond to this situation with acute stress reactions, for example, an increase of glucocorticosteroid concentrations in their serum, which can be determined by taking a blood sample after a predetermined time (e.g., 15 rnin). As many studied have shown, animals living in a stressful situation show higher glucocorticosteroid levels in subsequent challenge tests than do control animals. The transfer of laboratory animals to an open field is not always a sufficiently strong stressor to elicit maximal release of glucocorticosteroids. Thus, following maximal stimulation of their release by high amounts of ACTH, serum cortisol values in male guinea pigs are about three times higher after 240 min than they are 240 rnin after the transfer to an open field (Sachser, 1994a). Nevertheless, there is a very good correlation between the cortisol values of the individuals in the open field and those in the ACTH test. This indicates that challenge tests, such as transfer to a novel room or cage, give a suitable measurement of an animal’s adrenocortical activity or secretory capacity. In the field, the procedure of catching the animals or of the anesthesia necessary for blood sampling in larger species has been successfully used as a standard challenge to determine the adrenocortical capacity of individuals. A particularly simple challenge for the determination of the secretory capacity of the adrenal cortex in tree shrews in the laboratory, which so far has not been used by other researchers, is the blood sampling procedure itself (von Holst, 1986b). To this aim the animals were brought in their sleeping boxes to a laboratory and blood samples were taken 1, 5, 15, and 30 min after the room had first been entered. Between sampling the animals were returned to their sleeping boxes, but remained in the laboratory for the entire period. This challenge test is a strong stressor for all tree shrews: Their heart rates are elevated for the entire experimental period and the levels of catecholamines, glucocorticosteroids, and glucose in the blood increase greatly. Shortly after the test and transfer to the home cage, heart rate and all other parameters return to the initial levels. On average, the cortisol values of control animals increase within 30 min from less than 10 ng/ml serum to approximately 60 ng/ml serum, with substantial differences being observed between the individuals (for examples, see Fig. 9). As long as the animals live under constant conditions, repetitions of these challenge tests after periods ranging from 1 week to several months result in almost identical (and individually different) values for all animals ( r > .92; p < .001;

28

DIETRICH VON HOLST

150

120

.-C

30 Male 21

d

0 ' , 0

10

20

30

Minutes after first disturbance

FIG. 9. Blood sampling challenge tests (BSCT): Adrenocortical responses of 3 males to 3 challenge tests separated by 3-5 months.

based on data from several experiments with more than 150 animals; see also Fig. 9). The blood sampling procedure elicits a maximal glucocorticosteroid release in tree shrews within the first 15 min, which cannot be further increased by injection of higher doses of ACTH. Accordingly, in vitro superfusion analyses of the adrenals from controls and stressed animals show an extremely good correlation between the in vitro cortisol production of the adrenals after maximal stimulation of their secretion through addition of ACTH to the superfusion medium, and the serum cortisol values obtained from the individuals earlier in a challenge test (Fig. 10). The individual differences of cortisol challenge values are therefore due to corresponding differences in the adrenal capacities of the individuals to synthesize and release glucocorticosteroids after stimulation. Chronic stress (e.g., transfer to a new room or a confrontation with a dominant rival) always leads to an increase in these challenge values by up to 200%. O n the other hand, the opposite is found when males habituate to a new room or become dominant in a confrontation (see also Fig. 22). It must be emphasized here, that an alteration of a challenge value must not be taken as an indication of an equivalent alteration of serum baseline levels of the glucocorticosteroid hormones. This is due to the fact that the secretory activity of the adrenal glands is dependent on the nominal value

STRESS AND ITS RELEVANCE FOR ANIMAL BEHAVIOR

29

120 1

Correlation coefficient r = .91; p<.oo1 v = 12.13 + 6 . 3 7 ~

20

7

0

,

3

,

,

6

,

,

9

,

,

12

,

,

15

,

I

18

In vitro cortisol secretion of adrenals (nglmin)

FIG. 10. Correlation between the serum cortisol values of 12 males 15 min after beginning of a BSCT and the in vitro cortisol release of their adrenals 15 min after ACTH has been added to the superfusion medium. The in v i m tests were performed 8 days after the challenge tests.

of the hormone levels as well as on the metabolism and clearance of the hormones, all of which are factors that may be influenced by stressors. Therefore, increased secretory capacities of the adrenal glands in animals are found especially during active stress, without simultaneously increased baseline adrenocortical hormone levels. In summary, while an increased adrenocortical capacity (as measured with a challenge test) does not necessarily have to be associated with increased levels of glucocorticosteroids in the blood, it is a sensitive measure of stress, which can also be used in field studies. b. Sympathetico-Adrenomedullary System. In contrast to the cortex, the adrenal medulla arises embryologically from neural tissue, and remains a functional part of the autonomic (vegetative) nervous system. It may in fact be considered as a specialized sympathetic ganglion that, on activation of the sympathetic nervous system, discharges epinephrine and norepinephrine directly into the blood (Fig. 11). Epinephrine in the periphery is derived primarily or wholly from the adrenal medulla; norepinephrine, however, may be secreted from the adrenal medulla, or its presence may be due to the overflow of neurotransmitters from the noradrenergic sympathetic nerves into the circulation. In rats and cats, for example, approximately 60-70% of the peripheral serum norepinephrine is derived from sympathetic nerves (Kvetnansky et af., 1979; Stoddard, 1991). Both the activation of the sympathetic nervous system, as well as the release of adrenomedullary

30

DIETRICH VON HOLST

Autonomic nervous system Sympathetic system

Parasympathetic syster

nal rd Cervical Hair follicle muscle Blood vessel

Thoracic

Lumbar

Sacral

FIG. 11. Schematic diagram of the autonomic nervous system with the effector organs of its two subdivisions-the sympathetic and the parasympathetic nervous system.

hormones, therefore act upon different bodily functions in very similar ways and generally increase the capabilities of the organism (Cannon’s fight or flight response): Hence the sympathetic nervous system and adrenal medulla are usually summarily referred to as the sympatheticoadrenomedullary system. Apart from its catabolic effects on peripheral organs, peripherally released epinephrine (but not norepinephrine) also affects the central nervous system by eliciting general arousal and subjective feelings ranging from restlessness to anxiety. Furthermore, there is also evidence that the release of epinephrine in situations eliciting fear enhances

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31

the learning of avoidance behavior, while high doses apparently produce the reverse effects (McGaugh and Gold, 1989). The biosynthesis of the catecholamines epinephrine (E) and norepinephrine (NE) from tyrosine progresses in several steps in both the noradrenergic neurons of the sympathetic nervous system and in the adrenal medulla; the first step-the conversion of tyrosine to dopa-is catalyzed by the enzyme tyrosine hydroxylase (TH), which is the pacemaking step of the catecholamine biosynthesis. For the subsequent two steps-the formation of dopamine and norepinephrine-enzyme levels are not usually limited. The final conversion of norepinephrine to epinephrine is catalyzed by the enzyme phenylethanolamine-N-methyltransferase(PNMT). PNMT activities in sympathetic neurons are very low, thus norepinephrine is the main end product of catecholamine biosynthesis (the transmitter), while in adrenal medullary tissue PNMT activities are high, resulting in epinephrine as the main adrenomedullary hormone. The TH levels in the medulla are controlled mainly by neuronal influences from the sympathetic nervous system (Thoenen et al., 1969; Ungar and Phillips, 1983). Repeated stimulation of catecholamine release in stressful situations leads to an adaptive increase of the TH levels in the adrenal medullary tissue, which in turn increases the capacity of the adrenal gland to synthesize and release its hormones. In laboratory rats, for example, there is a doubling of adrenal T H activities within less than one week of daily immobilization stress (Fukuhara et al., 1992; Kopin, 1980; Kopin et al., 1988). PNMT levels, on the other hand, are usually not influenced by sympathetic (splanchnic) nerves, but by the glucocorticosteroid hormones of the adrenal cortex, leading to a 50% increase under persistent stress (Ciaranello, 1978; Kopin, 1980; Wurtman and Axelrod, 1966). There are, however, differences in the regulation of PNMT activities even between different strains of rats, as has been demonstrated recently by Lemaire and colleagues (1993). According to these authors, the increase of PNMT activity in male Wistar rats, kept in unstable mixed-sex groups, is dependent on the activation of the sympathetic nervous system, as it can be completely abolished by denervation of the adrenals. Assessment of sympathetico-adrenomedullary activity. Each capture and handling of an animal for the collection of blood samples immediately activates the sympathetico-adrenomedullary system and triggers the release of catecholamines into the blood, thus making the collection of baseline values impossible without prior introduction of cannulas into the blood vessels (Stoddard, 1991). Therefore, with one exception (Fokkema, 1985), I am not aware of investigations dealing with catecholamine baseline values in animals in social situations.

32

DIETRICH VON

nouT

CHALLENGE TESTS. It appears that challenge tests are useful techniques to gain information on the adaptive state of the sympathetico-adrenomedullary system. As already mentioned, the adrenal medulla adapts to an increased production and secretion of its hormones by increasing the activity of its pacemaking enzyme, tyrosine hydroxylase. Accordingly, the stimulated adrenal gland increases its secretion of the two hormones epinephrine and norepinephrine. In our research on tree shrews, we use their emotional response to handling during blood sampling as the challenge. We found that catecholamine levels in the blood of tree shrews are maximally raised as rapidly as 20 s after the first disturbance. When we take several blood samples from an individual within a period of 1-15 min, serum catecholamine levels remain more or less the same, while differences of several hundred percent are found between different individuals. The mean value of the norepinephrine and epinephrine concentrations in several blood samples from a given individual taken during a challenge test is therefore considered to be an indication of the adaptation state of its sympathetico-adrenomedullary system. This suggestion is supported by the following results: Between repeated challenge tests separated by at least 1 week, the individually different values of norepinephrine (as well as of epinephrine) are highly correlated (Fig. 12). Stressful situations, such as transfer to a new cage or a confrontation with a rival, increase the norepinephrine and epinephrine values by up to

12

55

0

10

. F a

-

0

z

E

f

.-C

a,

r = .85

r = .91

2

2

6 8 1 0 1 2 NE (nglml serum)

4

2

3

4

5

6

7

TH (nmollh adrenals)

FIG. 12. Correlations between BSCT values of serum norepinephrine (NE) of male tree shrews at two sampling points separated by 8 days (left) as well as between serum norepinephrine values and adrenal tyrosine hydroxylase activities (TH) of animals. Unpublished data: after Hutzelmeyer (1987).

STRESS AND ITS RELEVANCE FOR ANIMAL BEHAVIOR

33

100% within 1-3 days. After habituation to the new situation (room or cage) or removal of the rival, challenge values approximate the initial individual levels (see also Fig. 5). Finally, a very good correlation is found between the norepinephrine and epinephrine challenge values of tree shrews and their adrenal tyrosine hydroxylase activities (Fig. 12). Thus, challenge levels of catecholamines are a relevant measure of the activity or adaptive state of the sympathetico-adrenomedullary system, which can be used in the laboratory as well as under more natural conditions in large enclosures, as has been shown in studies on tree shrews, guinea pigs, and wild rabbits. In guinea pigs, for example, catecholamine challenge values are clearly related to individual behavioral strategies in situations of social conflict: Plasma norepinephrine levels were significantly higher weeks before as well as directly after a 10-min agonistic encounter in offensive “fighters” compared to nonaggressive “nonfighters” (Sachser, 1987). Thus, norepinephrine responsiveness is of predictive value for the behavior of guinea pigs in contest situations. Similar results are also known from tree shrews (e.g., Fig. 16). Excretion rates of catecholamines and their metabolites are frequently used in psychosocial stress studies in humans, and less frequently in psychophysiological studies in monkeys, but not in behaviorally oriented studies on other animals. This has many reasons, some of which have been already mentioned (see discussion on glucocorticosteroid excretion). Even more important is, however, the fact that catecholamines and their metabolites are rapidly metabolized and/or destroyed after urination. To avoid degradation, the urine must therefore be strongly acidified, which makes its collection more or less impossible, except from animals kept in a restraint chair. ENZYME ACTIVITIES. As mentioned above, TH and PNMT levels in the adrenal medulla adapt to stimulation with an increase in their activities. The determination of these two enzymes as a measure of the activity of the sympathetico-adrenomedullary system was introduced by Henry and colleagues (1972; Kvetnansky et al., 1970) in their work on social stress in mice. In the meantime, this method is widely established and is being applied to many different animal species by other research groups. As the increased change in enzyme activity appears only several hours after the application of stress, these methods are equally applicable in the field and the laboratory. However, since the animals have to be sacrificed, the range of application of this method is restricted. HEART RATE AND BLOOD PRESSURE. As the sympathetico-adrenomedullary system induces strong physiological reactions in the organism, indirect measures are sometimes used as indicators of sympathetico-adrenomedullary activity. A particularly important measure in psychobiological research is the heart rate and-mainly for medical purposes-blood pressure. Both

34

DIETRICH VON HOLST

parameters usually increase rapidly with every activation of the sympathetic nervous system. While the heart rate is activated by the sympathetic nervous system (including the catecholamines of the adrenal medulla) and inhibited by the parasympathetic nervous system, and correspondingly changes in both parts of the autonomic nervous system can influence heart rate, blood pressure is activated only in the short term by the sympatheticoadrenomedullary system and the renin-angiotensin system. Chronic emotional responses, however, can result in structural (arteriosclerotic) changes of the cardiovascular system, which may lead to persistent hypertension (Folkow et al., 1958, 1973). The heart rate is particularly well suited for the detection of acute activations of the sympathetico-adrenomedullary system in socially or otherwise stressful situations, and for the monitoring of chronic stressors and their potential pathophysiological consequences. Implantable radio transmitters have been used for many years to record heart rate telemetrically. Since the working life and range of transmitter systems depend on battery size, the weight of the transmitters usually determines their working life. An extremely small radio transmitter (weight including battery <1 g) with a range of above 30 m and a working life of 4-6 months has been developed at our institute (Stohr, 1988). This transmitter enabled us to record heart rate (and body temperature) continuously in animals differing greatly in size (Mongolian gerbils, laboratory mice, tree shrews, and wild rabbits) (Eisermann, 1992; Eisermann and Stohr, 1992). The weights of most commercially available transmitter systems used in stress research in nonhuman mammals are in the range of 5-10 g, with a lifetime of 1-2 weeks and a range of a few meters. This usually restricts their application to laboratory conditions and animals with body weights of over 200 g. The working life is also usually too short to allow any more than the acute effects of specific stressors to be recorded. This problem is exaggerated by the fact that, depending on the species and the size of the transmitters, a period of 1-2 weeks is necessary after implantation before the animals have recovered and stable, low levels of their heart rate can be recorded. In technically advanced systems, a magnetic on/off switch is used to transmit the signals only at certain times, which conserves power and may extend the working life up to several months. Blood pressure has mainly been measured in animals in chronic emotionally arousing situations, by direct arterial or indirect tail-cuff techniques at weekly time intervals. For both techniques, the animals must be handled and restrained before measurements can be taken. To reduce emotional stress responses, the measurements are often performed after slight narcosis. Nevertheless, these procedures elicit stress responses in the animals, which may influence the measurement. In laboratory animals, however,

STRESS AND ITS RELEVANCE FOR ANIMAL BEHAVIOR

35

the tail-cuff technique in particular has been proven to provide reliable results, when the animals are used to this procedure (Bunag, 1984). Over the past few years, the direct and continuous measurement of blood pressure has also become possible in small mammals using implanted radio transmitters; in one of the first studies using this method, Henry and co-workers showed parallel blood pressure changes in rats during chronic stress, measured by radiotelemetry and by indirect tail-cuff techniques. The latter, however, revealed levels 10% higher than the former (Henry et al., 1995). c. Pituitary-Gonadal System. Apart from the generative testicular and ovarian functions (the development of spermatozoa and ova, respectively), these organs also have endocrine functions. They produce sex hormones, the production of which is independent of spermatogenesis in males, but closely linked to oogenesis in females. Testes. The testes secrete hormones, which are collectively termed androgens, of which testosterone is the main physiologically active one. Depending on the species and organ, testosterone sometimes has to be converted enzymatically to dihydrotestosterone (17-DHT) in target cells in order to become biologically effective. Before birth, the testes of mammals release large amounts of androgens, which are responsible for the development of male-specific sex organs during fetal life, and for the organization of the central nervous structures responsible for the expression of male-specific behavior in later life (e.g., Breedlove, 1994; Collaer and Hines, 1995; Goy et al., 1988; Gustafson and Donahoe, 1994; Huhtaniemi, 1994; McCarthy, 1994; Phoenix et al., 1959; Turner, 1984). In the absence of the male sex hormones, female-specific sex organs and behavior patterns develop. These pre- or perinatal effects of androgens are usually characterized as “organizing effects” (Phoenix et al., 1959). Following puberty, testosterone (and DHT) are necessary for the differentiation and activation of spermatozoa during spermatogenesis, induce growth and function of accessory sexual glands, and modulate sexual, aggressive, and other testosterone-dependent behaviors. This occurs to a greater or lesser extent depending on the species (e.g., Arnold and Breedlove, 1985; Baum, 1992; Beach, 1975; Matochik and Barfield, 1991; Monaghan and Glickman, 1992; Thiessen and Rice, 1976). These peri- and postpubertal effects of androgens on morphology, physiology, and behavior are usually called “activating effects.” Furthermore, marking behavior and the secretory activity of glands, involved in marking or the characterization of the hormonal state of a male, are modulated by testosterone. Thus, decreased testosterone levels in a subordinate male can reduce the intensity of its aggression-eliciting signals and thus reduce attacks against it.

