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Principal Stress and Behavior
Stress and BehaviorAnders Pape Møller, Manfred Milinski and Peter J.B. Slater (Eds.)
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Advances in THE STUDY OF BEHAVIOR VOLUME 27 Advances in THE STUDY OF BEHAVIOR Edited by PETER J. B. SLATER JAY S. ROSENBLATT CHARLES T. SNOWDON MANFRED MILINSKI Stress and Behavior A Volume in Advances in THE STUDY OF BEHAVIOR VOLUME 27 Edited by ANDERS PAPEMOLLER Lahoratoire d'Ecologie UniversitP Pierre et Marie Curie Paris, France MANFRED MILINSKI Zoologisches Institut Ahteilung Verhalten-sokologie Universitat Bern Hinterkappelen, Switzerland PETER J . B. SLATER School of Environmental and Evolutionary Biology University of St. Andrews Fife, United Kingdom ACADEMIC PRESS San Diego London Boston New York Sydney Tokyo Toronto T h k book Copyright ir, p r i n t e d on acid-free paper. @ 0 1998 by A C A D E M I C PRESS A l l Rights Reserved. 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Copy fee\ for pre-199X chapters are as shown on the t i t l e pages. If no fee code appears on the title page, the copy fee i s the hame as for current chapters. 0065-3454/9X $25.00 Academic Press (I dii,i.\;mi o/'Iioi.c~orrrt Brui.i. & C'onilitrii? 525 B Street. Suite 1900. San Diep, California 97_101-4495.USA http:/Jwww.apnet.com Acadeniic Press Limited 24-2X Oval Road, London N W I 7DX. U K http://www.Iibuk.co.uk/ap/ International Standard Book Number: 0-1 2-004527-3 PRINTED IN THE UNITED STATES OF AMERICA 9X 99 00 01 01 03 Q W 9 8 7 6 5 4 3 2 I Contents Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix xi ... Xlll The Concept of Stress and Its Relevance for Animal Behavior DIETRICH VON HOLST I. I1. I11. IV . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Concept of Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Social Stress in Mammals . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2 42 106 109 Stress and Immune Response VICTOR APANIUS 1. I1. I11. IV. V. Vl . VII . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Nature of Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Nature of Immunocompetence . . . . . . . . . . . . . . . . . . Neurological Linking of Stress and Immunocompetence . . Endocrine Linkage of Stress and Immunocompetence . . . . Why Stress Alters Immunocompetence . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 134 136 140 142 145 149 150 Behavioral Variability and Limits to Evolutionary Adaptation under Stress P. A . PARSONS 1. I1. I11. IV . V. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Energy Limits to Adaptation . . . . . . . . . . . . . . . . . . . . . . . Variability and the Survival of Variants . . . . . . . . . . . . . . . Extending the Limits of Adaptation . . . . . . . . . . . . . . . . . . From Stress-Resistance Genotypes to a Connected Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V 155 158 164 165 169 174 174 vi CONTENTS Developmental Instability as a General Measure of Stress ANDERS PAPE M@LLER I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Genetic and Environmental Determinants of Developmental Instability . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Directional Selection and Developmental Instability . . . . . IV . Fitness Correlates of Developmental Instability . . . . . . . . . V . Practical Uses of Developmental Instability . . . . . . . . . . . . VI . Conclusions and Prospects for Future Studies . . . . . . . . . . VII . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181 186 190 192 193 206 208 208 Stress and Decision-Making under the Risk of Predation: Recent Developments from Behavioral. Reproductive. and Ecological Perspectives STEVEN L . LIMA I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Behavior of Feeding Animals: Classical Motivations . . . . . 111. Patterns of Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . After an Encounter with a Predator . . . . . . . . . . . . . . . . . . V . Social Situations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII . Long-Term Consequences of Decision Making . . . . . . . . . VIII . Ecological Influences and Implications . . . . . . . . . . . . . . . . IX. Additional Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . X . Conclusions and Summary . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 217 225 235 239 241 245 248 261 264 265 Parasitic Stress and Self-Medication in Wild Animals G. A . LOZANO I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Self-Medication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Prophylactic Se1f.Medication . . . . . . . . . . . . . . . . . . . . . . . . IV. Therapeutic Self-Medication . . . . . . . . . . . . . . . . . . . . . . . . V . Skepticism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Behavioral Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291 293 294 298 303 304 CONTENTS VII . Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII . Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii 308 310 311 Stress and Human Behavior: Attractiveness. Women's Sexual Development. Postpartum Depression. and Baby's Cry RANDY THORNHILL AND F. BRYANT FURLOW I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Human Attraction and Attractiveness . . . . . . . . . . . . . . . . I11. Parent-Daughter Relations and Women's Sexual Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . Postpartum Depression . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Infant Crying as a Signal of Phenotypic Quality . . . . . . . . . VI . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319 321 332 341 352 356 359 Welfare. Stress. and the Evolution of Feelings DONALD M . BROOM I . Feelings. Their Role and Their Evolution . . . . . . . . . . . . . I1. Welfare. Stress. and Feelings . . . . . . . . . . . . . . . . . . . . . . . . I11. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371 394 400 401 Biological Conservation and Stress HERIBERT HOFER AND MARION L . EAST I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Stress in a Conservation Biology Context . . . . . . . . . . . . . . I11. Designing a Conservation Study to Measure Stress and Its Impact . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. The Natural History of Stress . . . . . . . . . . . . . . . . . . . . . . . V . Effects of Anthropogenic Stressors . . . . . . . . . . . . . . . . . . . VI . Conservation Research and Management Activities as Stressors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII . The Equivalence of Natural and Anthropogenic Stressors 405 407 420 428 452 473 486 ... Vlll CONTENTS VIII . Minimizing Occurrence and Impact of Stress in Conservation Research and Management . . . . . . . . . . . . . . IX . Conclusions: How Important Is Stress in Biological Conservation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 488 494 496 497 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 527 Contents of Previous Volumes . . . . . . . . . . . . . . . . . . . . . . . . . . . 549 Contributors Numbers in parentheses indicate the pages on which the authors' contributions begin. VICTOR APANIUS (133),Department of Biological Sciences, Florida International University, University Park, Miami, Florida 33199 DONALD M. BROOM (371), Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 OES, United Kingdom MARION L. EAST (405), Max -Planck -1nstitut f i r Verhaltensphysiologie, 0-82319 Seewiesen Post Starnberg, Germany BRYANT FURLOW (319). Department of Biology, University of New Mexico, Albuquerque, New Mexico 87131 HERIBERT HOFER (405). Max -P/anck -1nstitut far Verhaltensphysiologie, D 82319 Seewiesen Post Starnberg, Germany DIETRICH VON HOLST (1). Department of Animal Physiology, University of Bayreuth, 95440 Bayreuth, Germany STEVEN L. LIMA (215), Department of Life Sciences, Indiana State University, Terre Haute, Indiana 47809 G. A. LOZANO (291), Department o,f Biology, University of California, Riverside, Calljornia 92522 ANDERS PAPE M0LLER (181), Laboratoire d'Ecologie, UniversitP Pierre et Marie Curie, Paris Cedex 5, France P. A. PARSONS (155), School of Genetics and Human Variation, La Trobe University, Bundoora, Victoria 3083, Australia RANDY THORNHILL (31Y), Department of Biology, University of New Mexico, Albuquerque, New Mexico 87131 ix This Page Intentionally Left Blank Preface The aim of the Advances in the Study of Behavior series remains as it has been since the series began: to serve the increasing number of scientists who are engaged in the study of animal behavior by presenting their theoretical ideas and research to their colleagues and to those in neighboring fields. We hope that the series will continue its "contribution to the development of cooperation and communication among scientists in our field," as its intended role was phrased in the preface to the first volume in 1965. Since that time, traditional areas of animal behavior research have been given new vigor through the links they have formed with related fields and by the closer relationship that now exists between those studying animal and human subjects. Beginning with Volume 25, which was on the special topic of Parental Care, we departed from the previous policy of publishing articles on varied subjects in every volume. This volume, titled "Stress and Behavior," is the second thematic volume. The next volume will again be a broad-ranging one, as was the last, and it is our intention to continue the series with this mixture of wide-ranging volumes of eclectic interest and occasional volumes focusing on particular themes that appear timely to us. Although volumes such as this do represent a new initiative, they do not, we believe, violate the basic principles underlying the series. The specific theme of Stress and Behavior was chosen because it is an especially exciting and active area of research at present, and one to which researchers with a wide range of approaches and backgrounds are making important contributions. We have invited as contributors leading experts across this range, thus giving a truly multidisciplinary perspective on the topic. For this volume we have been fortunate to be joined by Dr. Anders Pape Mflller as guest editor, and his expertise in the area has been of immense help to us. Sadly, this will also be the last volume for which Dr. Manfred Milinski will act as an associate editor: he has made a valuable contribution to the series, for which we are