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DIETRICH VON HOLST

Production and release of androgens by the testes is controlled by the hypothalamus. Gonadotropin-releasing hormones (GnRH) induce the release of follicle-stimulating hormone (FSH), which stimulates spermatogenesis, and luteinizing hormone (LH), which induces synthesis and the release of androgens from the Leydig cells of the testes. Ovaries. In contrast to the male androgens, female sex hormones have no organizational functions in mammals. Following puberty, dependent on the development of the follicles and subject to the influence of the two gonadotropins LH and FSH, the ovaries produce two main classes of sex hormones-estrogens and progestins, which are responsible in female mammals for their sex-specific morphology and physiology (e.g., Carter, 1992; Takahashi, 1990). Both classes of female sex hormones are also produced placentally in gravid females, as well as by the adrenal cortex in both sexes. Estrogens (especially the most effective estradiol) stimulate growth of the uterine wall and have a variety of other functions contributing to the maintenance of the condition of the female reproductive system. The biologically relevant progestin is progesterone, which is secreted in most mammalian species after ovulation by the corpus luteum of the ovary, and is necessary for maintaining pregnancy. In small rodents no corpus luteum develops during the estrous cycle and progesterone is synthesized by the interstitial tissue of the ovary. Estrogens (in many rodents in combination with progesterone) are responsible for female sexual receptivity, although their effect differs very much between species. In addition, estrogens can also enhance the attractivity of females by inducing the production of odors (sex pheromones) or other signals. Most stress situations apparently inhibit the release of GnRH, thus modifying fertility and the sex-hormone-dependent behavior of both sexes (e.g., Kime et al., 1980; Moberg, 1987; Orr and Mann, 1992; Rabin et al., 1988). Because of their specific receptors, the Leydig cells of the testes can also directly be influenced by glucocorticosteroid hormones (e.g., Evain et al., 1976; Stalker et af., 1989). Thus, 2 hr of immobilization induces a fall in serum androgen concentrations of rats without detectable changes in serum LH values. The chronic stress of a daily 2-hr immobilization for 10 days, however, results in decreased serum levels of both testosterone and LH (Maric et al., 1996). Correspondingly, in vitro corticosterone directly inhibits testosterone production by purified Leydig cells of rats, although only at high concentrations (Gao et al., 1996). In contrast to long-lasting stressful situations, acute stressors sometimes cause a transient elevation of plasma testosterone concentrations, despite the suppressed LH levels that precede the subsequent decline in testosterone levels. The reason for this effect has not been clarified yet, although increased testicular blood flow as a general consequence

STRESS AND ITS RELEVANCE FOR ANIMAL BEHAVIOR

37

of heightened sympathetico-adrenomedullary activity or of testicular nerves, as well as testicular pro-opiomelanocortin-derived peptides, may be involved in this function (e.g., Mann and Orr, 1990). Assessment of gonadal activity MORPHOLOGICAL AND HISTOLOGICAL DATA. As mentioned before, the testes have endocrine as well as generative functions that are not necessarily closely correlated. In order to obtain information on the generative functions, data collection in animal studies on social stress focuses on the weights of the testes, epididymides, and secondary sex glands, and in particular on histological investigations on the testes and epididymides for the proof of normal spermatogenesis, although the presence of intact spermatozoa does not rule out functional damage. In females, the weights of the ovaries provide a rather more superficial indication of their function; histological investigations on the ovaries, however, provide relatively differentiated information on maturing follicles, the number of ova released into the uterus, or impeded follicular maturation (e.g., increased follicular atresia). In addition, investigations on the uterus can provide information on implantation and also on abortions. All of these methods, however, are unsatisfactory in connection with studies on social stress and its consequences for the gonadal system, as they necessitate the sacrifice of the animals. HORMONE MEASUREMENTS.The determination of sex hormones in the blood, urine, or feces is possible in both sexes. The problems involved in the collection of these data (methods of blood sampling, time factors, methods of sample collection) are equivalent to those involved in the collection of glucocorticosteroids. Relatively reliable data on the influence of social and other factors on endocrine activity in the testes can be collected by blood sampling in males. In females, this is usually not possible except in extreme conditions, as the changes in hormone values are dependent on the stage of the cycle or pregnancy and daily hormone determination would be required in order to pinpoint stress-related changes, especially in animals with estrous cycles of a few days. This would be possible by determining selected hormones and/or their metabolites in the urine or feces. However, due to the many difficulties involved, very little information is as yet available on the quantitative influences of social and other stressors on the female endocrine system. Extreme changes, such as those during estrus and pregnancy can, however, be assessed by determination of hormones in urine or feces (e.g., Schaftenaar et al., 1992). d. Immune System. The immune system has two functional divisions: the innate (unspecific) system and the adaptive (specific) system. The innate immune system acts as a first line of defense against infectious agents

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DIETRICH VON HOLST

(viruses, bacteria, fungi, and parasites) and clears them before they establish an overt infection. The adaptive immune system produces a specific reaction to each pathogen (antigen), which normally eradicates that particular agent. The adaptive immune system also remembers the infectious agent with the aid of B and T memory cells, causing an immunity of variable duration against the pathogen by inducing a much enhanced specific response at the next contact with this antigen. Both divisions of the immune system consist of a large number of humoral (molecules) and cellular (leucocytes) factors, which circulate with the bloodstream and are distributed throughout the entire body (Table 11). During infections, both systems are usually activated and combat the infectious agents in an integrated way: Following clonal activation after contact with an antigen, T lymphocytes produce several soluble molecules (cytokines), which stimulate the phagocytes to destroy the infectious agents more effectively, and also stimulate antibody production by the B lymphocytes. These antibodies then also help the phagocytes to recognize their targets. Depending on the antigen, the various parts of the immune system are differently involved in the response pattern. Although the immune system displays a certain degree of autonomy, the research of the last two decades has revealed multiple channels of communication between the central nervous system and the immune system. Emotionally stressful situations are particularly associated with altered immune function, and in some instances with altered health status, although these two processes have not been linked causally (e.g., Adams, 1994; Ader and Cohen, 1985; Ader et al., 1991; Dunn, 1989; Glaser and Kiecolt-Glaser, 1994; Kelley et al., 1994; Laudenslager and Fleshner, 1994; Monjan, 1981; Solomon and Amkraut, 1981). In his first publication, Selye (1936) described thymolymphatic involution as one of the most conspicuous signs of stress,

TABLE I1 MAJORCOMPONENTS OF THE INNATEA N D THE ADAPTIVE I M M U N ESYSTEM Innate (unspecific) system Humoral factors Lysozymes Complement system Acute phase proteins (e.g., Creactive protein) Interferons Cellular factors Phagocytes (polymorphs and monocytes) Natural killer (NK) cells

Adaptive (specific) system

Antibodies (produced by B lymphocytes)

T lymphocytes (e.g.. cytotoxic cells, helper cells)

STRESS AND ITS RELEVANCE FOR ANIMAL BEHAVIOR

39

and the connections between the hypothalamo-pituitary adrenocortical axis and the immune system are among the best examined so far (e.g., Dhabhar et al., 1995; Heinjen et al., 1991; Keller et al., 1991; Munck and Guyre, 1991; Zwilling, 1994). A strong immunosuppressive effect is also exerted by the catecholamines, but virtually every hormone investigated so far has some effect on the immune system (such as growth hormone, prolactin, and sex hormones), although not all of the effects are direct (e.g., Bernton et al., 1991; Grossman, 1984; Kelley, 1991; McCruden and Stimson, 1991; Olsen and Kovacs, 1996; Paavonen, 1994; Rabin et al., 1994). Furthermore, lymphoidal tissues (e.g., bone marrow, thymus, spleen, and lymph nodes) are innervated by sympathetic and sciatic fibers and hence the central nervous system can directly influence immune function (e.g., Ackerman et al., 1991; Felten and Felten, 1991; Madden and Livnat, 1991). On the other hand, the immune system is capable of modulating both neuroendocrine responses and the behavior of mammals by nervous influences from lymphoidal structures and by lymphokines produced by leucocytes, which act on hypothalamic and other central nervous structures (e.g., Anisman et al., 1993; Bateman et al., 1989; Besedovsky and del Rey, 1991; Blalock, 1988; Carr and Blalock, 1991; Hall et al., 1991; Madden and Felten, 1995; O’Grady and Hall, 1991; Sternberg, 1988). These responses may improve the defense reaction of the body against pathogens. Thus, increased glucocorticosteroid levels may help to suppress an overly strong immune response, which could in itself be dangerous (as can be seen in allergic reactions) (e.g., Munck et al., 1984). Behavioral changes, such as lethargy, anorexia, or reduced grooming, which are typically observed in sick animals, can also be elicited by cytokines produced during the immune response of an organism against an infection (e.g., Crnic, 1991; Kelley et al., 1994; Myers and Murtaugh, 1995). Assessment of immunological functions. There are two fundamentally different-but not mutually exclusive-approaches to gaining information on the activity and capacity of the immune system in stressful situations: RELATIONSHIP BETWEEN STRESS A N D DISEASES

Diseases in natural populations. The first indications of the influence of psychosocial factors on the functions of the immune system were provided by investigations that demonstrated a relationship between certain social situations and the outbreak of diseases (e.g., at high population densities in rodents or after the death of a partner in human beings). This epidemiological approach is of particular relevance for the human situation, as it provides the only means of assessing the relevance of social influences on disease. However, this approach usually requires large amounts of data and provides no information on the specific underlying immunological

40

DIETRICH VON HOLST

processes associated with the increased morbidity (e.g., Gentry, 1984; Weiner, 1977; Wenar, 1983). Induction of diseases. In this case, pathogens (such as parasites, bacteria, or tumor cells) are injected into animals housed under different stress conditions and the outbreak and progress of the disease is correlated with the intensity of the stress (in some cases also the rejection of skin transplants is used for the characterization of immune capacity). In general today, those immunological parameters are also assessed that are capable of providing information on the action of the stress-induced changes in resistance to disease. This approach is of undeniable importance to the explanation of the relationship between stress and resistance to disease, but is methodologically complicated ( e g , knowlege of suitable pathogens, complicated housing conditions) and hence has so far been used only in a small number of investigations on standard laboratory animals (especially rats and mice) (for details see Section III,B,Z). Direct assessment of immunological parameters. No general statements can as yet be made on the effect of social and psychological influences on the immune system, as it is composed of many different subunits that can exhibit synchronous or antagonistic changes, depending on the situation or subsystem. We also have very little current understanding of the relevance of changes in specific immunological subsystems to disease in an individual. Therefore, the monitoring of many different parameters is necessary, in order to obtain a picture as informative as possible of the reaction of the immune system. This is, however, not the case in most experimental research carried out on animals. Immune measures are usually selected on the basis of financial or methodological constraints (e.g., availability of antibodies and laboratory facilities). Studies based on a limited selection of immune parameters may, however, result in a failure to detect immunological changes. Premature conclusions that a situation has no immunological effects must therefore be avoided. In the rest of this section, the most common immunological parameters used in animal behavior studies are mentioned. A reduction of one or several of these parameters is usually interpreted as a measurement of a reduced immune function. HUMORAL FACTORS AND LEUCOCYTE NUMBERS IN THE BLOOD. Very little blood is required for the determination of humoral factors (e.g., serum concentrations of immunoglobulins or C-reactive protein) as well as of the numbers and types of leucocytes in the blood (e.g., total leucocyte number, leucocyte subcategories such as neutrophils, eosinophils, basophils monocytes, lymphocytes), and subsets of lymphocytes (e.g., T and B lymphocytes, cytotoxic T lymphocytes, T helper cells, natural killer cells). All these parameters can therefore be determined at regular intervals from blood samples of

STRESS A N D ITS RELEVANCE FOR ANIMAL BEHAVIOR

41

individuals. For the differentiation of the lymphocyte subsets and the determination of the different immunoglobulins specific antibodies are necessary, which are currently available only for human beings and a few experimental animal species. The activity of the complement system in serum samples can easily be measured by in v i m bioassays (e.g., CH5[)hemolysis test), by determining the volume of serum necessary to induce hemolysis in 50% of red blood cells (usually of sheep) used as antigens. The measurement can be carried out repeatedly even in small mammals, as this test requires very little serum. IN VITRO TESTS. The in vitro proliferative response of lymphocytes after stimulation with mitogens is widely used as a functional test of the activity of the cellular part of the immune system. Different mitogens can be used, such as concanavalin A (Con A), that stimulates in most species predominantly T lymphocytes, or pokeweed, that is more specific to B lymphocytes. These in vitro tests can be performed on cell cultures of lymphocytes from the blood or from the spleen, as well as on unmanipulated blood samples. In addition to the proliferation rate, the production of the different cytokines (interferons, interleukins) by the lymphocytes after stimulation with mitogens can be determined in the supernatant of the cell suspension. Phagocytic activity of blood cell suspensions (mainly of monocytes [macrophages] and neutrophil leucocytes) can be determined after contact with an antigen (e.g., cymosan A) and the hereby induced release of reactive oxygen molecules. All tests mentioned so far can be performed on the cells of all animal species, so long as the laboratory facilities are available. Most studies so far have been done on animals living under constant laboratory conditions: the application of some of these methods under field conditions is, however, also possible. Natural killer (NK) cell activity is usually determined in v i m by bioassays using the destruction of certain tumor cell lines by the NK cells; the currently available tumor cell lines are, however, specific to only a few laboratory animal species. Although originally most functional tests in animals (especially mice and rats) used lymphocyte cultures received from the spleen of the animals, studies on human beings are naturally based on cells from the blood. The relationship between the activities of these cells of different origin is not clarified. It is, however, accepted that changes in the blood reflect changes in other immunological organs (such as spleen or lymph nodes). Of course, tests on blood cells have the great advantage that changes in immunological functions under different conditions of stress can be monitored on an individual level. These tests are therefore being used increasingly in animal research. For most of these tests, however, large numbers of blood cells

42

DIETRICH VON HOLST

are needed, which means that sacrifice is necessary for animals below the size of a rat. I N VIVO ANTIBODY PRODUCTION AFTER A N ANTIGEN CHALLENGE. In these tests the capacity of an intact organism to produce specific antibodies against antigens (e.g., sheep erythrocytes or keyhole limpet hemocyanin [KLH]) is determined. The specific antibody concentrations can be measured indirectly (by bioassays such as in sheep erythrocytes) or directly by immunological methods, when specific antibodies against the antibodies produced by the individual are available. For antibody determination very little blood is needed, and therefore these functional tests can be performed even in small animals. Since antibody concentrations do not change quickly due to handling processes, these tests can be used in laboratory and field conditions. To determine the time course of antibody production, several blood samples must usually be taken over a period of several weeks, which may raise some problems in field studies. 111. SOCIAL STRESS IN MAMMALS

A. INTRODUCTION While the research of Selye and his contemporaries was mostly concerned with the effects of physical stressors, after 1950 many ecologically oriented studies were published, indicating that social factors participate in pituitaryadrenocortical regulation. This work originated from the hypothesis, advanced by John J. Christian in 1950, that regulation of population densities of small mammals might be achieved by mechanisms intrinsic to the population itself: Increasing population density, according to his hypothesis, results in qualitative and quantitative changes in the behavior of the animals, which in turn stimulate pituitary-adrenocortical activity and decrease pituitarygonadal activity. As a consequence of these endocrine stress responses, the mortality of the animals increases and their natality decreases, thus counteracting the increase of population density. In his first paper (1950), Christian suggested adrenocortical exhaustion and, as a direct consequence, mortality as the major cause of cyclical fluctuations in the population numbers of small mammals. In subsequent years, however, it became evident that increased susceptibility to infectious and parasitic diseases, brought about by increased adrenocortical activity and decreased reproduction, is much more important (e.g., Christian, 1963, 1971, 1975; Christian et al., 1965). 1. Self-Regulation of Mammalian Populations

The growth of mammalian populations usually stops at a more or less stable level below the environmental capacity. Exceptions are the popula-

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43

tions of many small rodents of the arctic regions, which undergo marked fluctuations with a periodicity of 3-4 years. The causes of these cycles have interested ecologists since the beginning of this century (Elton, 1942). Some authors proposed extrinsic factors such as weather, food, predation, and diseases as the predominant or sole factors influencing animal numbers in natural populations. Without doubt all these extrinsic factors act on populations, either alone or in combination, and can cause a population decline or even an extinction in particular instances, but it is unclear whether they can limit population increases under natural conditions (Krebs and Myers, 1974). Therefore, alternative hypotheses were proposed, claiming population regulation by intrinsic factors. According to a hypothesis proposed by Chitty (1958,1960), the behavior of animals changes with density as a consequence of selection on genetically different behavioral types. Aggressive individuals might be at a selective advantage as the population increases. Aggressive behavior among individuals could then be the direct cause of the population decline, even though the exact reasons for the mortality during the decline are so far unknown. Several studies have demonstrated genetic changes associated with density changes (using mostly gel electrophoretic variants as markers), but the question still remains as to whether these genetic changes are the cause or the result of the demographic changes (Krebs, 1996; Krebs and Myers, 1974). The hypothesis that they are the cause has never gained wide acceptance. In contrast, from the outset, Christian’s hypothesis of a self-regulation of mammalian populations by social stress was a subject of wide debate (e.g., Krebs, 1996; Krebs and Myers, 1974), resulting in intensive stress research in the laboratory as well as in the field (mostly on voles, mice, rats, and rabbits). The strength of this concept is based on the results of experimental crowding of small mammal populations in the laboratory, which demonstrated all the changes in reproduction, growth, and mortality typically found in natural populations of high densities. The relationships between behavior, stress, and density under natural conditions, however, remain far from clear. This is for many different and often methodological reasons. In my opinion, the most important of these is the lack of detailed behavioral studies. This is not surprising in the case of the generally cryptic small mammals. Most studies have collected data only on density-dependent changes in agonistic behavior, and even then these data are often inferred indirectly by counting skin wounds on animals living in different population densities. Such an approach to the determination of relevant behavioral changes may be misleading, however, as some studies have demonstrated that no relationship exists between the number of wounds and population densities in rodents (e.g., Batzli and Pitelka, 1971; Christian, 1971; Krebs,

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1964). As a consequence, Christian (1963) introduced the term “social density” to explain discrepancies between animal numbers per unit space and stress responses. This term, however, is far from clear and may include any combination of aggressive behavior, social interactions, space, and number of individuals. In addition, population densities in the field have usually been estimated on the basis of trappings, which are subject to a great number of potential shortcomings, such as differences in their success rate, depending on the individuals’ trap experience, their social status, or their food situation (e.g., Krebs and Myers, 1974). Statements on population densities and changes therein are therefore affected by gross and possibly density-dependent errors. Furthermore, the indices used in the measurement of stress in individuals have usually been provided by data on adrenal weights or by other indirect measurements of adrenocortical activity, both of which give only rough indications of the endocrine state of the animals. Finally, data on the sympathetico-adrenomedullary activities are generally lacking. Thus, many contradictory conclusions on relationships between population densities and stress may have been based on inadequate measurements or interpretations of the data. For these reasons I will not go any further into the question of selfregulation of densities of individuals in mammals. In addition, several reviews on the causes of population cycles in small mammals have been written over the past 30 years and the understanding of these causes has not subsequently improved (e.g., Christian, 1978; Krebs, 1996; Myers et al., 1971; Nowell, 1980; Snyder, 1968; Watson and Moss, 1970).

2. Behavioral Stress Research in Biomedicine While initially ecological questions were central to research on stress, and factors relevant to populations such as fertility and mortality were investigated, since the 1960s, interest has shifted into the biomedical area. Three developments were responsible for this shift. 1. The development of modern techniques in hormone analysis, allowing the recording of endocrine stress reactions in humans and revealing the general correspondence between many different animal species including humans.

2. The proof that in animals, as well as in humans, psychological processes are decisive in the triggering of stress reactions. Psychosocial stressors are capable of long-term modification of both the sympatheticoadrenomedullary and the adrenocortical systems. 3. The epidemiological evidence that social challenges (life events) and individual traits in personality (e.g., type A personality, characterized

STRESS AND ITS RELEVANCE FOR ANIMAL BEHAVlOR

45

after Rosenman, 1986, by enhanced competitiveness, aggressiveness, impatience, and a chronic sense of time urgency) in humans are involved in the development or outbreak of specific diseases. Experimental investigations on animals are now primarily directed at recording physiological processes potentially involved in the development of disease in humans. The relevance of heart and circulatory diseases to our society has resulted in most research focusing on this area. In recent years, psychoneuroimmunological research has increasingly analyzed the importance of social stress reactions in the development of infectious diseases and tumors. Up to the present, research has concentrated almost entirely on laboratory animals kept in highly standardized conditions. It is only in recent years that a few ecologically oriented field research projects have reappeared, such as on dasyurid marsupials in Australia, rabbits in Europe and elsewhere, and mongooses, cheetahs, and several monkey species in Africa (see Section 111,BJ). Although the relevance of social stress for the “regulation” of populations is still controversial, nowadays it is generally accepted that social interactions or situations may result in strong stress responses in mammals (including humans), which in certain circumstances can greatly reduce the vitality and fertility of individuals and even lead to their death. Based on results of our studies on tree shrews and wild rabbits, I shall present in the following section my view of the relevance of recent stress research for the understanding of animal behavior. B. SOCIAL CHALLENGES THAT MAYRESULT IN STRESS RESPONSES 1. Social Conpict

a. Adrenocortical Activity and Fights. Fights over limited resources such as food, shelter, territories, and/or rank are among the most conspicuous behaviors in animal societies. All mammals react to acute stress, such as is certainly induced by a fight, by immediate activation of their sympatheticoadrenomedullary and hypothalamo-pituitary-adrenocortical systems. If such fights occur only infrequently, they have no consequences for the fertility and health of an individual: For instance, tree shrews can be subjected to a 30-min confrontation daily over a period of several weeks without suffering any detrimental physiological effects. If these daily fights occur more frequently, however, they can have grave detrimental effects on the fertility and health of the animal and even-as described at the beginning of this chapter in Antechinus-rapidly result in death. One or two aggressive conflicts per hour, not unusual in a natural environment, may be sufficient to have a damaging effect, as the adrenocortical hormones require one to

46

DIETRICH VON HOLST

several hours, depending on the species and the interaction, before they (and hence many other parameters) revert to their original concentrations. As is the case in Antechinus, and also in many other species with seasonally restricted reproduction, this phase of increased aggression is characterized by correspondingly increased adrenocortical activity in individuals, as was shown in the early 1960s by Bronson (1963, 1964) in his studies on natural populations of woodchucks (Marmora monax). The same is also true for other species (Saad and Bayle, 1985; Saboureau eta/., 1977; Schiml el al., 1996), as shown later in more detail, based on our investigations on European wild rabbits. European wild rabbits (0ryctoIagu.y cnniculus) live in small territorial groups of 1-3 males and approximately double the number of females. The territories are intensively defended by the males against external rivals during the reproductive period. Within the groups both sexes exhibit separate intrasexual linear ranking systems. Aggression in both sexes is particularly high at the beginning of the reproductive period, whereas for the rest of the year wild rabbits live together largely in peace (e.g., Brambell, 1944; Cowan, 1987; Lockley, 1961; Marsden and Holler, 1964; Myers and Poole, 1959; Mykytowycz, 1958; Southern, 1940). In order to gain information on the influence of agonistic behavior on physiological parameters, adult wild rabbits of a large natural population on the North Sea island of Sylt were investigated at the beginning of the reproductive period, when aggression was maximal (end of March), and again after the end of the reproductive period (October/November). At these two times of the year a total of 500 animals were shot between 18:OO and 19:OO hours over a period of 3 years, and within less than 3 min of their death blood samples were taken and different organs extracted for endocrinological and other investigations. As food availability, temperature, and day length were more or less equivalent during the two hunting seasons in spring and late autumn, differences in physiological stress parameters should be due largely to differences in aggression. As expected, we found greatly increased adrenocortical and sympathetico-adrenomedullary activities as well as many other changes in both sexes in spring, indicating high levels of stress. That is, under natural conditions, wild rabbits of both sexes show endocrine stress responses of the same magnitude as those demonstrated mainly in rodents under laboratory conditions (Fig. 13). We have closely investigated these relationships between social behavior and physiological stress responses over a period of 10 years in a population of wild rabbits living in a seminatural environment in an enclosure covering an area of approximately 22,000 m2 (e.g., Eisermann et a/., 1993; Kiinkele and von Holst, 1996). In these conditions, both sexes also start fighting

STRESS AND ITS RELEVANCE €OR ANIMAL BEHAVIOR

Adrenal gland

Corticosterone

400

I

(ng/ml serum)

Cholesterol 32

300

24

200

16

100

8

0

(nmollh adrenals)

(mall00 ml . serum)

PNMT activity

Adrenal medulla 500

(Cell nucleus volume in LP)

36

6.0

400

27

4.5

300

18

3.0

200

9

1.5

1

0 TH activity

7.5

47

Males Females

1no

1

(nmollh adrenals)

n

I

Males Females

Spring: 89 males, 76 females

Males Females

Autumn. 25 males, 20 females

FIG. 13. Indices of adrenocortical and sympathetico-adrenomedullary activities of adult male and female wild European rabbits in spring and autumn. The volume of the cell nuclei of adrenal medullary cells was histologically determined from 10 males and females in each season; their enlargement indicates a markedly increased adrenomedullary activity in spring. All data means ( + SEM). Significant differences between the spring and autumn data: * p < .05, * * p < .01.

heavily in spring and many injuries result (Fig. 14). In the males these fights are over territories and ranking positions and decrease only slightly in the course of the reproductive period. Females exhibit a bimodal pattern of aggression with maxima at the beginning and the end of the reproductive period. At the beginning of the reproductive period, fights usually occur over ranking positions within their groups as well as with external females, while in autumn most aggression is directed against young animals attempting to enter the groups (Fig. 15). In concert with these behavioral changes, there are marked changes in adrenocortical activity in both sexes. Conflict avoidance by the restriction of aggression mainly to the reproductive period is therefore a useful means of avoiding such stress reactions.

48 4.0

1

DIETRICH VON HOLST

[7 sexual following

Male sexual behavior (interactionshour)

urination

3.0 2.0 1.o

0.0 2.4

Fresh wounds (number)

1.8

1.2

0.6 0.0

250

Corticosterone

200 150 100

50 Dec

Feb

Apr

Jun

Aug

Oct

Dec

FIG. 14. Sexual behavior (medians), wounds, and serum corticosterone ( M _f SEM) of wild European rabbits, kept in a 22,000-m2 enclosure under natural conditions. Data from about 25 males and 50 females, observed over a 4-year period. Blood samples forcorticosterone determination were taken monthly from the animals 1 hr after maximal stimulation of their corticosterone release by an injection of ACTH. For behavior analysis each individual was observed about 8 hr per month. Unpublished data; after Schonheiter (1992).

b. Physical versus Psychosocial Processes. At the onset of mammalian stress research some authors suggested that intensive physical effort during fighting and wounds resulting from these fights might be the principal factor in adrenal enlargement; however, most studies found no correlation between injuries from fights and adrenal weight, and suggested psychological (psychosocial) factors as underlying causes (e.g., Christian, 1963). One of the first studies supporting this hypothesis was carried out by Davis and Christian (1957), who demonstrated a significant correlation between

STRESS AND ITS RELEVANCE FOR ANIMAL BEHAVIOR

49

Aggressive behavior (interactions per hour) 9.0

Males

6.0

3.0 0.0

Females

0adult group members

Jan

Mar

May

Jul

Sep

Nov

Jan

FIG. 15. Aggressive behavior of adult male and female wild European rabbits toward juveniles and adults (medians; for animal numbers see Fig. 14).

adrenal weight and social rank in groups of male house mice. Another study by Barnett (1958) found increased adrenal weights only in subordinate wild Norway rats (Rattus norvegicus) in a resident intruder paradigm. Furthermore, Bronson and ElefthCriou (1965b) showed that even the mere exposure without physical contact of subordinate mice to fighters-if they had previously been defeated in a confrontation-produces adrenocortical responses of the same magnitude as those observed in mice actually attacked and defeated. Similar findings have been obtained from research on Syrian hamsters (Mesocricetus auratus) (Human et al., 1992). Furthermore, research by Fokkema and Koolhaas, using chronically catheterized laboratory rats, showed that defeated males exhibited over twice the increase in blood pressure during brief dyadic encounters than their superior rivals (Fokkema, 1985; Fokkema and Koolhaas, 1985). If an animal had previously been defeated and was then threatened by exposure to the victor, while penned in a small wire mesh cage, the mere presence of the victor raised the former victim’s blood pressure to the same level as during the direct defeat. Extreme psychosocial stress can even rapidly cause death, as has been demonstrated in wild Norway rats (Barnett, 1958, 1964, 1988; Barnett et al., 1975), tree shrews (von Holst, 1972a,b, 1985a), rhesus monkeys (Hamil-

50

DIETRICH VON HOLST

ton and Chaddock, 1977), and humans (Stumpfe, 1973). Death in these cases is always associated with behavioral impairment, indicating a state of helplessness or loss of control, and extremely heightened adrenocortical activity (as shown in rats and tree shrews). An example of these mechanisms is given in the following section. c. Social Stress in Tree Shrews. Tree shrews (Tupaia belangeri, order Scandentia) are small diurnal mammals distributed throughout Southeast Asia. In the wild, tree shrews live in pairs in territories that they defend vigorously against intruders of their own sex. In the laboratory, adult males (and females) also immediately attack intruders of their own sex and normally defeat them within a few minutes. A short time after the fight, the winners show no further signs of arousal and pay virtually no attention to the defeated animals. The losers, in contrast, creep into any hiding place, which they leave only to eat and drink. During the following days, fights between the animals are extremely rare or nonexistent. Nevertheless, the losers die within a few days. Death is not a result of physical exertion during the fights, nor are wounds the cause of death, as the animals usually inflict only superficial scratches and bites on one another. Death is rather more the consequence of the continual presence of the winner, as was shown by the following experiments: An adult male was placed in the cage of a male conspecific (an experienced fighter), which usually immediately attacked the intruder and subdued him in less than 2 min. Afterward, both animals were separated either by a nontransparent partition or by a wire mesh partition, so that the loser could no longer be attacked but could continually see the threatening winner. Short fights were repeated every 1-3 days. Losers separated by a nontransparent partition from the winner recovered from the fights just as fast as the winners and did not die prematurely, even when they were subjected to short daily fights over weeks. The situation of the losers within sight of the winners, however, was completely different: As from the first subjugation, all the submissive animals sat in a corner of their part of the cage or in the sleeping box attached to the cage for practically the whole day, hardly responding to external stimuli. During confrontations, they did not even attempt to escape the attacks of the dominant animals, but usually suffered them without any attempt t o defend themselves or to flee. After the first subjugation their body weight decreased daily at an individually stable rate of 2-8% of their initial weight and all animals died within 2-20 days, if not separated earlier. As these results show, death of the submissive animals is not a direct result of the fights and their physiolgoical consequences, but is rather a result of central nervous (emotional) processes in the defeated animal, induced by the constant presence of the threatening winner.

STRESS A N D ITS RELEVANCE FOR ANIMAL BEHAVIOR

51

In all submissive animals dramatic stress responses were seen: Among other responses, serum glucocorticosteroid levels rose to more than six times their initial levels, serum concentrations of both thyroidal hormones (thyroxine and triiodothyronine) decreased to less than 40%, and those of testosterone to less than 10% within a few days. As shown by histological examinations, within a few days spermatogenesis ceased completely and all animals became sterile. In addition, a dramatic drop in the numbers of lymphocytes as well as of basophil and eosinophil granulocytes to less than 20% of their initial values indicates strong immunosuppression (von Holst, 1985a). The physiological cause of death differed among the animals. In animals dying within 8 days of the confrontation, urea nitrogen (and creatinine) levels in the serum rose to more than ten times their initial values, leading to death by uremia, while in those animals surviving longer, these increased only slightly. The cause of death in the latter is not known (von Holst, 1972a,b, 1985a). (Of course, the introduction of an individual into the cage, or “territory,” of an experienced fighter without any possibility of avoiding the rival is an unnatural and extremely stressful situation, especially in a territorial animal such as the tree shrew.) To obtain information on a less severe form of stress or even on adaptation to it, two male tree shrews unknown to each other were put together in a cage with two separate sleeping boxes, water bottles, and feeding dishes. In this situation the animals did not start to fight immediately, but first hesitantly explored and marked the cage. Slight fights usually began within the first few hours, leading in most cases to clear dominance relationships within 1-3 days. While the behavior of all males was more or less comparable before the fights, it changed considerably after the dominance relationship was established, depending on the social position of the animals. Although both animals continued to live together in the relatively small cage, the winners more or less ignored the losers and attacks on the latter were rare or even completely absent. The losers, on the other hand, drastically moderated their behavior. On the basis of behavioral differences, two types of losers could be distinguished: subdominant and submissive animals. Submissive animals corresponded to those of the first experiment: They crouched in a corner of the cage or in a sleeping box and left their hiding place only to drink and eat hastily. They even tolerated the infrequent attacks of the winners without any attempts to defend themselves or to flee. They ceased grooming completely and their fur became rough and dirty. To the human observer they gave an apathetic or depressive impression. All submissive animals died within 2 weeks.

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DIETRICH VON HOLST

Sudominant animals, in contrast, showed greatly increased locomotor activity, watching the movements of the dominants continually and attempting to avoid possible confrontations by giving way or fleeing. If a confrontation could not be avoided, they even defended themselves. Under these conditions, subdominants were capable of living in the presence of the dominants for weeks, albeit with a reduced freedom to move. In concert with these behavioral changes, body weights and many different physiological parameters in the animals changed drastically. In accordance with the stress concept, the confrontation had the immediate effect of activating the sympathetico-adrenomedullary and the pituitaryadrenocortical systems in both rivals: Accordingly, the serum concentrations of catecholamines and glucocorticosteroids as well as the heart rates of all animals were greatly increased (Fig. 16). As soon as the dominance relationship was clearly recognizable in the behavior of the tree shrews, all stress reactions in the dominant animals disappeared in spite of continued occasional fights. Moreover, glucocorticosteroid concentrations in the blood dropped marginally below the original values, their body weights increased, and their gonadal functions improved: After about 3 weeks, the dominant animals were significantly heavier than before the confrontation and serum testosterone concentrations had increased by approximately 100% (Figs. 17 and 18).

1

Cortisol

6.0

Epinephrine

Norepinephrine

(ng/ml serum)

I

20

5.0

8.0

15

4.0

6.0

10

3.0

4.0

5

D

SD

SM

2.0

D

SD

SM

(nglml serum)

10.0

7

D

SD

SM

FIG. 16. Serum concentrations ( M -+ SEM) of cortisol and catecholamines o f , minant (D), subdominant (SD), and submissive (SM) tree shrews 8 days before (light bars) and two days after (cross-hatched bars) start of a confrontation. Blood samples were always taken 2 hr before the activity period. Cortisol data (20-30 animals per group) are initial values, catecholamine data (10 animals per group) are BSCT values. Significant differences to initial values: * p < .05; * * p < .01; ***p < ,001.

53

STRESS AND ITS RELEVANCE FOR ANIMAL BEHAVIOR

Controls 40

cortisol (nglml serum)

20 301

B 2 10 20

!

Dominants

B 2 10 20

Subdominants

B 2 10 20

Submissives

6 2 10 20

FIG. 17. Serum concentrations (M t SEM) of cortisol and testosterone of male tree shrews before (B) and at different days after start of the experiment. 20 controls remained in their living rooms, but were treated as the other animals (handling for weighting and blood sampling) and 25-40 males per group that confronted each other. See text for further details.

Subdominants Submissives

0

6

12

18

24

Days after start of experiment FIG. 18. Body weight changes of the animals after the start of the experiment ( M for animal numbers see Fig. 17. Initial body weights of the males: 190-220 g.

?

SEM):

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DIETRICH VON HOLST

Defeated tree shrews were characterized by reduced body weights and a number of hormonal and other physiological changes; among other effects, the serum concentrations of testosterone, insulin, and thyroid hormones decreased drastically. Overall, these effects were the same in subdominant and submissive animals, differing only in their extent (Fig. 19). Qualitative differences were, however, to be found in their sympatheticoadrenomedullary and pituitary-adrenocortical systems. Active and passive stress reponses in tree shrews. In dominant animals, the activity of the sympathetico-adrenomedullary system reverted back to Testosterone 9.0

30

8.0

20

7.0

10

6.0

0

5.0

1.0

1

Triiodthyronine (ng/ml serum)

36

j

Protein (g/100mlserum)

4

1

Kidneys

*

Insulin (ng/ml serum)

150

0.8

27

0.6

18

100

0.4

9

75

0

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0.2

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Hemoglobin

20

1 (g/lDo

ml blood) !joo

18

400

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300

14

200

3I 7L

c

D SD SM

C

D SD SM

ion

1 i

1

Triglycerides (mg/l00 ml serum) I

I

Epididymides ;mg)

C

D SD SM

FIG. 19. Several physiological measures and organ weights ( M ? SEM) of controls and experimental male tree shrews 10 days after start of the experiment. Animal numbers as in Fig. 17. Significant differences to controls: * p < .05: **p < .01; ***p < ,001. C: controls: D: dominants: SD: subdominants: SM: submissives.

STRESS AND ITS RELEVANCE FOR ANIMAL BEHAVIOR

55

its original state after the dominance relationship had been established, and the serum catecholamine levels even showed a tendency to levels lower than those before the confrontation. In contrast, subdominant animals exhibited a continually increased sympathetico-adrenomedullary activity. While, correspondingly, the heart rate in the dominant animals returned to normal once dominance was established, it remained high in subdominant animals, not only throughout the day (when an attack by the dominant animal was always possible), but also at night when they were sleeping in their own sleeping box. Nocturnal heart rates in subdominant animals were almost equivalent to diurnal values, thus abolishing the original day-night rhythm (Fig. 20). The tyrosine hydroxylase activity in their adrenal glands-an index of sympathetic activity-also increased on average by loo%, in comparison to dominant or control animals (Fig. 21). Contrary to the Selyean concept, the serum levels of the glucocorticosteroids decreased to initial levels; their adrenocortical secretory capacity, however,

500

d

E6=

n

u)

300 200

100

iii

500

a E u) c

400

al

m

E 3 L

g 2

5

0)

72

I

Male dominant

400

300 200 100 500

1

Male submissive

400 300 200 100

Days before and

I

after start of confrontation

FIG. 20. Heart rates of a dominant, a subdominant, and a submissive male tree shrew before and after the start of the confrontation.

56

DIETRICH VON HOLST Cortisol

40

(nglmlserum) *** I

60

Corticosterone

Adrenal weight

**

***

nglrnl serum)

I

I

30

45

20

30

32

10

15

28

0

0 Urea nitrogen

100

(mg/l00 ml serum)

80

***

I

60 40 20 0

36

24 irosine hydroxylase

10.0

:nmol/h adrenal)

PNMT

* I

adrenal)

7.5

2.4

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0.0

0.0 Eosinophils

Lymphocytes

Erythroblasts

2800

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540

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180

0

C

D SD SM

0

*

(n/ pl blood)

C

D SD SM

0

C

D SD SM

Data 10 days after start of experiment

FIG.21. Several measures of the adrenocortical and adrenomedullary systems as well as some blood cell numbers ( M i SEM) of controls and confronted male tree shrews 10 days after start of the experiment. Animal numbers and abbreviations as in Figs. 17 and 19. Significant differences to controls: * p < .05: **p < .01: ***p < ,001. See text for further details.

increased to the same level as in submissives, indicating a heightened cortisol release and elimination under active stress (Figs. 17, 21, and 22). Submissive animals exhibited the opposite reactions: a tendency to a decreased sympathetico-adrenomedullary activity (Fig. 21), as indicated by their lowered adrenal tyrosine hydroxylase activities; and a substantial increase in their serum levels of glucocorticosteroids, as well as of their adrenal capacities (Figs. 17, 21, and 22), which was probably responsible

STRESS AND ITS RELEVANCE FOR ANIMAL BEHAVIOR

***

57

*** .L

D

SD

SM

D

SD

SM

FIG.22. Serum cortisol levels ( M t SEM) 15 min after start of blood sampling challenge tests of dominants (n = 11). subdominants (n = 11) and submissives (n = 6 ) before (left) and 10 days after (right) start of the confrontation. Significant differences: * p < .05: ***p < .001. Note the significantly lower initial values of the males that became submissive during the confrontation.

for the increased loss of muscle and adipose tissue, leading to a dramatic loss of weight averaging 5% daily (von Holst, 1986a, 1994; Stohr, 1986). Interestingly, the adrenal capacities of prospective submissive animals were significantly lower before the confrontation than those of the other two groups (Fig. 22). Confrontation also triggered marked immunological changes: No significant change was observed in leucocyte numbers and subsets in the blood of dominant and subdominant animals. In contrast to dominant animals, however, the efficiency of lymphocytes and phagocytes in the subdominant animals was clearly reduced (Fig. 23). In submissive animals, substantial changes were also found in those types of leucocytes indicative of strong immune suppression (e.g., Fig. 21: lymphocytes and eosinophil granulocytes). Accordingly, the proliferation capacity of their lymphocytes was reduced to less than 20% of original values following a 10-day confrontation period. Subordination and presence of the dominant animal therefore affected the immune system in subdominant and submissive animals. These effects were qualitatively equivalent in both categories of animals as far as their inhibiting effect on proliferation of lymphocytes was concerned, but different regarding the distribution of the different types of leucocytes in the blood: General statements on the possible qualitative differences in immunological reactions in these ethologically and physiologically distinct subordinates are not yet possible based on available data.