very grateful. His place will be taken by Dr. Tim Roper, of the University of Sussex, and we look forward to benefiting from his broad interests and well-honed editorial skills in future volumes. P. J. B. Slater xi This Page Intentionally Left Blank Stress & Behavior Introduction All organisms suffer from a deficiency of one or more resources during their lifetime, and conditions for development, growth, survival, and reproduction are rarely, if ever, optimal. This was realized by Charles Darwin, who used competition among conspecifics for limiting resources as a cornerstone of his theory of natural selection. Although suboptimal living conditions are widespread, the relationship between environmental conditions and behavior has not attracted much attention from scientists (for exceptions, see Hoffmann and Parsons, 1989; Maller and Swaddle, 1997). The conditions under which organisms live are frequently suboptimal, and the difference between suboptimal and optimal conditions is often perceived by individuals as causing a change in their state. Stress is an appealing but illusive concept in biology, with definitions being almost as numerous as the different fields of research. Although stress can be defined explicitly in terms of concentrations of biochemicals involved in metabolism (Ivanovics and Wiebe, 1982), a general but still operational definition is that provided by Hoffmann and Parsons (1989): the state caused by any agent that results in suboptimal performance and potentially causes permanent damage to an individual. Life has evolved under stressful conditions, although exceptions exist, such as the relatively constant environments experienced by organisms living in caves and hot springs. The fact that organisms generally live and reproduce under adverse environmental conditions has not been well appreciated by the majority of the community of biologists. Theoretical evolutionary biology has not considered stress to be of overriding importance. For example, Sewall Wright's famous shifting balance theory is based on the concept of a fitness landscape with multiple valleys and peaks. The unappreciated fact that different phenotypes may be expressed under poor and optimal conditions may have enormous effects on the ability of species to evolve from one peak to another. Empirical evolutionary biologists have performed most of their studies on fruit flies, mice, and rats under benign laboratory conditions where stress is much reduced compared to natural situations. Interestingly, phenotypic and genetic parameters are not congruent or even comparable in such situations, as revealed by recent studies of animals reared under more and less benign conditions (Bijlsma and Loeschcke, 1997; Maller and Swaddle, 1997). This discrepancy between conditions under which animals are studied and conditions under which they live also applies to behavioral research. ... Xlll xiv INTRODUCTION Laboratory studies are generally performed under benign conditions with ad libitum availability of food and a virtual absence of predators and para- sites. Field studies are predominantly conducted in optimal habitats with high population densities, whereas much less is known about behavior of animals in low-quality, marginal habitats where stress is predominant. Behavior under adverse environmental conditions is a topic of general interest for two reasons. First, such studies may provide us with a better understanding of the conditions under which most evolution has taken place. Second, studies of behavior under stressful conditions may give us a better understanding of the consequences of global change, as well as provide us with important information on conservation biology. In the first chapter, von Holst describes how social relationships, especially aggressive ones, can influence the physiological state of individuals in many positive and negative ways. This chapter introduces the neuroendocrinology of the stress response. Stressed organisms are easy targets of infectious diseases. The complex strategic use of the immune system sometimes affords suppression of immune defenses under stress as discussed by Apanius in the second chapter. In the third chapter, Parsons presents an energetic approach to the fitness of organisms that are challenged by biotic and abiotic stress. Under stressed free-living conditions, his environmental model suggests that favored "good genotypes" are likely to be stress resistant and heterozygous. In the fourth chapter, Mgller discusses developmental stability as a reflection of the ability of organisms to buffer their developmental trajectories against disturbance. The inability to fulfill this goal can be assessed in terms of fluctuating asymmetry, i.e., random deviations from perfect symmetry, and used for studies of, for example, environmental monitoring, animal welfare, and human medicine. In the fifth chapter, Lima discusses the many ways in which behavioral decision-making alters the nature of predator-induced stress. In the sixth chapter, Lozano discusses the growing evidence that wild animals use self-medication in response to parasitic stress. In chapter 7, Thornhill and Furlow examine how stress interacts with