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DIETRICH VON HOLST

Before

After

Before

After

FIG.23. In vitro lymphocyte proliferation (LP) after stimulation with the mitogen concanavalin A (Con A) and in vitro phagocytosis of monocytes and granulocytes ( M ? SEM) of 16 subdominant male tree shrews before and 10 days after start of the confrontation (for details see Section 11,C: direct assessment of immunological parameters). While their lymphocyte numbers during the confrontation were not different from initial values, their proliferation capacity decreased markedly ( p < ,001).

d. Assessment of Dominance between Rivals. As the results of research on tree shrews show, prospective winners and losers exhibited differences in body weights and cortisol values after only 2 days (Figs. 17 and 18), even though it was not usually possible to predict the outcome of the confrontation based on the animals’ behavior at this point. Similiar results were found in domestic guinea pigs (Sachser and Lick, 1989): At the age of 30 days, the authors removed juvenile males from their breeding colonies and housed each of them with a female in a 1-m2 enclosure. At the age of about 8 months, two males were confronted by removing the partition between their enclosures. In all cases these confrontations escalated into fights between the two opponents, which declined to low levels after the first day. However, dominance relationships between the rivals could not be distinguished before day 4 of confrontation. Nevertheless, at day 2-3, prospective winners and losers already differed significantly in nearly all physiological parameters measured: Prospective losers exhibited a higher loss of body weight, their serum concentrations of cortisol and catecholamines were two to three times higher than those of prospective winners, while their testosterone levels decreased to about 30% of their original value. Commencing with the fourth day of controntation, the physiological changes became so dramatic that the losers died within a few days. As these data show, the outcome of the agonistic encounters could be

STRESS AND ITS RELEVANCE FOR ANIMAL BEHAVIOR

59

confidently predicted from physiological data 1-2 days before it could be derived from the behavior of the individuals. These results of research on tree shrews and guinea pigs demonstrate the animals’ capability of predicting the outcome of a confrontation while it is taking place and of producing the corresponding physiological reactions. How quickly endocrine changes occur during the changing assessment of the situation by the animals is demonstrated by Schuurman’s results (1981) with laboratory rats, which had been provided with chronic jugular vein cannulae for frequent blood sampling. Schuurman introduced a male rat into the home cage of an aggressive male conspecific. At first both animals were separated by a wooden partition, which was removed after a habituation period of 2.5 hr for a l-hr confrontation. During the first quarter of the confrontation fierce fighting took place, but with no apparent dominance relationship between the two rivals: Both showed offensive as well as defensive behavior, and their plasma corticosterone levels rose to about five times their initial values. After about 15 min a dominance relationship developed, which resulted in distinctly different adrenocortical responses in both animals: Although the victors continued offensive fighting, their plasma corticosterone levels declined. On the other hand, plasma corticosterone levels of losers continued to rise and the animals exhibited much defensive and submissive behavior, but no longer any offensive behavior. At the end of the encounter, plasma corticosterone level in losers was more than twice the level found in victors. Once a dominance relationship has been established in mammals, fights between rivals usually decrease. Nevertheless, as shown for tree shrews (see earlier discussion), the permanent presence of a dominant rival can cause dramatic stress responses in subordinate animals and ultimately even lead to their death within a few days. In spite of these highly negative consequences of subordination, fights for dominance are often astonishingly slight or even completely absent in tree shrews. This is apparently due to the fact that male tree shrews are able to recognize the potential “strength” or “dominance” of a rival before the first physical interaction, as is shown by the following experiments. Two animals were transferred to an experimental room and housed in one cage, but separated for 20 days by a wooden partition. Before the transfer into the experimental room, and on days 10 and 20, blood samples were taken from all animals for the determination of several endocrine and immunological parameters. On days 21-23, the two animals confronted each other daily for 10 min, while the rest of the time they were separated by the wooden partition. Depending on the results of these confrontations, the animals were designated prospective “dominants” and “subordinates.”

60

DIETRICH VON HOLST

From the first day in the experimental cage both animals apparently recognized the presence of the rival: They sniffed intensively at the wooden partition and marked it. Animals that later turned out to be subordinate in the confrontation seemed more alert and exhibited more locomotor activity, as was also evident from their slightly decreased daily resting times compared to their prospective dominant rivals. While few overt behavioral differences were observed between the two groups, there were significant immunomodulatory effects (Fig. 24). These effects were in opposite directions in the animals of the two groups: Tree shrews that later became subordinate showed indications of an immunosuppression, while in prospective dominants the activity of the immune system improved. In contrast to these immunomodulatory effects, we found only very slight changes in adrenocortical or sympathetico-adrenomedullary activities. Amazingly, the immunosuppressive effects in subordinate animals before any physical contact between the rivals were of the same magnitude as those in direct confrontations with constant physical presence of the rival (Fig. 25). To exclude the possibility that these opposite immunomodulatory reactions were consequences of differences in the constitution of the rivals, the

200

Lymphocyte proliferation

Interleukin 1

150

I 3

100

7

50

m ._ c ._ ._ c

o

-

-

0

Interferon gamma

400

c ._

***

111 300

5 200 0

100

0

H

10 20

H

10 20

Dominants Subdominants

H

10 20

H

10 20

Dominants Subdominants

FIG.24. Several immunological measures taken from male tree shrews housed in the same room with a potential rival ( M 2 SEM); data 8 days before (H) as well as 10 and 20 days after transfer into the experimental room: 20 prospective dominant and 20 prospective subdominant males. Significant differences: **p < .01; ***p < ,001. See text for further details.

STRESS AND ITS RELEVANCE FOR ANIMAL BEHAVIOR

Lymphocyte proliferation 120 -

80 40

(lo3 cpm/lo5 cells)

+ r *

6.0

-

0-

3.0

10

Home

2 12 Exp

2 12 Conf

00

1

61

Interferon gamma

10 Home

2

12 Exp

2 12 Cod

Experimental conditions and days of blood sampling FIG.25. In v i m lymphocyte proliferation after stimulation with the mitogen Con A and interferon production of 20 male subdominant tree shrews in their home room (Home: days 0 and lo), 2 and 12 days after transfer into an experimental room with a rival (Exp), and 2 and 12 days after start of the confrontation (Conf); significant differences to initial values (= Home 10): * p < .05: * * p < .01. See text for further details. Unpublished data from Vitek (1996).

tests were repeated 3 months later with all experimental animals in different combinations: Previous dominants were confronted with previous dominants, and previous subordinates with previous subordinates. In all cases, the animals formed new dominance relationships, with identical immunological consequences for the final winner and loser, just as in the first experiment. Thus, the differences in these immunomodulatory reactions of prospective dominants and subordinates were only dependent on the situation, that is, on the “quality” of the prospective rival. As preliminary data show, male tree shrews are capable of obtaining information on the potential strength of a rival through olfactory signals, which originate from urine and glandular areas used for territorial marking. It is entirely unknown, however, what determines this olfactorily communicated “strength” of a rival. As our experiments with tree shrews have shown, body size or body weight are of no predictive value for the outcome of a fight between unknown rivals; the same is true for differences between animals in testosterone serum concentrations or excretion rates.

2. Dominance Relations and Stress Responses in Other Mammals a. Pituitary-Adrenocortical and Sympathetico-Adrenomedullary System. As shown in the previous section, subordinate tree shrews exhibit behaviorally dependent differences in their stress responses, with only the behaviorally inactive and apathetic submissive animals corresponding physiologically to the Selyean concept. These findings strongly support the

62

DIETRICH VON HOLST

concept of Henry and associates, which proposes that mammals exhibit two types of stress with differing neuroendocrine responses and differing ultimate disease states (e.g.? Ely and Henry, 1978; Henry and Meehan, 1981; Henry and Stephens, 1977): Cannon’s flight or fight response, accompanied by heightened sympathetico-adrenomedullary activity, which may eventually lead to cardiovascular deterioration (“active stress”); and Selye’s stress response, characterized in particular by heightened pituitaryadrenocortical activity (“passive stress”). In species such as the tree shrews, which live in territorial pairs in natural conditions, adult individuals of the same sex cannot be kept together for any length of time, since, depending on their passive or active coping behavior, this may result in the death of subordinate animals within a few days to weeks. For this reason, our confrontation experiments with males were always terminated after 3 weeks. Following this time span, subdominant animals also succumbed to apparent signs of coronary insufficiency. The situation is different in animals that normally live in groups with idiosyncratic yet relatively stable social roles. In most species, hierarchical systems with dominant and subordinate animals develop in such a way that subordinates can usually live in the presence of dominants with only very slight or no signs of stress at all. In situations of continuing conflict that result from social instability within the group, life may be stressful for subordinates as well as for dominants, even in the absence of overt aggression, as was shown by Henry and associates in their extensive studies on laboratory mice (e.g., Ely, 1981; Ely and Henry, 1978; Henry and StephensLarson, 1985). After weeks of isolation, the authors introduced several male and female laboratory mice into colony cages consisting of small boxes connected by tubular runways and designed to induce frequent social interactions. This always resulted in intensive fights among the males, which led within a few weeks to a relatively stable social system with one dominant male and several subordinates in each colony. During the time of colony formation the physiological responses of all animals were typical of general nonspecific arousal, with increased activities of their adrenocortical and adrenomedullary systems. Once the social hierarchy was established, overt aggression and strong stress responses decreased. Nevertheless, the behavior and physiological response patterns of dominants and subordinates differed: Dominant animals were significantly more active and vigilant than the subordinate animals; they constantly visited the boxes of the cage system and tried to exert control over all other animals. The subordinate males, on the other hand, were restricted to very small areas of the population cage and showed behavioral withdrawal, which according to the authors minimized aggressive encounters with the dominants. As demonstrated previously by other au-

STRESS A N D ITS RELEVANCE FOR ANIMAL BEHAVIOR

63

thors in crowding experiments on mice and other species (e.g., Bronson, 1973; Chapman et al., 1969; Louch and Higginbotham, 1967; Popova and Naumenko, 1972), even in this more naturalistic experimental design subordinate mice maintained slightly elevated adrenocortical activities. In contrast, dominant mice developed heightened adrenomedullary activities and a moderate hypertension, which apparently permitted the animals to maintain the vigilant patrolling behavior necessary to the control and stabilization of the colony. Testosterone levels in subordinate mice decreased to about 30% of that of controls kept in standard laboratory cages, while the levels in the dominants did not differ from those in controls, in spite of their heightened sympathetico-adrenomedullary stress responses. All these physiological response patterns typical of dominants and subordinates could be reversed by experimentally induced changes of the social positions of individuals (e.g., removal of dominant males). When the animals were separated before 5 months of social conflict, all these stress values returned more or less to those of control animals. If, however, the situation persisted, within a few months pathophysiological changes began to develop in all animals: Fixed hypertension and increased PNMT values, increased heart weights, and histopathological deterioration, such as interstitial nephritis, aortic arteriosclerosis, intramural coronary arteriosclerosis, and myocardial fibrosis developed, which after 9 months of colony living remained irreversible even after months of isolation, and led to the premature death of many individuals (Henry and StephensLarson, 1985; Henry et al., 1971; Vander et al., 1978). As shown by these data, in mice kept in complex population conditions, dominant individuals show predominantly active stress responses, while the subordinate animals show slight passive stress responses. These results are in contrast to our data on dominant tree shrews, which show no sympathetico-adrenomedullary activation. However, this is probably due to the striking differences in the social organization of these two species. In tree shrews, which live in territorial pairs in the wild, a defeated rival is apparently no longer threatening, and therefore, even in small cages, does not elicit any behavioral or physiological arousal. In contrast to dominant mice, dominant tree shrews even show slightly decreased adrenocortical activity. Lundberg and Frankenhaeuser (1980) particularly emphasize this “bidirectional nature of the pituitary-adrenal response.” In their study on humans, adrenocortical suppression was demonstrated in conditions characterized by high levels of control and predictability. According to the authors, this is consistent with the conclusion by Levine and associates (1979), that reinforcement is an important cognitive factor mediating suppression of the adrenocortical system. This also seems to be the case in dominant tree shrews. Wild mice in natural conditions, however, live in

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DIETRICH VON HOLS’I

groups containing one dominant and many subordinate males. The dominant male must constantly control and keep in check the subordinate males and this apparently necessitates heightened adrenomedullary activity (Crowcroft, 1955; Lloyd, 1973). Thus, it is not the social position that determines the physiological state of an animal, but the effort of achieving and maintaining the status (i.e., whether the position is endangered or not). This has been clearly demonstrated by Lundberg and Frankenhaeuser (1980) in the experiments on humans, which demonstrate that pituitary-adrenocortical activation is associated with negative feelings of distress, and sympathetico-adrenomedullary activation with feelings of alertness and a readiness to act. A similar conclusion was drawn by Ursin and associates (1978), who identified a “cortisol factor” and a “catecholamine factor” in their analysis of data collected in a study of trainee parachutists. It has to be emphasized, however, that in these and many similar studies with mice chronic high levels of stress were induced by using males that were housed singly after weaning for many weeks before colony formation (“unstable social situation”). In contrast to these socially deprived individuals, mice that had been raised in groups were able to live together in stable social groups without overt aggression and stress responses (Fig. 26). As pointed out already (Section II,B,3,c), these results again demonstrate the crucial role of social experience after weaning for an animal to cope with social conflict in a more or less stress-free way. These results lead to the conclusion that, depending on species, group composition, and group stability, dominant individuals may be characterized by lower or higher hypophyseo-adrenocortical and/or sympatheticoadrenomedullary activities than their subordinate conspecifics. Our knowledge of the effects of social positions and behavior on differing endocrine stress responses in mammalian species, however, is scanty, as most researchers have chosen to work with one system only (usually the adrenocortical). Accordingly, countless publications on many different species have demonstrated that repeated subjugations or a subordinate social position result in increased adrenocortical and decreased gonadal activities. However, little is known about the species-specific and context-dependent positive or negative consequences of dominant positions for the sympatheticoadrenomedullary system (including heart rate and blood pressure). An exception to this is provided by several studies on rats and monkeys (mainly on the effects of psychosocial stress on blood pressure) and, as mentioned earlier, to some extent also by studies on wild rabbits, tree shrews, and guinea pigs. An overall analysis of our wild rabbits housed in large enclosures revealed a distinct relationship between the social rank of males and females and

STRESS AND ITS RELEVANCE FOR ANIMAL BEHAVIOR

Systolic blood pressure I

65

Tyrosine hydroxylase

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(oh of controls)

,

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Renin

Corticosterone

1 (nglml plasma)

1(nglml plasma x hr) 10

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FIG.26. Effects of chronic psychosocial stress on several physiological measurements of male mice. Systolic blood pressure, adrenal tyrosine hydroxylase activity, and plasma values of renin and corticosterone of mice housed singly (light bars) in small cages as well as of mice housed in mixed-sex groups for 6 months. Significant differences between animals housed in unstable (cross-hatched bars) social groups and the other housing conditions (stable groupsstriped bars) are indicated: **, < .01; ***, < .001. See text for further details. Adapted from Henry (1992). with kind permission from Transaction Publishers, New Brunswick.

their adrenocortical activities during the reproductive season (Fig. 27). At the group level, however, marked differences existed (Fig. 28): Generally, males living without rivals within their groups had the lowest corticosterone challenge test values. Depending on the number of subordinate rivals in a territory (and hence the social instability within their groups), the adrenocortical activities of dominant males rose to the values of subordinate males; thus no rank-related differences were evident. Furthermore, the number of females living within a group had some influence on the adrenocortical activities, especially of the dominant males. Dominant females, in contrast, had generally the lowest adrenocortical activities of all animals within their groups, although their activities differed to some extent among the groups. Taken together, these data indicate that the adrenocortical activities of dominant wild rabbits depend on the composition and stability of their

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DIETRICH VON HOLST

Corticosterone values

Heart rates (bpm)

I

250

240

200

200 monthly mean)

1

2

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>3

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Social rank of animals

FIG.27. Relationship between social ranks, adrenocortical activities, and heart rates of wild European rabbits, which lived in large field enclosures. Corticosterone measures of four reproductive seasons from animals from a 22,000-m2 field enclosure. Blood samples for hormone analysis were taken from all animals once every month 1 hr after maximal stimulation of their corticosterone release by an injection of ACTH; corticosterone values of the males are absolute serum concentrations; since corticosterone measures of females show marked variations during the reproductive season (see Fig. 14). their values are given as deviations from the monthly mean of all females. All data are means ( 2 SEM) of the mean of 4-6 values of each animal from one reproductive season. Heart rates of the animals were determined telemetrically by transmitters implanted ( M 2 SEM of 30-60 days of measurement per individual) in animals that lived in about 150-m2 enclosures. Social ranks were determined by behavioral observations (> 40 hr per animal). Animal numbers are shown at the bottom of the bars.

groups, which may explain that, especially under unstable social conditions in wild rabbits as well as in other species, no rank-dependent differences in adrenocortical activities are found. The heart rate in dominant individuals of both sexes, living in smaller enclosures (about 150 m2) in groups of 2-3 males and as many females, was also lower compared to that in subordinates, and every change of rank resulted in a corresponding change of heart rate (Fig. 27; see also Eisermann, 1992). Most stress research has been performed on laboratory rats. Rats are highly social and intensive fighting is present only for as long as the animals are unfamiliar with each other and no stable hierarchies have developed

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Two or more females per group 1-male groups

5

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FIG. 28. Relationship between social ranks and corticosterone challenge values of wild European rabbits in relation to their group composition. Data from animals living without male rivals in their territories are shown in top and bottom parts. See Fig. 27 and text for further details.

(Barnett, 1975; Calhoun, 1963). During this period all animals show typical stress responses including elevated blood pressure. Once a stable hierarchy with a dominant male has developed, fighting more or less ceases and blood pressure decreases (Henry et al., 1993). Nevertheless, differences between dominants and subordinates persist, as has been shown by Dijkstra and colleagues (1992) in male Wistar rats housed in mixed-sex groups in complex colony cages. Compared to pairwise housed controls, the dominant animals exhibited significantly heightened testosterone plasma levels, while those of the subordinates were in the range of the controls; corticosterone plasma levels were increased in both ranks, but in subordinates the increase was about 150%, three times higher than in dominants. If the composition of a mixed-sex group of rats is changed regularly, thus preventing the establishment of a hierarchical social system, this persistent stress can result in a progressive rise of systolic blood pressure over a period of months (Fig. 29). There are, however, marked differences between different strains of rats in their cardiovascular response to chronic

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DIETRICH VON HOLST

h

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FIG. 29. Effects of housing conditions on the systolic blood pressure of male Long-Evans rats. Significant differences to initial levels: *p < .OS; **p < .01; ***p < ,001 (see text for further details). Adapted from Henry er af. (1995). with kind permission from LippincottRaven Publishers, Philadelphia.

stress, which is found to correlate with their aggressiveness: The very aggressive Long-Evans rats show a great increase of blood pressure, the less aggressive Sprague-Dawley rat, a modest increase, and no change is observed in the peaceable Wistar-Kyoto (hyperactive) strain (Henry ef al., 1993). The same relationships found between aggressive behavior and blood pressure responses also seem to apply to individual differences within a strain (Bohus et al., 1987; Fokkema, 1985, Fokkema and Koolhaas, 1985; Fokkema et al., 1988; 1995). These authors tested the aggressive behavior of male laboratory rats (strain TMD-S3) in several resident-intruder tests. Following these precolony tests, 10 males together with 5 sterilized females were transferred into a large colony cage, which was fitted with small boxes in which the animals could find shelter. Cannulas were attached to most males for intermittent direct blood pressure measurements and blood sampling. In this seminatural situation, the levels of aggressive behavior in individuals correlated with those levels determined in the precolony resident-intruder tests: The more aggressive in the precolony tests the more competitive were the rats during confrontations in the colony, whether they became dominant animals exhibiting offensive behavior, or subdominant animals exhibiting defensive behavior or flight. However, blood pressure as well as plasma corticosterone levels in dominant animals tended to be lower than those in equally competitive subdominant animals.

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In contrast to the actively competing dominant and subdominant animals, lower blood pressure was observed in nonaggressive rats (“subordinates”) as well as in formerly dominant animals that had lost their position after severe defeat (“outcasts”) and made no further attempts to defend themselves. Barnett (1975) termed such frequently defeated males “omegas”; behaviorally they correspond to submissive tree shrews: They are inactive, socially withdrawn, and die after a few weeks without having suffered any wounds. This was demonstrated by the study by Fokkema (1985), mentioned earlier, as well as in a life span study of laboratory rats under seminatural conditions (Blanchard et al., 1988). An even more dramatic decline in blood pressure in the course of chronic conflict has been reported by Adams and Blizard (1987) in S/JR rats (a salt-sensitive strain), which were repeatedly exposed to the presence of a trained fighter rat (Long-Evans) and subjugated by it. Thus, chronic high blood pressure is the result of continuous attempts of socially active animals to adapt to an environment that is both threatening and demanding; on the other hand, loss of control (as also seen in submissive tree shrews) results in decreased blood pressure. The relevance of the stability of a social position for the physiological status of an animal is also evident from studies by Russian scientists on hamadryas baboons (Papio hamadryas) and rhesus monkeys (Macaca mulatfa).The heart rates of these monkeys were recorded telemetrically using a transmitter placed on the monkeys’ backs in the pocket of a jacket. Dominant males of both species, kept in groups of 2 males and 1-2 females, always had lower heart rates than the subordinates and these differences could be reversed after experimentally induced changes of the social positions of the individuals. The higher heart rates in subordinate monkeys were not related to increased locomotor activity, but, according to the authors, reflected the degree of emotional tension (Cherkovich and Tatoyan, 1973). Any challenge to the stable position of a dominant male results in dramatic cardiovascular stress responses. An example of this was shown in dominant hamadryas baboons, which had lived for months with a harem of several females and their young. If the dominant male was not allowed access to his former group, which now lived with a rival male in an adjacent enclosure, the former harem owner at first tried fiercely to attack the new harem owner through the bars again and again. This behavior, however, ceased after a few weeks. Nevertheless, over a period of several months, hypertension, coronary insufficiency, myocardial infarction, and other somatic diseases developed, leading to the death of many former harem owners (Lapin and Cherkovich, 1971).

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Social stress as a determining factor in coronary artery disease has also been implicated by Hamm and associates (1983) in Java monkeys (Macaca fascicularis), which were kept in groups of 5 males for nearly 2 years. Subordinate individuals in stable groups had significantly heavier adrenal glands and more extensive coronary artery stenosis than did their dominant counterparts. In repeatedly reorganized “unstable groups,” dominant males developed greater blood pressure and arteriosclerosis of coronary arteries, but this occurred only in the more aggressive and highly competitive individuals, which retained dominant status over the whole study (Kaplan et al., 1982, Manuck et af., 1983; Shively and Kaplan, 1984). Subsequent studies have demonstrated that individuals of both sexes, exhibiting a heightened cardiac response to a standard stressor (threat of capture), probably sympathetic in origin, also develop the most extensive coronary lesions (Manuck et al., 1986; 1989, 1995). This is consistent with observations in humans on relationships between behavioral reactivity (“type A behavior”), sympathetic arousal, and cardiovascular disease (Dembroski et al., 1983; Houston, 1992). Similar results have also been found in field studies on monkeys. Dominant male olive baboons (Pupio anubis) living in stable groups in the East African savannah exhibited lower “initial” levels of cortisol (10 min after darting), but responded relatively faster and more strongly following stress due to anesthesia. In this way, differences between high- and low-ranking males were compensated (Sapolsky, 1982), as with findings in rhesus monkeys (Sassenrath, 1970). Additionally, subordinate olive baboons were less responsive to dexamethasone-induced cortisol suppression than were dominant males, which was due to a selective decrease of glucocorticosteroid receptors in the hippocampus (Brooke et al., 1994; Sapolsky, 1983, 1990). Since dexamethasone resistance is a typical indicator for reactive depression in humans, these results may indicate a similar state in animals with a long history of social instability and lack of control. Finally, there were indications of cardiovascular pathologies following prolonged periods of subordination. Compared to dominant individuals, subordinate animals exhibited significant reductions in high-density cholesterol, which can promote arteriosclerosis and coronary heart disease (Sapolsky and Mott, 1987). In studies on male rhesus monkeys, placed in groups of four in large cages for several months, Hamilton and Chaddock (1977) even demonstrated death of apathetic (“submissive”) individuals after a rank order had developed among the males. In contrast to all other males, the two animals concerned neither battled for dominance nor did they flee from attack. In general they crouched in the corner of the cage and, although attacked on occasion, they were not grossly maltreated. They appeared

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helpless in dealing with the situation and did not display the “fight or flight” syndrome. In New World monkeys, rank-related differences have also been demonstrated. Dominant squirrel monkeys (Saimiri sciureus) in stable heterosexual colonies as well as in newly formed groups have lower plasma cortisol levels than subordinate individuals (Candland and Leshner, 1974; Manogue et al., 1975). Furthermore, during group formation, concentrations of urinary catecholamines increased only in the midranking individuals who successfully fought to maintain their status, but decreased in those animals who were unsuccessful and became further subordinated. Similar endocrine findings have also been described for the African talapoin (Miopifhecustalapoin) (Eberhart et al., 1983; 1985). Sandra Vellucci (1990) manipulated the behavior of dominant and subordinate male talapoin monkeys with drugs that are used in the treatment of human psychiatric disorders, such as anxiety and depression. In order to maximize the number of interactions between dominant and subordinate animals, groups of males were allowed to interact daily for a period of 50 min with females. This led to intense fights for control between dominant individuals, while subordinate individuals retreated, huddled in corners, moved very little, and showed high levels of visual monitoring. As her results indicate, the behavior of dominant individuals is more susceptible to drugs that are known to decrease levels of anxiety in humans, whereas subordinate individuals appear more susceptible to treatment with antidepressant drugs. This clearly demonstrates different emotional states in the individuals, depending on their social position within this stressful situation. Overall, these results indicate lowered adrenocortical and sympatheticoadrenomedullary activities in dominant monkeys living in stable groups, while in unstable situations, heightened activities of both stress systems are present in high-ranking individuals actively trying to attain control and/ or dominance. There are, however, contradictory results even in closely related species of primates. Thus, Shively and Kaplan (1984) found that in Java monkeys, dominant males in well-established mixed-sex groups exhibited higher blood pressure and more advanced arteriosclerosis than subordinates, while the latter had heavier adrenal glands, indicative of heightened adrenocortical activity. Furthermore, McGuire and associates (1986) failed to detect a clear relationship between cortisol levels and dominance status in established colonies of vervet monkeys (Cercopifhecus aethiops sabaeus), while during competition for dominance, plasma cortisol increased in all males. The same has been demonstrated for baboons living in natural conditions (Alberts et al., 1992; Sapolsky, 1990).

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In contrast to males, data on relationships between social status and adrenocortical and adrenomedullary stress responses in females are largely missing. As shown by Christian (1980) in an extensive review, subordinate females in most rodent species are characterized by larger adrenal glands, indicating an increased adrenocortical activity. This was also demonstrated by Schuhr (1987) through direct measurement of plasma corticosterone in female laboratory mice housed in groups. As mentioned previously, in our study on wild rabbits, we found lower adrenocortical activities and heart rates in high-ranking males and females, but only when stable group compositions and a sex ratio of about 1 male to 1-2 females prevailed. In one of the few studies on female monkeys, Gust and colleagues (1993b) examined the relationship between specific social behavior and serum cortisol concentrations in rhesus monkeys. The subjects were 9 females living in an established long-term (8 years) mixed-sex group with their young, while a second group of 9 females was formed 5 months prior to the onset of the study and made up of animals initially unfamiliar to each other. During the 1-year study, the rank of the females correlated significantly with cortisol levels in the established group, with higher serum levels exhibited by subordinate individuals. This was not the case in the recently formed group. In addition, the authors demonstrated that cortisol levels were not only negatively influenced by aggressive interactions, such as receiving bites, but also positively influenced by sociopositive interactions, such as being groomed. In contrast to most studies, Creel and associates (1996) described higher fecal glucocorticosteroid levels in dominant African wild dogs (Lycaon pictus) of both sexes, as well as in samples of urine of dominant female dwarf mongooses (Helogale parvula), both living under natural conditions in the wild. Although measurements made on urine and feces samples must be interpreted cautiously, these data indicate higher adrenocortical activities in dominant females in both species, and also in males in African wild dogs. Details on group composition and stability are, however, not provided by the authors. To summarize, group formation in primates as well as in other species requires the establishment of a social structure. This process is typically characterized by high levels of aggression, particularly among males, with a return to baseline levels within a few days or weeks (e.g., Bernstein and Mason, 1963). As demonstrated by the data given above, the process of establishing a dominance hierarchy represents a potent psychosocial stressor in all mammalian species, and usually affects lower ranking animals more greatly. Social subordination and defeat in aggressive encounters usually leads to increased adrenocortical activity and this relationship has been found in both recently formed and established social groups. Further-

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more, active coping with social subordination in stable social systems, or active efforts to maintain a dominant rank in unstable groups, may eventually have health-impairing consequences when effects extend to the cardiovascular system. Such stress-related pathological conditions are evident even in those primate species in which overt fighting and injury are infrequent in the maintenance of dominant-subordination hierarchies. It must be emphasized once more that social hierarchies do not always result in rank-dependent stress states. In some species, life in well-established social systems is possible without any negative physiological stress effects on any group members, as has been shown by Sachser (1994b) in guinea pigs, and the same may apply to other species as well. b. Gonadal System. As already mentioned in the earlier sections, the effects of social stress on reproduction are profound and in extreme instances can result in sterility in both sexes within a few days. In their classic studies on small mammals housed in stationary and in freely growing populations, Christian and many others have demonstrated that the various endocrine responses of animals in crowded situations decrease natality at every possible physiological level: Crowding inhibits growth and development of reproductive organs in males and females; in addition to inhibiting spermatogenesis in males, it inhibits estrus and ovulation in females and it may delay or inhibit implantation. induce fetal reabsorption, and cause damage to or the loss of litters. Furthermore, changes in the endocrine state of stressed females may influence the physiology and behavior of their progeny (Sachser and Kaiser, 1996). Thus, prenatal stress has been associated with feminized sexual behavior in males and altered behavior in females, as well as with various changes in exploratory behavior, cognitive performance, and aggression. In 1958, Christian and LeMunyan described the effects of crowding of pregnant female laboratory mice on two generations of their offspring! These results have been repeatedly confirmed in recent years. Furthermore, after birth, reduced lactation and the retarded growth of progeny may delay their maturation and increase their morbidity. As these data have been reviewed in many excellent papers (e.g., Christian, 1978, 1980; Christian et af., 1965; Collaer and Hines, 1995; Krebs, 1978; Krebs and Myers, 1974; Lee and McDonald, 1985; Ward, 1984), I shall deal here only with some more recent results. Dominant male sugar gliders (Petaurus breviceps), living in stable colonies consisting of four males and one female, are heavier than socially subordinate males, have significantly higher plasma testosterone and lower cortisol levels, are more active, and are the only males that exhibit scentmarking behavior. When transferred into a foreign stable colony, former dominant males became subordinate and exhibited a reduction or loss of behavioral measures associated with dominance and a concomitant de-

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crease in plasma testosterone and rise in cortisol over a period of 3 weeks (Mallick et al., 1994). The same relationships between high plasma levels of testosterone and dominance have also been observed in males of many other species (e.g., humans: Booth et al., 1989; Elias, 1981; Mazur and Lamb, 1980; McGrady, 1984; nonhuman primates: Alberts et al., 1992; Coe et al., 1979; Keverne et al., 1982; Leshner and Candland, 1972; Mendoza et al., 1979; Rose et al., 1971, 1974, 1975; Sapolsky, 1982, 1983, 1985a,b; Schiml et al., 1996; rats, voles, and mice: review, Christian, 1980; guinea pig: Sachser, 1994a; Sachser and Prove, 1986; tree shrews: von Holst, 1969; see also Fig. 17). As plasma testorterone levels as well as the weights of testosteronedependent organs are usually correlated with dominance rank and sometimes also with the frequency of aggressive behavior in stable social systems (e.g., monkeys: Alberts et al., 1992; Rose et al., 1971; laboratory rats: Koolhaas et al., 1980; Monder et al., 1994), it is sometimes assumed that individuals with higher initial testosterone levels and therefore heightened levels of aggression will gain dominant rank positions. This conclusion is, however, not justified. Mendoza and associates (1979) housed male squirrel monkeys either alone or in groups of three males with or without a female. While prospective dominant males housed alone had the lowest plasma testosterone levels compared to the subordinate individuals, their testosterone levels were highest in all-male groups, and this effect became even more pronounced in the presence of females (Fig. 30). Gonadal endocrine activity changes very quickly during dominance interactions, as was shown already in 1973 by Bronson and associates in their

0Males alone

;ii 280

5m -

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. _n E

m c c

2 Q) 5 0

210 140

70

I

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FIG. 30. Relationships between social rank, housing conditions, and plasma levels of testosterone in male squirrel monkeys; 3 males per rank. Increase of testosterone levels in dominant and decrease in subordinate males in the different test situations significant a t p < .01. Adapted from Mendoza er al. (1979), with kind permission from Elsevier Science Ltd, The Boulvard, Langford Lane, Kidlington OX5 IGB. United Kingdom.

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studies on laboratory mice (Bronson, 1973; Bronson and Marsden, 1973; Bronson et al., 1973). The authors grouped 4 adult males per cage for periods of time ranging from 1 hr to 14 days. During the first hour all males fought intensively to establish dominance orders; at the same time, their plasma levels of corticosterone increased by about a factor of five and their gonadotropin levels decreased by about 20% for FSH and by more than 90% for LH. Plasma corticosterone levels returned to baseline levels between days 1 and 3 in dominants and between days 3 and 6 in the case of subordinates. In dominant mice, the most conspicuous effect was the increase in weight of their preputial glands, which produce an aggressionprovoking pheromone, while preputial glandular weight decreased in subordinates by about 30% within 14 days. The same relationship between preputial glandular weight and rank has also been found in laboratory rats (Dijkstra et af.,1992). In this study the authors also demonstrated a strong increase in testosterone plasma levels in individuals as a consequence of a successful fight for a dominant position. In their elegant studies on laboratory rats, Koolhaas and associates (1980) followed the endocrine changes in males during and after confrontations, by repeated blood sampling using cannulas inserted into blood vessels. During the 1-hr encounters, plasma testosterone concentrations rose in victors as well as in losers, but the rise was significantly greater in victors than in losers. About 30 min after the start of the confrontation, plasma testosterone concentration in both victors and losers started to decrease. Victors regained their original baseline levels about 90 min after the end of the confrontation, whereas testosterone levels in losers continued to decline, reaching about 20% of initial levels 4 hr after the end of the confrontation, and most defeated rats maintained these lowered baseline levels for several days (Schuurman, 1981). In summary, increased testosterone levels, such as are usually found in high-ranking males, are the consequences rather than the cause of high rates of aggression, as exogenous manipulations of testosterone concentrations within the physiological range do not cause parallel changes in rates of aggression or other testosterone-modulated behaviors (e.g., Booth et af., 1989; Dixon, 1979 Mendoza ef al., 1979; Monaghan and Glickman, 1992; Rose, 1985). On the other hand, loss of control as evident in subordinate individuals or loss of a dominant status is associated with suppressed testosterone levels, and can even lead to sterility within a few days (see also Rose 1985; Rose et al., 1972; 1974). As is the case in the adrenocortical and sympathetico-adrenomedullary systems, the gonadal endocrine system is not activated by the physical exertions of successful fighting but by the emotional processes induced by it. Accordingly, Mazur and Lamb (1980) have shown in human males that

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only those that win a contest (leading them to perceive that their status is thereby improved) show an elevation of testosterone levels. This is apparently also the case in nonhuman mammals, as can be deduced from the findings of our research on tree shrews (Kaiser, 1996). In order to differentiate between the physical and psychological effects of confrontations on dominant and subordinate male tree shrews, 2 males confronted each other for 10 min daily over a period of 14 days, in an experimental cage that could be divided into two identical subdivisions by a wall. The confrontations always led to low-key fights, which resulted right from the beginning in definite dominance relationships. Outside the confrontation periods each animal was separated from its rival by a wooden partition (“without visual contact”) or a wire mesh partition (“visual contact”). Outside the confrontation periods, dominant individuals in visual contact with their rivals were less active and rested more compared to the days before the confrontation period. In addition, their daily excretion rates of cortisol decreased after the start of confrontations, while the excretion of testosterone and the in vitro proliferation rate of their lymphocytes increased (Fig. 31). By contrast, dominants without visual contact with their rivals showed no changes in behavior or physiological parameters in comparison with initial values. Thus, only constant visual contact with the subordinate opponent and the emotional process of “elation” probably thereby induced modulated the behavior and physiology of dominant individuals. As expected, subordinates in visual contact with their dominant rivals showed opposite reactions to those of their opponents: a slightly increased locomotor activity and urinary cortisol excretion, as well as a decrease in testosterone excretion and the in vitro proliferation rates of their lymphocytes (Fig. 31). Surprisingly, subordinate animals without visual contact with their rivals showed qualitatively similar reactions to dominant animals in visual contact with their subordinate rivals (Fig. 31). Thus, low-key fights during the daily confrontations had no negative effects on the behavior or physiological parameters of the subordinates. Our data even point to an improved physiological state of these individuals, which may be due to the high level of control and predictability which these animals perceive in this situation (Fig. 31). In female mammals, social subordination is associated with a diminished number of ovulatory cycles and hence also with impaired reproductive success (Dittus, 1979; Drickhammer, 1974; Sade et al., 1976; Silk etal., 1981; Walker et al., 1983; Wilson et al., 1978; Wise et al., 1985). Most studies have been carried out on rodents and, as they have been reviewed extensively,