human behavior in relation to physical attractiveness, the development of women's sexuality, and parental investment in babies. The biological function of feelings such as pain and fear may be to affect an organism's behavior in such a way that it maximizes the chances that good things will happen and minimizes the chances that bad things will happen. Broom discusses welfare, stress, and the evolution of feelings in chapter 8. The last chapter, by Hofer and East, reviews why stress has important implications for biological conservation and considers practical ways in which conservationists can identify and tackle problems due to stress. INTRODUCTION xv We believe that a concerted research effort concerning behavior of animals living under adverse environmental conditions will add considerably to our understanding not only of the role that behavior plays in the general coping strategies of animals, but also of the flexibility of behavioral strategies under variable environmental conditions. The authors of the chapters of this thematic volume of Advances in the Study of Behavior have shown some ways in which this may be achieved. A. P. M@ller M. Milinski References Biljsma, R. and Loeschcke, V. (eds.) (1997). "Stress. Adaptation. and Evolution." Birkhauser. Basle. Hoffmann, A. A. and Parsons, P. A. (1989). "Evolutionary Genetics and Environmental Stress." Oxford University Press, Oxford. Ivanovics, A. M. and Wiebe, W. J. (1982). Toward a working definition of stress: A review and critique. In "Stress Effects on Natural Ecosystems" (G. W. Barrett and R. Rosenberg, eds.), pp. 13-27. Wiley, New York. M@ller,A. P. and Swaddle, J . P. (1997). "Asymmetry, Developmental Stability and Evolution." Oxford University Press, Oxford. This Page Intentionally Left Blank 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 Copyright 0 1998 by Academic Press All rights of reproduction in any form reserved 0065-3454198 $ZS.MI 2 DIETRICH VON HOLST 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. OF STRESS 11. THECONCEPT 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 STRESS AND ITS RELEVANCE FOR ANIMAL BEHAVIOR 3 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. OF THE STRESS CONCEPT B. DEVELOPMENT 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 Decreased clotting time I I I I 1 ventilation increased Increased blood flow to brain, heart, and skeletal muscles Lipolysis I 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). I STRESS A N D ITS RELEVANCE FOR ANIMAL BEHAVIOR 5 "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 6 DIETRICH VON HOLST 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 STRESS AND ITS RELEVANCE FOR ANIMAL BEHAVIOR 7 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 8 DIETRICH VON HOLST 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 STRESS AND ITS RELEVANCE FOR ANIMAL BEHAVIOR 9 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 I t-7 Nonshock 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. 10 DIETRICH VON HOLST 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 11 STRESS AND ITS RELEVANCE FOR ANIMAL BEHAVIOR 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). Perceived stimulus Threat to cont ss of control Active behavior @ -, Passive behavior (Fight -flight) (Nonaggressive) Behavioral arousal with challenge to status Inhibition of spatially organized behaviors and status Defense reaction Territorial or status control with mobility, display, and Defeat reaction Loss of territorial or status control, low sex drives I I YAnger") Control Striving ("Fear") Loss of control ("Depression") Norepinephrine Epinephrine t t tc tc t cf Glucocorticosteroids cc t Sex hormones t 4 t 4 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. 12 DIETRICH VON HOLST 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 13 STRESS AND ITS RELEVANCE FOR ANIMAL BEHAVIOR 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 700 1 I I Males raised -f in colonies -0 individually - m s m - a .500 E c 0 100 -1 0 -5 0 5 10 15 20 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. 14 DIETRICH VON HOLS'I 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 STRESS AND ITS RELEVANCE FOR ANIMAL BEHAVIOR 15 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 16 DIETRICH VON HOLST 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 -a, -a, 150 12 males per sampling point u) Q 125 .*0 .. -0- * * 75 -: 0 --c COrtlSOl - Norepinephrine .-m. Testosterone 5 10 15 Days after transfer into a new room 20 FIG. 5. Some endocrine responses of male tree shrews after a transfer into an unfamiliar room. Significant differences to initial values: * p < .05. STRESS AND ITS RELEVANCE FOR ANIMAL BEHAVIOR 17 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 21 -u=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. STRESS A N D ITS RELEVANCE FOR ANIMAL BEHAVIOR 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 - c 0 2 L Male 475 dominant Daily confrontations and visual contact 5 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 STRESS A N D ITS RELEVANCE FOR ANIMAL BEHAVIOR 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 0 55 - 0 z 10 . F a 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 ha