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Locomotory activity

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I *

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FIG.31. Effect of visual contact on locomotory behavior (medians), testosterone excretion ( M 2 SEM), and in vitro lymphocyte proliferation after Con A stimulation ( M 2 SEM) of 14 dominant and 14 subdominant male tree shrews. After 10 days of habituation to the experimental room all animals were daily confronted for 10 min over a 2-week period; at other times they were separated by either a wooden partition (without visual contact) or a wire mesh partition (visual contact). Blood samples were taken 1day before the first confrontation and 1 day after the last confrontation. Urine was collected over the whole period and the individual means of each animal’s excretion rates over 8 days before the confrontation were used as initial values. Locomotory behavior was determined daily for 3 hr. Significant differences: *p < .05; **p < .01.

they will not be covered here (e.g., Christian, 1978, 1980; Christian et a!., 1965; Krebs, 1978; Krebs and Myers, 1974; Lee and McDonald, 1985). The same relationships between rank and reproductive success were demonstrated in primates as well as in other mammalian groups. Among macaques

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living in natural or seminatural environments, subordinate females are less likely to become pregnant, and their pregnancies are more likely to result in abortion, stillbirths, or neonatal death than are those of dominant females. Furthermore, in free-ranging populations of rhesus monkeys at Cay0 Santiago and La Parguera and in wild populations of toque macaques, subordinate female genealogies were found to exhibit a lower intrinsic rate of natural increase than those of dominant females (Dittus, 1979; Drickhammer, 1974; Sade et al., 1976). This was also demonstrated in groups of adult female Java monkeys housed in harem groups consisting of one adult male and five to six females (Adams et al., 1985). To induce social instability and social disruption in three groups, the females were redistributed every 12 weeks for a period of 24 months (unstable groups), while the remaining groups served as stable controls for the duration of the study. Compared to socially dominant females, subordinate individuals had fewer ovulatory menstrual cycles, more cycles with deficient luteal plasma progesterone concentrations, increased adrenal weights, and increased heart weights. Social instability, however, influenced none of these variables. These results indicate that impaired reproductive success observed in subordinate female macaques may be related, at least in part, to changes in ovarian function. The same relationship between rank and reproductive success has been found in many studies on European wild rabbits under seminatural conditions (e.g., Garson, 1979; Myers and Poole, 1962; Mykytowycz, 1959a,b). In our studies on wild rabbits we also found higher reproductive success and individual fitness in females, depending on their rank at the time of their insemination and pregnancy: Compared to subordinate females, dominant individuals gave birth to more litters per year, the weight of the young was higher at birth and at weaning, and mortality during the nest period was lower (Fig. 32). This last feature is due mainly to decreased milk production in females of subordinate ranks, which leads to the starvation of their young. The lower number of litters produced by subordinate females is apparently not due to sterility or delayed implantation, but results from a high rate of resorption and abortion of entire litters during pregnancy, as was verified by hormone analysis. As a consequence of this higher reproductive success in dominant females, there are also rank-dependent differences in the fitness of the individuals (Fig. 33). A particularly interesting effect of dominance on reproductive success has been demonstrated in dwarf mongooses. In these group-living carnivores, the oldest male and female dominate reproduction, while the younger and subordinate group members are reproductively suppressed and provide care for the offspring of the oldest pair. Nevertheless, subordinate males from several wild populations in Tanzania exhibited urinary testosterone

STRESS A N D ITS RELEVANCE FOR ANIMAL BEHAVIOR

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.

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>3

FIG.32. Relationships between social rank and reproduction of female wild European rabbits living in a 22.000-m2 field enclosure. Data from about 50 females and 4 years; the numbers of young are indicated in the bars of the bottom figure. Data in figure “Mortality” are means; other figures means ? SEM. See text for further details. Unpublished data from H. Draxler. 1996.

levels corresponding to those of dominants. They were, however, apparently prevented from mating by dominant male aggression. In contrast, subordinate females exhibit a decreased ovarian function (Creel et al., 1992). As Keane and associates have shown in a subsequent paper (1994), subordinates of both sexes mate and about 20% of all young had subordinate mothers or fathers. Those subordinates that reproduced were of higher rank than those that did not. Among the primates, marmoset monkeys and tamarins demonstrate an extreme form of rank-dependent fertility. In laboratory colonies, as well

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Social rank and reproductive success of males and females in % 100

139 animals in 7 years

75 50 25

0

1

2 3 Rank of females

1 >I Rank of males

FIG. 33. Relationships between social rank and reproductive success of male and female European rabbits. Data are percentages of all young that survived until the reproductive season following the year of their birth. The mothers of the litters were determined by observations, and the fathers were determined by multilocus DNA fingerprinting. See text for further details. Unpublished data: after Zobelein (1996).

as under natural conditions, only the socially dominant female of each group reproduces, while ovulation in subordinate females is always suppressed. This infertility is immediately reversed when subordinate females are removed from their group and housed singly. As shown by Abbott and associates (1988), the social suppression of fertility in the subordinate females is apparently mediated by impaired hypothalamic GnRH secretion. The most impressive example of socially induced contraception is known from naked mole rats (Heterocephalus glaber), which live in colonies of up to 300 animals entirely underground in the semiarid regions of East Africa. In the wild as well as in captivity, there is only 1 breeding female, the “queen,” and 1-2 breeding males in each colony, while all other animals are infertile workers or play defensive roles within the colonies. Suppression of reproduction in nonreproductive females appears to be induced by ovulatory failure due to insufficient gonadotropin secretion from the anterior pituitary gland and the same suppression of gonadotropin release is also evident in the nonreproductive (subordinate) males (Abbott et al., 1989; Sherman et al., 1991). It is probable that in all of the cases mentioned so far, neural responses associated with psychosocial stress operate through the hypothalamopituitary-gonadal axis, to induce ovarian dysfunction and subsequent infertility or pregnancy failure, such as has been demonstrated in small mammals (Christian, 1980). Support for this is provided by the findings of elevated plasma prolactin concentrations and failure of the estrogen-induced LH

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surge in socially subordinate ovariectomized talapoins (Bowman et al., 1978; Keverne et al., 1982), and lower fecal estrogen levels found in subordinate wild yellow baboons during the luteal phase of ovarian cycles, when compared to females of high rank (Wasser, 1996). Furthermore, Packer and associates (1995) found some of the strongest evidence for the advantage of high rank in primates in their 30-year study of olive baboons (Papio cynocephalus anubis) at the Gombe National Park: Dominant females had shorter interbirth intervals, improved infant survival, and accelerated maturation of their daughters. It must be mentioned in this context, however, that the authors also reported negative effects on several aspects of female reproductive success, which they interpreted as the reproductive cost of dominance. However, as pointed out by Altmann and colleagues (1995), this conclusion is not supported by their data and might have resulted from inadequate interpretation of external signs of early pregnancy. c. Zmmune System. Most earlier research into the relationship between the social behavior of animals and their immune system and resistance to disease stemmed from crowding experiments and was conducted mainly on mice. Davis and Read (1958) conducted a series of experiments on the influence of daily fighting on wild-stock house mice that were infected parenterally with about 125 Trichinella larvae. Each mouse was housed in a separate cage and from day 3 through 11 after infection half of the mice were placed in groups of 5-6 animals for 3 hr a day, while the other half were left separated. All mice were killed on the 15th day after infection. About 25% of the singly housed mice were infected with an average of 9 worms apiece, whereas all grouped mice were infected and had an average of 32 worms. Furthermore, severe and prolonged fighting among crowded male mice impeded the development of acquired immunity to the dwarf tapeworm and also increased the reinfection rate in mice with wellestablished acquired immunity (Weinmann and Rothman, 1967). The effects were clearly rank dependent: Four days after a second dose of tapeworm eggs (3500 eggdmouse) the dominant mice had an intestinal cysticercoid count of 27, comparable to that in nonstressed mice exposed to the same infection; however, the counts in subordinate individuals ranged from 108 (rank 2) to 685 cysticercoids (lowest rank of the 8 males). In a similar study, Tobach and Bloch (1958) demonstrated a significantly reduced survival time to an acute tuberculosis infection in socially stressed mice (20 individuals per cage) in comparison to singly housed controls. Furthermore, Edwards and Dean (1977) found that laboratory mice of both sexes kept at high animal numbers (30 and 60 animals per cage) exhibited reduced antibody production (against typhoid paratyphoid vaccine) and reduced resistance to disease. This was evident from the significantly higher mortality rate following an injection of Salmonella typhimurium, in compar-

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ison to groups with lower animal numbers (2 or 10 animals per cage). The inflammatory response to subcutaneous implants of cotton pellets moistened with turpine as well as the formation of granulation tissue is also reduced in grouped mice (Christian and Williamson, 1958). Finally, Temoshok and Peeke (1988) found differences in induced tumor growth in two experiments on adult female Syrian hamsters placed in groups of ten: Females ranked as dominant by the authors exhibited reduced tumor growth compared to the subordinate individuals. These results indicate an influence of social disturbances on disease susceptibility due to immunomodulatory processes. One of the earliest experimental proofs of this stems from research carried out by Vessey (1964), who examined the antibody production against bovine serum in male laboratory mice. Previously isolated mice were placed together in groups of 6 each for 4 hr daily. They were injected with bovine serum 5 days after grouping and were found to have significantly lower titers of circulating antibodies than isolated control mice. Vessey (1964) also provided the first indication of rank-dependent immunological changes: The winners of confrontations exhibited substantially higher titers of antibodies than did the losers; likewise, T lymphocytes of subordinate mice showed a distinctly reduced in v i m response to mitogenic stimulation and reduced interleukin 2 production compared to their dominant counterparts (Hardy et al., 1990). Correspondingly, Ebbesen and associates (1991) found a lower incidence of virus-induced leukemia in dominant mice compared with subordinates. Similar suppressive effects of defeat or subordinate social rank on immunological parameters have also been found in many other species. In one of the earliest studies on rats, Raab and associates (1986) found higher tyrosine hydroxylase activities in both dominants and subordinates compared to individually or pair-housed rats (controls) after 10 days of chronic cohabitation. Only subordinates, however, lost body weight and they exhibited plasma corticosterone levels more than twice as high as those in dominants and controls. In addition, they had smaller thymus glands and a reduced lymphocyte response to in vitro mitogenic stimulation, while the values of dominants did not differ from those of controls. In laboratory rats housed in colonies, rank-dependent alterations in various components of the cellular and humoral systems have also been demonstrated (Bohus et al., 1992). Taken together, the results of these authors indicate an improvement of the immune system in dominant rats and to a lesser degree also in actively coping subdominants, while most immunological parameters in subordinates and outcasts are clearly suppressed. In piglets housed in mixed-sex groups in large pens for 80 days, Hessing and associates (1994) demonstrated clear relations between rank and sus-

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ceptibility to disease and immune reactivity. Based on dominance fights and a food competition test, piglets were divided into high-, middle-, and low-ranking groups. Dominant individuals showed a higher in vitro lymphocyte response to an Aujeszky disease virus, less severe clinical signs of disease, and threefold lower mortality rates compared to the individuals of lower rank. Similar findings have also been described in farmed red deer hinds (Hanlon et al., 1995). Corresponding results have also been demonstrated for primates. Gust and associates (1991) studied the immunological consequences of social stress associated with the formation of a new group of 8 unfamiliar adult female rhesus monkeys, introduced into an outdoor enclosure along with 1 adult male. The establishment of a stable dominance hierarchy, apparent within 48 hr, was accomplished without serious fighting and in a complete absence of wounding. While humoral components of the immune system (IgG, IgA, IgM) were not significantly influenced over the period of colony formation, within 24 hr all females generally showed a significant increase in cortisol plasma levels and a 30% decrease in absolute numbers of total lymphocytes as well as CD4+ and CD8+ T cells. These changes were significantly greater in the 4 lowest ranking females compared to those with higher ranks. After 1 week the T cell subsets of the high ranking females had returned to initial levels or exhibited even higher levels; however, values of the low-ranking females returned more slowly to baseline levels and were still low 9 weeks after group formation. This was in spite of the fact that there were no significant differences in aggressive (offensive or defensive) or affiliative behaviors between the two groups, with the exception of grooming: High-ranking subjects were groomed significantly longer than the subordinates. Recent studies by Gust et al. (1996) on female pigtail macaques (Macaca nernestrina) confirmed these results. Furthermore, highranking males in small stable groups of male rhesus monkeys exhibited significantly higher lymphocyte proliferation than middle- or low-ranking individuals. Regrouping of the animals led to an increase in aggressive behavior and plasma cortisol levels and a decrease in the lymphocyte proliferation response to a mitogen (Clarke et aZ., 1996). The same effects have been found in male Java monkeys, with particularly strong immunosuppressive effects among those monkeys showing high levels of fear behavior (Line et al., 1996). In a field study, Alberts and colleagues (1992) found significantly lower lymphocyte counts and a higher basal cortisol concentration in an adult male that had entered a stable group of olive baboons, as well as in those individuals that were victims of the intruder’s aggression, than in noninvolved individuals. The same suppressive effects of crowding stress, repeated regrouping or defeat, and social subordination on resistance against

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parasites, bacterial and viral diseases, tumor growth, as well as on humoral and cellular immunological parameters have also been found in many other studies, conducted mainly on mice, rats, and rabbits (e.g., 1994; Brayton and Brain, 1974a,b; Edwards and Dean, 1977; Edwards et al., 1980; Fleshner et al., 1989; Hardy et al., 1990; Hoffman-Goetz et al., 1991; Mykytowycz, 1961; Plaut et al., 1969; Stefanski and Ben-Eliyahu, 1996; Stefanski and Hendrichs, 1996; Stefanski et al., 1996; Tecoma and Huey, 1985; for reviews, see also Ader and Cohen, 1985; Monjan, 1981; Plaut and Friedman, 1981; Riley, 1981). Amazingly, the odor from stressed laboratory mice alone can induce an altered immune function in conspecifics after 24 hr of odor exposure, and lead to a decrease in the number of cells forming antibodies to sheep red blood cells (Zalcman et al., 1991), as well as to a decrease in production of interleukin 2 by Con A-stimulated spleen cells, and decreased activity of natural killer cells (Cocke et al., 1993). In contrast to the observed suppression in cell-mediated responses, stress-odor exposed mice had an enhanced humoral immune response to KLH. Thus, even in a given strain, stressors do not necessarily affect all immune measures unidirectionally, which cautions against premature conclusions based on a limited selection of cellular or humoral immune parameters. The general conclusion can be drawn from these and many other studies, that “stress” as determined by adrenocortical activation can increase susceptibility to infectious diseases. However, there are exceptions to this generalization (e.g., Moynihan et al., 1994). In their study on male and female laboratory rats that were submitted for 4 weeks to different forms of regrouping, Klein and associates (1992) found clear indications of heightened adrenocortical activity (adrenal enlargement and increased basal corticosterone levels) and thymus involution. However, compared to undisturbed controls, neither natural killer cell activity, splenocyte reactivity to mitogens, nor the rate of spontaneous development of antibodies against a common pathogen of the respiratory tract of mice (Mycoplasma pulmonis) were changed in the stressed animals. This could be due to genetic differences in immunological responsiveness, as was demonstrated in 1955 by Tobach and Bloch. These authors used strains of rats and mice that varied in degree of “emotionality,” and found that the most “emotional” strains had the shortest mean survival times after a standard dose of intravenously administered tuberculosis bacilli. Similarly, Friedman and Glasgow (1973) found, depending on mouse strain, that grouped laboratory mice are more susceptible to Plasmodium berghei than individually housed animals. Likewise, Fauman (1987) demonstrated that, relative to subordinate animals and isolated controls, dominant laboratory mice have a reduced anti-

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body response to an antigen (keyhole limpet hemocyanin). The extent of the reduction of the antibody response in dominant mice was related to the intensity of their aggressive behavior during confrontations. Similar results have also been found in male laboratory rats (Bohus et al., 1993) as well as in female chimpanzees (Pun trogfodytes) living in captive colonies (Masataka et al., 1990). Furthermore, according to Hausfater and Watson (1976), high-ranking individuals in groups of free-living yellow baboons (Papio cynocephafus) of both sexes exhibited a higher fecal parasite ova emission than more subordinate individuals. Subadult individuals generally occupy lower ranks and have lower egg counts than older ones. However, examination of the mean egg counts in adult individuals only continued to show a correlation between egg output and dominance rank in males, but not in adult females. There are many possible reasons for these and other contradictions. In relation to investigations carried out on laboratory animals, some of these contradictions may be due to differences between the various strains. They could also, however, be due to differences in housing conditions and in the duration and type of stress. Very often the necessary information needed to assess this question is missing, as are appropriate control groups. In addition, the detailed observations required to supply reliable information on social ranking and stress levels in individuals have often not been carried out (see also Bohus and Koolhaas, 1991). Furthermore, data on the activity of the sympathetico-adrenomedullary system, which could indicate the presence of social tension, are completely lacking. Finally, all of these studies are based on a rather limited selection of immune parameters, which makes general conclusions on the function of the immune system impossible. In spite of these shortcomings, all investigations do indicate a strong response of immune parameters to socially stressful situations. d. Physiological Costs of Male Dominance. In general, engaging in social conflict exposes individuals to the risk of injury and attacks by predators, diverts precious energy from reproductive activities and feeding opportunities, and may enhance vulnerability to disease. In the long term these costs are weighed against potential benefits for the dominant individual of ready access to mates with high reproductive success (e.g., Huntingford and Turner, 1987; Maynard Smith and Price, 1973; Riechert, 1988). In fact, one of the most prominent views on subordinate animals is that they have less access to mates and consequently leave fewer offspring than do dominant animals, an idea that was advanced by Zuckerman (1932) and Maslow (1936) in the 1930s for primates. This concept is widely accepted today and its validity has been demonstrated for many species in the wild as well as in captivity (e.g., Ellis, 1995; Miczek eta!., 1991). Several studies in laboratory conditions have also shown that females, when given a choice, tend to

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associate and mate with dominant males (e.g., lemmings: Huck and Banks, 1982; rats: Carr et al., 1982; bank voles: Hoffmeyer, 1982; Shapiro and Dewsbury, 1986; hamsters: Brown et al., 1988; White, 1986; vervet monkeys: Keddy, 1986). The relationship between social status and susceptibility to disease is currently of great interest among some evolutionary biologists, as parasite burdens may influence several aspects of social and sexual behavior (e.g., Barnett and Sanford, 1982; Dobson and Hudson, 1986; Edwards, 1988; Edwards and Barnard, 1987; Freeland, 1981; Kavaliers and Colwell, 1995; Moore and Gotelli, 1990; Rau, 1983; Read, 1990; Toft and Karter, 1990; Wedekind, 1994). In 1982, Hamilton and Zuk proposed that because of the genetically based interactions between parasites and their hosts, females are expected to choose mates based on their resistance to pathogens. Male secondary sex characters or ornaments were supposed to have evolved at least in part as indicators of this resistance. According to the authors, females should prefer males with fewer parasites, an indication of which is given by the degree of the development of secondary sex characters. Many, though not all, tests designed to prove this hypothesis have been supportive. On the basis of the higher susceptibility of human males compared with females to a variety of bacterial, viral, and parasitic diseases, Marlene Zuk proposed in 1994 that high-ranking males are more vulnerable to diseases: “The deleterious effects of testosterone may be an unavoidable price paid by males for achieving reproductive success in a competitive environment.” This hypothesis, however, seems rather unlikely, at least for mammals. As shown in the previous sections, it is absolutely possible that under conditions of social instability, dominant individuals fighting actively for control may develop cardiovascular diseases that may shorten their life. Their immunological resistance and therefore their resistance to bacterial, viral, and parasitic diseases is, however, usually higher than that of subordinates, especially in stable social situations. Furthermore, the hypothesis is based on rather doubtful premises. The author writes: “Social dominance has also been demonstrated to be testosterone dependent, with experimental castration generally reducing aggression and subsequent testosterone injections usually causing its return.” Although these effects of castration and subsequent testosterone replacement have been demonstrated in several mammalian species, the results are of limited relevance to the hypothesis put forward by Zuk. Behavioral endocrinology has shown that aggressive behavior in male mammals is predominantly determined by genetic influences, which apparently modify prenatally through testosterone those central nervous structures involved in the expression of aggressive behavior (e.g., de Ruiter et

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al., 1993). Postnatally, these modifications are based on experience, so long as certain minimal testosterone levels are present, and this is probably the case in most mammals in natural conditions (e.g., Monaghan and Glickman, 1992; Sachser et al., 1994; Scott and Fredericson, 1951). Testosterone levels are indeed usually higher in dominant males, but it must be emphasized again that these increased testosterone levels are the consequence, rather than the cause of the high-ranking positions. There are, at least to my knowledge, no conclusive published data indicating that intact males of a given species (or strain) with higher initial testosterone levels are more aggressive and successful in confrontations than rivals with lower levels. Rather, the opposite seems to be true (see also Figs. 17 and 30) and, accordingly, it is not possible to increase aggression or other testosteronemodulated behaviors in male mammals with normal serum androgen levels by means of testosterone treatment (e.g., Clarke et al., 1996; Leshner, 1981; Monaghan and Glickman, 1992; van Oortmerssen et al., 1987; Rose et al., 1972; 1975). Additionally, Zuk states in this context: “No one could be more stressed than the males of many vertebrate species during the mating season, when courtship displays are exhausting, the environment must be constantly scrutinized for competitors and those competitors fought off. . . .” Although this statement applies to most species, a decrease in the response of immune parameters during the mating season has not been demonstrated so far, apart from in the highly stressed marsupial, Antechinus, mentioned in this chapter’s introduction (for a recent review, see also Nelson and Demas, 1996). In our wild rabbits there is definite evidence of an improved immunological state during the mating season compared to the nonmating season (Fig. 34). The same objections have to be raised to the postulation by Zuk of a relationship between testosterone levels of fertile males and their immunological resistance. In general, increased testosterone levels during fetal life as well as in adult males after puberty are thought to reduce cell-mediated immunological resistance, although it must be pointed out that these conclusions are based on studies on very few laboratory animal species. Furthermore, there is considerable controversy concerning the effects of sex hormones on antibody formation and unspecific biological resistance (e.g., Grossman, 1984; Madden and Felten, 1995; McCruden and Stimson, 1991; Olsen and Kovacs, 1996; Schuurs and Verheul, 1990). Nevertheless, castration of adult males of those species examined so far does at least lead to increases in their cellular immune resistance compared to that of fertile males. However, Zuk’s conclusion that this relationship also applies to fertile individuals is not supported by the literature. In contrast, most data pre-

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0.5

1

Testosterone I

(ng/ml

Lymphocyte proliferation

immunoglobulin G

36 32 20

24

20 Reproductive season

Nonreproductive season

FIG. 34. Serum testosterone levels (40 males) and immunological parameters (117 males) ( 2 SEM) from the reproductive period (April-September) and the nonreproductive period (October-March). Differences between the seasons were always significant at p < ,001.

of wild European rabbits living in a 22,000-m2 field enclosure. Means

sented in the previous section indicate an improved immunological resistance for dominant males, along with increased testosterone plasma levels. Preliminary data from our laboratory collected on European rabbits, tree shrews, and Long-Evans laboratory rats also contradict this hypothesis: Injection of fertile males with physiological doses of testosterone over a period of 2 weeks had no recognizable immunosuppressive effects in any of these 3 species; on the contrary it even increased several cellular immune parameters in rabbits that were kept under constant laboratory conditions. e. Summary. In a stable dominance hierarchy, the dominant individuals can predict and actively control the outcome of social interactions, they have priority of access to food, mates, and other resources. On the whole, this situation increases the fertility and health of dominant individuals, while the opposite is usually true for subordinate individuals. This endocrinological advantage of a dominant social position may, however, be very small or even nonexistent, depending on the social system and the species (Table 111). The contrasting situation of instability occurs in the wild, when new animals migrate into a social group and destabilize the status quo, or when individuals die. In captivity, such instability is evident when social groups are first formed. In this case, the situation is very different for dominants when compared to stable systems. Typically, the rates of aggressive interactions are elevated, and are focused on animals in high-ranking positions. Rank shifts may occur repeatedly and unexpectedly. This is a situation that

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TABLE I11 RELATIONSHIP BBI'WEEN SOCIAL STATUS, COPING STYLE, A N D PHYSIOLOGICAL RESPONSE PATTERN Dominant Control of situation or position Coping style Pituitaryadrenocortical function Sympathicoadrenomedullary function Pituitary-gonadal function Immune function

Subordinate

low, rank threatened active (offensive) slightly elevated

low

loss of control

active (defensive/ offensive) slightly elevated

passive and apathetic markedly elevated

unchanged

markedly elevated

greatly elevated

elevated

elevated

reduced

unchanged or often improved

?

reduced

unchanged or even reduced markedly reduced greatly reduced

high active (fights usually not necessary) unchanged or decreased

exhibits anything but control and predictability. All individuals, especially the top-ranking individuals, are therefore experiencing stress (Table 111). These relationships between control and predictability of a social situation and the physiological response pattern of an individual may explain most of the discrepancies between studies on social rank, physiology, and stress-related diseases in different mammalian species. It must be emphasized again that, in order to gain reliable information on the physiological state of an individual, it is not sufficient to focus on one stress system only, as is the case in most studies so far conducted, but information has to be collected at least on pituitary-adrenocortical as well as on sympatheticoadrenomedullary systems. Furthermore, conclusions about the influence of social situations on the immune status of an individual must be based on a large number of different immune parameters, to avoid the premature conclusion that a situation is without immunological effects.

3. Disruption of Social Bonds a. Introduction. Dominance has been used in the preceding text in a very general way, as a shorthand term that indicates the outcome of agonistic or competitive interactions between two individuals. Dominance relationships are usually more pronounced in males and are of overwhelming importance to all aspects of the life of group-living mammals-they influence their behavior, reproductive success, and health.

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Dominance hierarchy has often tacitly been assumed to be an equivalent term to social organization. However, dominance hierarchies based on agonistic behavior are only one aspect of social systems. Although much less conspicuous, social bonds, usually based on attachment between individuals, are at least as necessary for the establishment and stability of social systems as are dominance relationships. Attachment between mothers and their infants is usually a precondition for the survival of infants in mammals, and later social bonds to peers and mates, as well as to adults of the same sex, may develop, which profoundly influence the behavior of the animals. Such bonds, in terms of social support, can also play a positive role in health by presumably altering the way in which a potentially stressful situation is perceived (House et al., 1988; Levine, 1993a; Unden et al., 1991). It is, therefore, not surprising that the loss of a social bond and/or lack of social support may result in strong stress responses and an increased risk of mortality, as is indicated by numerous epidemiological studies in humans (e.g., Berkman and Syme, 1979; Broadhead et al., 1983; Dyer et al., 1980; Gilman et al., 1982; House et al., 1982; Kannel et al., 1987; Perrson et al., 1994; Schoenbach et al., 1986). Although social relationships play an important role in most mammalian societies, research into the relevance of social bonds and the stress-buffering effects of sociopositive interactions are usually neglected in stress research on nonhuman mammals. b. Mother-Infant Bond. Mammalian young are born defenseless and are highly dependent on their mothers for a relatively long period of time. Therefore, they have to learn to bond to their mothers, which is essential for their nurture and social development and has been dramatically demonstrated in rhesus monkeys in Harlow’s classical studies (e.g., Harlow and Suomi, 1974; Harlow et al., 1971; Rosenblum and Plimpton, 1981; Suomi, 1976). The mother-infant bond is crucial for the infant to learn to overcome fear of novel stimuli and to control aggression in social settings in later life. Maternal separation from infants is one of the most profound stressors for monkeys, and usually results in the death of the young in the wild (e.g., Thierry et al., 1984). In the laboratory, it has been used as an animal model for separation and depression. Extreme passive stress responses characterized by increased excretion of urinary 17-hydroxycorticosteroids and plasma cortisol levels, as well as strong immunomodulatory responses occur in infant rhesus and squirrel monkeys in response to separation from their mothers (e.g., Coe and Scheffler, 1989; Hrdina and Henry, 1981; Levine, 1993b; Levine et al., 1985; Wiener et al., 1992). Infants (age around 30 weeks) of macaques also showed behavioral changes and depressed in vitro lymphocyte proliferative responses to T-cell-specific mitogens over the 14-day separation period, while response to a B-cell-selective mitogen was not significantly affected. In addition, decreased natural killer cell

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activity and significant alterations of different lymphocyte populations occurred (Lubach et al., 1996). Following reunion, both behavior and immunological parameters returned to initial values. Maternal responses to separation were usually similar to those of their infants (Laudenslager et al., 1982; Reite et al., 1981). The behavioral responses of infants to separation from their mothers differ among species, even when they are as closely related as the pigtail and bonnet macaques. Both species exhibit an initial agitation phase, characterized by distress vocalizations, high levels of locomotion, and other behavioral attempts by the infant to relocate and reestablish contact with its mother. Only pigtail macaques, however, exhibit a second depressive phase in this response (Boccia et al., 1995). This difference is apparently due to the different social environments of the young of these two species. Because bonnet macaques exhibit lower levels of aggression and higher levels of social contact than pigtail macaques, mothers are less restrictive and permit their infants to freely interact with other group members. As a consequence, when separated from their mothers, these infants are adopted by one of the other females. Boccia and collegues (1995) tested the effect of this different socialization directly. They examined the behavior of two infant pigtail macaques, who grew up in an environment of elevated aggression induced by a feeding paradigm, which allowed the individuals to feed only one after another. As a consequence, the high-ranking mother always had free access to the food, while the subordinate mother became restricted. This situation had striking effects on the social relationships of the infants of these two mothers. The infant of the unrestricted dominant female exhibited close attachments to four other group members, representing over 80% of the social interactions, whereas the infant of the subordinate mother restricted his social interactions to the mother. When the two infants were separated from their mothers, the second infant without alternative attachments, but not the first, became profoundly depressed and spent nearly 50% of its time during the separation exhibiting a depressive slouched posture (Boccia et al., 1991). Furthermore, the authors showed that social support from older peers can be protective. In a social group containing six infants, three had significant attachments with three older juveniles in the group, while the others were attached only to their mothers. The authors removed all mothers and juveniles from the group except for the three previously identified juveniles. Thus, out of the six remaining infants, only three retained social relationships with the juveniles with whom they had already had relationships prior to the separation. As a measure of social support, the authors took the number of affiliative behaviors directed by the juveniles to each infant. This measure demonstrated the strong protective effects of social

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support after mother-infant separation. Behaviorally, infants with social support showed less evidence of depression, as was reflected in play and eating behaviors. They also exhibited no change from baseline function in natural killer cells, while infants without support showed a 40% decrease from baseline function 2 hr after separation. Similar studies have been performed on only a few nonprimate species, such as the laboratory rat. Removal of mothers of laboratory rats at an age of 2 weeks markedly decreased the heart rates of their infants to about 60% of the normal rate during the following 2 days, which was followed by leveling off and recovery during the next few days (e.g., Hofer, 1981, 1994). In addition, increased plasma corticosterone baseline levels and adrenal responsivity to acute stressors were evident even several days after a single 24-hr period of maternal deprivation (Rosenfeld et al., 1992; Takahashi, 1991). The social bonds between mothers and their infants have also been evaluated in guinea pigs. Guinea pigs are capable of coordinated locomotion almost immediately following birth. Maternal care is minimal. When infants at an age of about 2 weeks were separated from their mothers for 30 min and transferred to an unfamiliar cage in an unfamiliar room, they exhibited high rates of vocalization and almost doubled plasma cortisol levels. The presence of their mothers reduced vocalization and plasma cortisol levels significantly, while the presence of an unfamiliar lactating female produced no effect over a period of hours (Hennessy and Ritchey, 1987). c. Bonds between Juveniles and Adult Individuals. The separation from peers can also result in strong physiological responses. The removal of squirrel monkeys from their companions resulted in a strong decrease of the lymphocyte proliferation to the mitogen Con A. The decrease reached significance within the first day, was maximal on the second day, and returned to initial levels within 7 days. As in common marmosets (Johnson et al., 1996) and most other primate species, plasma cortisol levels in squirrel monkeys peaked during the first day after separation, but took much longer to return to baseline levels than did the immune parameter (Coe, 1993; Friedman et al., 1991;Levine et al., 1989). The independence of adrenocortical activation and immune responses has also been demonstrated in juvenile rhesus monkeys, which were removed from their natal social group to peer housing at the age of 2 years. The highest plasma cortisol levels and greatest decrease of total blood lymphocytes and several T cell subsets (CD4+ and CD8+) were observed on the first day. While their adrenocortical activities returned to baseline levels within about 2 weeks, immune measures remained decreased for up to 2 months (Gust ef al., 1992). In contrast to juveniles, adult male rhesus monkeys showed no stress response to separation from their group (Gust et al., 1993a), while separation of adult individu-

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als from their group in rats and sheep causes a strong activation of the pituitary-adrenocortical system (cited from Toates, 1987). Ratcliffe and associates (1969) studied the psychological response of swine to separation after the social bonds of grouped animals had been established. Swine housed pairwise or in groups, responded to human visitors with grunts and squeals for a handout. Competition among the males was very low and was limited to pushing and shoving. By contrast, separated swine, especially the normally sociable females, failed to respond to visitors, lying unresponsive and refusing offers of added food. After a year of isolation the separated females showed a significantly greater development of arteriosclerosis than those that were grouped. These data suggest that the lack of social bonds may result in sustained emotional disturbance and pathophysiological changes. Kaplan and associates (1991) examined the relationship between the aggressive and affiliative behavior and cellular immune parameters in adult male Java monkeys living in small groups, whose members were periodically redistributed over months. While the authors did not find any influence of social status on the immune parameters, the in vitro lymphocyte proliferation in reaction to the two T-cell-selective mitogens concanavalin A (Con A) and phytohemagglutinin (PHA) was greatest in individuals that were both highly affiliative and exhibited low levels of aggression. Furthermore, natural killer cell activity was highest among highly affiliative males, regardless of their levels of aggression. These findings indicate that the cellular immune competence may be enhanced among monkeys that, in response to a disrupted social environment, spend large amounts of time in affiliation with other males, or in males that seek and find social support. 4. Social Support and Its Stress-Reducing Effects As these findings show, numerous factors influence the magnitude of the physiological stress response, and one of the most important variables in pairwise or group-living mammals appears to be the presence of a familiar social partner. Social support generally reduces the magnitude of stress responses and it has a stress-buffering effect, as was impressively demonstrated by Levine and associates in their studies on squirrel monkeys (Levine, 1993a,b). The authors exposed a well-established group of adult squirrel monkeys to a live Boa constrictor that was confined in a plastic box. Although direct physical contact between the monkeys and the snake was prevented, all monkeys showed increased levels of vigilance, agitation, and avoidance behavior. A strong adrenocortical activation, however, was observed only when the monkeys were tested individually, but not when tested together as a group. Surprisingly, this stress-buffering effect appears only when adult squirrel monkeys are exposed to this situation together

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with multiple partners. In pair-housed individuals no social buffering was evident, although the behavioral signs of arousal were reduced (Coe et al., 1982). In this context, the findings of Mendoza and Mason (1986) are of special interest. They compared the effects of intruders on behavior and adrenocortical activities of polygynous squirrel monkeys and monogamous titi monkeys (Cullicebus rnoloch), housed as heterosexual pairs. In titi monkeys, the presence of an intruder resulted in marked behavioral signs of agitation, especially in the subjects of the same sex as the intruders. Plasma cortisol levels of females showed no consistent changes to intruders of either sex, while those of males were always increased in the presence of a male rival. Squirrel monkeys of both sexes, on the other hand, responded to female intruders with a reduction in plasma cortisol to below baseline levels, whereas a male intruder had no effect. Maintenance of a monogamous social structure, such as in titi monkeys, is presumably based on a bond between the male and female of the pair and the exclusion of male rivals by the male. In squirrel monkeys, which usually live in large groups of both sexes, life as a pair is lacking in the usual companionship and could be improved by new individuals. Although this interpretation is not without contradiction, these results nevertheless demonstrate impressively that the social system of a species influences the behavioral and physiological responses of the individuals to conspecifics in very different ways. The relevance of the quality of the relationships between individuals on their stress-reducing effects are also evident from guinea pigs (Sachser et ul., 1998). In mixed-sex colonies male guinea pigs develop long-lasting and strong bonds to some females, while no such social ties exist to other females. When male guinea pigs are taken from such colonies and placed singly into unfamiliar cages their plasma cortisol levels increase for hours by about 100%compared to initial levels. Presence of an unfamiliar female from a different colony or a familiar female from their own colony to which no social bonds exist has no stress-reducing effects. There is, however, a sharp reduction in the endocrine stress response when each male is transferred into an unfamiliar cage together with a female with whom a social bond exists (Fig. 35). The relevance of social integration and social bonds to members of a group is especially evident in our work on wild rabbits. Throughout the reproductive phase, each female usually produces 5-6 litters at monthly intervals, resulting in up to 30 progeny per year. Depending on the number of adult females in our enclosure, up to 1000 animals are born each year. However, about 70% of the young are taken by predators (e.g., cats, martens, weasels, hawks) before the onset of the winter season and only an average of 5% of the original number actually survive the winter. The

STRESS AND ITS RELEVANCE FOR ANIMAL BEHAVIOR

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c

c

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175

u) 0 ._

r

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$ 125 h 100

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bonded

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FIG. 35. Cortisol values ( M -C SEM) of 10 male guinea pigs 2 hr after transfer into an unfamiliar enclosure alone (dotted horizontal line), together with an unfamiliar female, a familiar but unbonded female, or a bonded female. All data are percentages of initial levels. Significant differences between the effects of the presence of bonded and unbonded conspecifics: *p < .05; **p < .01. Adapted from Sachser et al. (1997).

number of surviving juveniles varies from one year to the next (from 0 to more than 40 individuals) and is approximately equivalent to the number of adults that have died. Consequently, the number of adult rabbits at the beginning of each reproductive season has remained surprisingly constant over the past 10 years (at 50-95 individuals). Predation plays only a minor role in mortality during the winter months (November to February). Death is usually due to an extreme loss of weight, based on the breakdown of all fat reserves as well as large quantities of muscle tissue, culminating in hypoglycemic shock. Although these findings point to starvation, a general lack of food cannot be the reason for death, as all the adults as well as those juveniles that survive the winter show no loss in body weight. Rather, the moribund juveniles are incapacitated, in spite of increased food intake, by extensive parasitic damage to their intestinal epithelium, which prevents the digestion and/or resorption of food. In addition, toxins produced by the changed intestinal flora probably also contribute to the death of the animals. Within the last few weeks prior to death, the number of oocysts and nematode ova in the feces of the moribund juveniles increases dramatically and parallel to the loss in weight (Fig. 36). In comparison to the surviving individuals, this parasitic infestation is probably due to a reduced immune resistance against the parasites, as indicated by a reduced in v i m lymphocyte proliferation (Fig. 37), a de-

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Body weight

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FIG. 36. Changes of body weight and numbers of nematode eggs (predominantly Trichostrongylus retorfaefornzins and Gruphidium strigosum) and oocysts of several Elimera species in the feces of 20 subadult European wild rabbits during the last weeks before their death in the winter period. All data are means ( 2 SEM). See text for details.

creased number of T lymphocytes in the blood, and a 50% reduction in the phagocytic capacity of the leucocytes. Based on our current findings, mortality during the winter months appears to be a result of socially induced immune suppression: Young animals usually leave their native groups in autumn and attempt to join other groups (Kunkele and von Holst, 1996). In this process, all immigrants are initially attacked and chased away by members of the group. However, some juveniles are tolerated after a while and integrated into the group, although most do not achieve this social integration. The successful integration of a juvenile is indicated by its spatial position within the group and by its behavior toward the adults: Integrated animals restrict their whereabouts more or less exclusively to the existing territory of a group of adults, while nonintegrated animals tend to roam over a wide area and from one group to the next. In addition, integrated animals are observed either in close proximity to or in direct contact with individuals of a group during 30% of observation time, while this is seldom the case in nonintegrated individuals (Fig. 38: Spatial integration). Although aggressive reactions by adults are directed against integrated and nonintegrated juveniles with almost equal frequency, integrated animals are more often involved in friendly interactions with adults. While in integrated animals two out of three interactions with adults are of a friendly nature, nonintegrated animals are only involved in one sociopositive interaction for every three aggressive ones (Fig. 38: Social integration). Finally, both groups also differ significantly from each other in immunological measures: nonintegrated individuals clearly exhibit lower values than integrated animals (Fig. 38: Immune measures).

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FIG. 37. Body weight, parasites in feces, and length of the intestinal villi of 20 subadult wild rabbits at their death during the winter (Died) as well as their food intake and in vitro lymphocyte proliferation (LP) after Con A stimulation 2-6 weeks before their death. All measures were also determined at corresponding times from 20 animals of about the same age that survived the winter period (Surv). All data are means (rf- SEM); significant differences: **p < .01; * * * y < ,001.

The change in integration state was followed during the winter season in several juveniles: Individuals that were first more or less integrated within a group were expelled from it, and nonintegrated juveniles were accepted. In all cases this also involved changes in immunological parameters: If integration status deteriorated, then lymphocyte proliferation was reduced; if integration status improved, that is, in the case of successful integration into a group, proliferation increased (Fig. 39). Based on these findings, an improved immune state and a reduced parasitic infestation in juveniles surviving the winter would appear to be the result of successful integration into the existing social group. Accordingly, out of more than 100 animals observed in detail over 5 years, only those animals capable of successful integration into groups during the autumn and winter months actually survived the winter. The number of juvenile wild rabbits is therefore regulated during the winter season, by giving only those individuals that have achieved integra-

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Local attachment

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FIG. 38. Spatial integration. social integration, and immune measures of about 20 integrated ( I N ) and 30 nonintegrated (NI) subadult wild rabbits during the winter period ( M 2 SEM). Immunological parameters: means of 1-3 measurements per animal; behavioral data: means of 8-24 hr of observations per animal. Significant differences between IN and NI: **p < .01; ***p < ,001. See text for further details. Unpublished data from M. Kaschei (1996).

tion into an existing group a chance of surviving the winter. As the acceptance of juveniles into an existing group of adults is apparently dependent on the size and composition of the group, this mechanism results in optimal group composition before the onset of the reproductive season. As mentioned previously, numerous epidemiological studies on humans indicate that social bonds, in terms of social support, can play a positive role in the health of an individual. The direct physiological mechanisms are, however, far from being clear. As shown by the various studies on nonhuman mammals described above, a breakdown of social bonds elicits strong passive stress responses, while the presence of a bonded partner or group has some stress-buffering effects. Furthermore, the development of a bond can exert strong physiological consequences even in individuals that apparently beforehand had lived an unstressed life. Thus, tree shrews can be housed singly for more than 10 years in captivity and be in excellent condition without any apparent signs of stress. Neverthe-

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FIG. 39. Changes of the in virro lymphocyte proliferation after Con A stimulation of 8 subadult wild rabbits before and within 4 weeks after change of their social integration. Unpublished data from M. Kaschei (1996).

less, the formation of a pair bond greatly improves their well-being, as indicated by physiological data. Tree shrews usually live in pairs in the wild. Putting a male and a female together, however, does not inevitably lead to the formation of a pair bond. In some instances it can result in intensive fights and-unless the animals are separated-in the death of one of the opponents (male or female). In most cases, however, especially in large enclosures, tree shrews of both sexes can coexist, although they suffer from a certain amount of social tension, as evident from occasional fights and avoidance behavior. At estrus, successful copulations may even occur, but the offspring are always cannibalized by the parents shortly after birth (von Holst, 1969). In all these unharmonious pairings, even if overt aggression is not evident, the heart rates of the animals are constantly increased, as is the case in subdominant males living together with a dominant male under constant active stress (Fig. 40). In about 20% of all pairings, however, contact between an unfamiliar male and female is characterized from the outset by amicable behavior, which conveys the strong impression of “love at first sight.” Both individuals “greet” each other frequently with

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long bouts of mouth licking (up to 90 min per 12-hr observation day), they move around in close contact, and mostly rest together. Copulations may occur on the first day, but are not a necessary prerequisite for such a harmonious pair bond. During the nights both animals always sleep together in the same nest box, which is never the case in the previously mentioned unharmonious pairs. In the laboratory harmonious pairs can live together for more than 10 years and breed successfully and regularly in the absence of any aggression. In all harmonious pairs we found a drastic reduction in serum levels of glucocorticosteroids and adrenocortical reactivity to standard stressors, and-even more surprising-a reduction in heart rates (Fig. 40). Furthermore, all immunological parameters that were measured indicate an improvement of the immunological state of both individuals. The opposite is true for unharmonious pairings. Amazingly, the quality of a pairing depends on personal “sympathy” or “antipathy” between the individuals. Thus, a male that has been fiercely rejected by one female can be accepted as a “loved” partner by another female. Accordingly, the physiological status of tree shrews kept as pairs changes depending on the quality of their pair bond, as shown in studies in which females were paired with different males. In standard tests females respond to males that they will accept as partners with high marking responses, and to those that they will not accept with low marking responses. Hence it was possible to pair females once with males that they accepted as partners, and once with males that they rejected (Fig. 41). As the results of these pairings demonstrate, both sexes exhibited low levels of aggression and high levels of sociopositive behaviors when females were combined with males to whose scent they had shown the highest marking responses (harmonious pairs). The opposite was the case when the females were unharmoniously paired with males whose scent stimulated their marking behavior very little (Fig. 41). Furthermore, harmonious pairings decreased serum levels of glucocorticosteroids and epinephrine, while increasing those of gonadal hormones as well as improving cellular and humoral immune measures. The opposite was true in the same individuals in unharmonious pairings (Fig. 42). Unfortunately, little is known of the physiological effects of pair formation in other species, with the exception of several studies on monogamous and polygamous species of vole. In the prairie vole (Microtus ochrogaster), long-term heterosexual pair bonds are formed, which are characterized by affiliative behaviors, such as side-by-side contact, and are independent of sexual behavior (Carter et al., 1988, 1995; Winslow et al., 1993). In contrast to tree shrews, however, prolonged mating of naive females with an unfamiliar male is necessary for the induction of a pair bond in this species (Insel et al., 1995). The

-1

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disharmonious pairing I

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harmonious pairing

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single

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il

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Days after beginning of pairing experiment FIG.40. Effects of an unharmonious and a harmonious pairing on the heart rate of a female tree shrew. Night periods are striped. Data higher and lower (unharmonious and harmonious pairing, respectively) than the mean of the last 3 days before the pairings are accentuated by black. Adapted from von Holst (1987). with kind permission from Gustav Fischer Verlag, Stuttgart, Germany.

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Marking behavior

Sociopositive behavior

Defensive behavior

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.b >

m

$ 30

3.0

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Harm Unharm

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FIG. 41. The marking activities of females in response to the scent of different males were used to create harmonious (Harm) and unharmonious (Unharm) pairings (for details, see von Holst, 1985b). The more that the scent of a male stimulates the marking activity of a female the greater is the probability that the pairing with the female will result in a harmonious pair bond. Each female was therefore paired for 14 days with that male whose scent elicited the highest, and after 4 weeks of single housing with that whose scent elicited the lowest marking response (“Marking behavior”). During the pairings the behavior of each male and female was recorded for a total of 12 hr (“Sociopositive behavior” and “Defensive behavior”). All data are means ( 2 SEM); significant differences are indicated: ***p < .001.

Tes

Cor

Nor

Epi

LTT

IgG

FIG.42. The effects of harmonious (striped bars) and unharmonious (cross-hatched bars) pairings on several physiological measures of 12 male tree shrews. All data ( M ? SEM) are given as deviations from the initial levels of the males before the pairings. Abbreviations: serum levels of testosterone (Tes), cortisol (Cor), norepinephrine (Nor), epinephrine (Epi), and immunglobulin G (IgG); in vitro lymphocyte proliferation after Con A stimulation (LTT). Significant differences are indicated: ***p < ,001. See text for further details.

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results of many studies indicate that centrally released oxytocin during mating may be critical to the formation of partner preferences in female prairie voles, while vasopressin appears to be more important to pair bonding in the males of this species (e.g., Carter er al., 1992; Insel and Hulihan, 1995; Williams et al., 1992,1994). Furthermore, plasma glucocorticosteroid levels may respond to and influence the development of social attachments. In naive female prairie voles, cohabitation with a male resulted in a dramatic decline in serum corticosterone levels, which facilitated pair bonding. When corticosterone levels were reduced via adrenalectomy, females developed partner preferences after 1 hr of cohabitation, while sham-operated and untreated females required 3 hr or more of cohabitation to establish partner preferences (De Vries et al., 1995). The role of oxytocin in the development of social bonds was first proposed by Klopfer (1971), who suggested that the increased oxytocin levels after birth facilitate the mother-infant attachment. In subsequent studies, the relevance of centrally released oxytocin for the development of maternal behavior and the development of mother-infant bonds was shown in sheep, rats, and some other species (e.g., Da-Costa et al., 1996; Kendrick et al., 1987; Pedersen and Prange, 1985; Uvnas-Moberg, 1994; Yu et al., 1996). Furthermore, many studies demonstrated the relevance of oxytocin for sexual behavior (in addition to sex hormones). Thus, injections of oxytocin in estrous rats stimulates sexual behavior in female rats, reduces aggression, and increases physical contact with the males (Arletti and Bertolini, 1985; Caldwell et al., 1986); similar results were also found in female Syrian hamsters (Whitman and Albers, 1995). These data indicate that oxytocin may be involved in the formation of social bonds between mothers and their infants as well as between males and females in mammals (Carter et al., 1990; Keverne, 1988). Shared sexual experience and the concomitant oxytocin release usually found in both sexes may thereby facilitate social bonds in mammals including human beings. In addition, stress-buffering effects of intracerebroventricular injections of oxytocin have been described. In laboratory rats, the development of gastric lesions induced by cold and restraint stress or by the administration of cysteamine was reduced by oxytocin treatment (Grassi and Drago, 1993). The physiological mechanisms involved are unsolved, but nevertheless, these results indicate that social bonds may improve the health of individuals by reducing their response to stressors (probably by reducing the stress-inducing properties of stressors, due to the presence of a security-providing partner), as well as by influencing the physiological state of the individual.

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C. CONCLUSIONS Social relationships based on agonistic and sociopositive behaviors play an important role in most mammalian societies. They determine not only the stability of social systems but greatly influence almost all behavioral elements of the individuals as well as their fertility and health. Disturbances of social relationships may lead to stress responses that differ greatly in quality and intensity, depending on the stressor and the coping behavior of the individuals. Disruption of social bonds usually initially elicits an alarm response characterized by heightened physiological and behavioral arousal. Particularly in infants separated from their mothers, this is followed by passive stress responses characterized by apathetic behavior, withdrawal, and eventually death. Information on the long-term consequences of disruption of social bonds for the health of adults is, however, lacking for nonhuman mammals. In human beings, the loss of partners can have very strong healthimpairing effects. Social conflict elicits an immediate acute alarm response in all animals, characterized by increased sympathetico-adrenomedullary and pituitaryadrenocortical activation. There is evidence that norepinephrine (the fight hormone) predominates in this first response to challenges, which is probably characterized by the feeling of anger. If the stressful situation cannot be resolved by behavioral responses (e.g., by fight or flight), differing chronic stress responses may result, the degree of which is dependent on the perception of the amount of control over a social situation, and which are therefore almost exclusively psychological phenomena. The perception that loss of control is either possible or probable appears to lead to a change from anger to fear, as is indicated by an increasing production of epinephrine (the flight hormone) and mainly active subordinate behavior. As the threatening situation continues, this active coping can shift to a more passive, apathetic mode, accompanied by greatly increased adrenocortical activity and associated with the feelings of helplessness and depression. The adrenomedullary epinephrine release can remain high or decrease by comparison to actively coping individuals. Gonadal activity (at least in males) may actually increase in early phases of successful responses to challenge (during anger), but eventually declines as loss of control threatens. The immune system responds extremely sensitively to social challenges. Every challenge to control (feelings of anger, fear, and depression) is usually accompanied by profound indications of immunosuppression. The relationships between social rank and stress response depend mostly on the stability and predictibility of the social relationships. In stable social systems the dominants are usually not, or only slightly, stressed: Com-

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pared to subordinate individuals, they exhibit lower adrenocortical- and sympathetico-adrenomedullary activities as well as higher gonadal activities and immune resistance. Subordinate individuals usually show active stress responses, but in some cases also passive stress responses, which usually lead to death within a short period of time. In socially unstable systems, which are characterized by immigration processes or dominance conflicts, all individuals usually show active stress responses of varying degrees of strength as they fight to regain high control and predictability. The effects on dominants and subdominants, however, may differ depending on the species and the social situation. In many species, the highest active stress responses are found in those individuals that are dominant and fight actively to maintain their high social ranks. This may even lead to higher incidences of cardiovascular diseases and premature death, in comparison to subordinate individuals. The neuroendocrine stress responses accompanying these subjective feelings have a bipolar aspect: According to a concept proposed by Henry in 1986, the anger-fear (fight-flight) response is opposed by the serenityrelaxation state, which is characterized by enhanced grooming and resting. The opposite pole to the depression, loss-of-control, and loss-of-attachment axis, is probably a subjective feeling of elation, such as in dominant tree shrews in the presence of clear subordinate individuals-and probably also in animals with strong bonds to partners (Fig. 43). Because social relationships can influence the physiological state of individuals in so many positive or negative ways, it is not surprising that social status alone cannot always predict stress-related measures in individuals, particularly in natural conditions containing many uncontrollable social influences. This means that, in order to understand the physiological consequences of social interactions, an integrated approach is required to assess what factors, including rank and social bonds, interact to affect an individual’s fertility and health (see also Fig. 44). O r as Sapolsky (1988) puts it in his discussion of individual differences in olive baboons and their stress responses: Thus, among these primates, who you are. what your place is in your society, and what sort of society it is appear to have everything to do with your physiology, both under basal and stressed circumstances. Furthermore, one may argue at this stage that these rank-related differences in physiology are of consequence, that the pattern observed in dominant males seems to be the most adaptive.

And these comments on baboons apply to all mammalian species, from rodents to humans, as pointed out as long ago as 1977 by Henry and Stephens.

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FIG.43. Schematic diagram of the stress-buffering emotional processes and their physiological consequences. Adapted from Henry (1986). with kind permission from Academic Press, Inc., New York.

IV. SUMMARY Contact with conspecifics not only influences the behavior of individuals, but is also associated with marked physiological changes, which can influence their vitality and fertility in positive or negative ways, depending on the type of interaction. The term used to describe the negative effects is

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+pq dominant

Acute stress Chronic

passive stress

FIG. 44. Schematic diagram of the two stress axes (PAS, pituitary-adrenocortical system, and SAS, sympathetico-adrenomedullarysystem) and their activation (+) or inactivation (-) depending on the social control perceived by the individuals and the associated subjective emotions. As an example, the physiological states of tree shrews in the differing social situation described in this paper are given in circles. See text for further details.

social stress. In this chapter, the many negative physiological consequences of social stress are addressed and the stress-reducing effects of sociopositive contacts with conspecifics are described. The changes that have taken place over the past 30 years in the concept of stress are an important prerequisite to understanding the effects of social interactions on the physiology of individuals. As the current concept of stress has been developed within the boundaries of psychology and medicine, largely excluding the field of zoology, a short synopsis of this development is given. According to this concept, physiological stress reactions are generally triggered by central nervous processes (emotions or feelings), which always occur when a situation is characterized by uncertainty or unpredictability, that is, when the individual’s control over the situation is endangered or impossible. Differing physiological stress reactions are induced, depending on the behavioral strategy used by an animal to either obtain control or to cope with the situation. Active attempts at obtaining control over a situation (e.g., fight or flight) are characterized initially by

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activation of the sympathetico-adrenomedullary system and in the long term by cardiovascular disease (active stress). Passive perception of defeat or loss of control is characterized initially by pituitary-adrenocortical activation and in the long term by negative effects on almost all bodily functions (passive stress). In a second section, such peripheral physiological processes are described as are essential to the understanding of stress reactions. Since various stress reactions differ depending on the situation and the coping behavior of the individuals, studies based on one or only a few measures can lead to misleading or false conclusions. An introduction is also given into the most frequently used methods in obtaining indications of the activities in the pituitary-adrenocortical and sympathetico-adrenomedullary systems, as well as in gonadal and immune functions. Particular attention is paid to the limitations of these methods. In a third section, based on our research on tree shrews, an overview of the relationships between the social position of an animal and its physiological state is given. General statements cannot be made due to the close relationships between social system, social rank and rank stability, and the impact of positive relationships with other conspecifics. However, in stable social systems, an overall dominant position can improve the fertility and vitality of the individual, while subordinate individuals exhibit no or only slight active or passive stress reactions, dependent on the species. In unstable social systems, dominant individuals are characterized by particularly high active stress reactions, due to their efforts of improving or retaining their position through increased levels of aggression; subordinate animals exhibit active or passive stress reactions of variable intensities. Finally, the importance of social bonds for the health of individuals is assessed: The loss of social bonds can provoke long-term stress reactions, while the presence of bonded partners has stress-reducing effects. Because social relationships can influence the physiological status of individuals in so many positive or negative ways, social status alone cannot always predict stress-related measures in individuals, especially in natural conditions containing many uncontrollable social influences. Consequently, in order to understand the physiological consequences of social interactions, an integrated approach is required to assess which factors, including rank and social bonds, interact to affect an individual’s fertility and health. Acknowledgments

This chapter is dedicated to my late friend James Henry whose scientific results and concepts were twenty years ahead of his time. I wish to thank Norbert Sachser for his critical comments, which greatly improved this manuscript. Thanks are also due to Debby Curtis for her most

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valuable help with the English version on this paper. 1 also appreciate the editors’ helpful remarks on the manuscript.

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