**5. Integration and regulation**

The stress response is characterized by the interplaying of several centers, mechanisms and systems, in order to reestablish the homeostatic conditions. Although it has been known for long time that the stress response is initiated by the activation of the Sympathetic-Chromaffin (SC) and the hypothalamus-pituitary-interrenal (HPI) axes, it was also known from the beginning that other regulatory systems are involved from the initial stages in the building of the stress response. Thus, it was already described by Selye that some pathological components were usually included in the events related to the General Adaptation Syndrome, such as gastrointestinal ulcers and thymolymphatic atrophy, clear signs of immunosuppressed status. Later on, an increasing number of evidences obtained during the last decades, both in lower and higher vertebrates, indicate that the stress response includes also the immune system. Even at the early life stages, the neuroimmunoendocrine interaction is active under stress. Hence, an important number of interconnections are established after stress, not only between the nervous and the endocrine system, but also between the endocrine and immune system, thus constituting a complex network of transmitters between the three regulatory systems. Although this aspect has not been extensively studied in all vertebrates (Verburg-Van Kemenade et al., 2009), it seems apparent that in all of them the nervous, endocrine and immune systems do not operate independently but rather they are part of the repertoire of the physiological

The Endocrine Response to Stress - A Comparative View 279

central stress system, with CRH and noradrenalin stimulating each other primarily through CRH type 1 and *α*1-noradrenergic receptors, respectively. Autoregulatory negative feedback loops are also present in both the PVN, CRH and brainstem noradrenergic neurons, with collateral fibers inhibiting CRH and catecholamine secretion via presynaptic CRH and *α*2 noradrenergic receptors, respectively. Both the CRH and the noradrenergic neurons also receive stimulatory innervations from the serotoninergic and cholinergic systems, and inhibitory input from the *γ*-aminobutyric acid (GABA)-benzodiazepine (BZD) and opioid peptide neuronal systems of the brain, as well as from the end-product of the HPA axis, the GCs (Charmandari et al., 2005). It is likely that dysfunctions of information processing across these circuits, resulting from environmental adversity and/or genetic factors,

In terms of the bidirectional relationships between endocrine and immune systems many of the hormones assessed so far can have an influence on immune agents or mechanisms, and even more some of them play significant roles in the regulation of the immune response. This is the case of the hypothalamic CRH and the adrenal hormone cortisol. The other axis related to stress, the sympathetico-chromaffin axis also influences immune response through catecholamines. In the reverse direction, the majority of evidence indicates that either direct or indirect stimulation of hypothalamic CRH secretion is the primary means by which cytokines (IL-1, IL-6, and TNF-α) activate the HPA axis. Thus, the neural pathways which IL-1 have been proposed to influence the neuroendocrine hypothalamus in mammals are numerous and diverse, including the inhibitory effects of either inhibitors of prostaglandin synthesis or disruption of catecholaminergic input into the hypothalamus

Moreover, a reduced number of family molecules appear consistently in the interconnection pathways in all vertebrates. For example, immunocytes can release Proopiomelanocortin (POMC)-derived peptides which have been shown to be involved in the regulation of the immune system. The Growth Hormone can stimulate the activity of the immune system. Sympathetic neurotransmitters can modulate the respiratory burst activity of trout phagocytes. Melanotropins, Melanocyte Stimulating Hormone (MSH), and Melanocyte Concentrating Hormone, (MCH) exert relevant stimulatory effects of the immune system. In mammalian species POMC is produced not only by neuroendocrine tissues, but also in lymphoid cells, which reinforces the neuroimmunoendocrine connection. More work has to be done to ascertain the regulatory mechanisms, and in particular, the role of the receptors and the role of putative paracrine mechanisms that may explain at some extent the

This may be the case of organs such the head kidney in fish, as it concentrates several secretory cells belonging to either immune or endocrine systems. It should be emphasized that in fish the head kidney or pronephros is an important centre of the endocrine response. In fact this organ becomes key player in the organization of the integrated response to stressors. Head kidney plays a substantial endocrine role in the secretion of the two main hormones of the hormonal axes, cortisol from interrenal cells, the major fish glucocorticoid and mineralocorticoid, and catecholamines, released by chromaffin cells which are components of the sympathetic nervous system releasing, and receiving specific connections from the central nervous system [30]. Moreover, not only the neural and the endocrine system meet in this organ, but also the immune and hematopoietic systems are found in the pronephros. Thus, this organ is the homologous to the bone marrow of higher vertebrates which involves that the production of lymphocytes, monocytes and neutrophils is localized

generate alterations that can culminate in maladaption.

interconnection mechanisms between regulatory systems.

(Turnbull and Rivier, 1999)

responses available to react in front of particular circumstances out of the normal physiological range, i.e.: disease, exercise, extreme environmental changes. Communication between these three physiological systems has had less attention in lower vertebrates compared to mammals, mainly because the amount of effort dedicated to such species is less and because of the lack of specific biochemical tools. However, such functional connections are present in all vertebrates and therefore this interconnection network may be an early mechanism in the evolution (see figure 2).

Fig. 2. Stress axis and main neuroimmunoendocrine interactions

These interactions start already at the central levels and there are multiple sites of interaction among the various components of the stress system. Neural control of stress is a complex process that requires the integration of information regarding both actual and potential outcomes. The organization of stress pathways further suggests that inputs on the physiological status of the animal can contribute to the eventual endocrine or autonomic response to the stressor. The majority of the pathways initiating physiological stress responses seem to be made at the level of limbic structures, which communicate information to subcortical sites positioned to interface with ongoing homeostatic feedback (Ulrich-Lai and Herman, 2009). The physical separation of autonomic and HPA stress effector circuits promotes some degree of independence of the two stress-modulatory cascades, allowing for appropriate tuning of neural and hormonal responses to specific demand characteristics of the actual or anticipated event. However, these two physiological systems also work together, both in terms of overlap in their underlying neural circuitry and in terms of their physiological functions. Charmandari et al., (2005) reviewed some of these connections. Reciprocal neural connections exist between the CRH and noradrenergic neurons of the

responses available to react in front of particular circumstances out of the normal physiological range, i.e.: disease, exercise, extreme environmental changes. Communication between these three physiological systems has had less attention in lower vertebrates compared to mammals, mainly because the amount of effort dedicated to such species is less and because of the lack of specific biochemical tools. However, such functional connections are present in all vertebrates and therefore this interconnection network may be an early

Hypothalamus

CRH ceruleus

GH CRH

These interactions start already at the central levels and there are multiple sites of interaction among the various components of the stress system. Neural control of stress is a complex process that requires the integration of information regarding both actual and potential outcomes. The organization of stress pathways further suggests that inputs on the physiological status of the animal can contribute to the eventual endocrine or autonomic response to the stressor. The majority of the pathways initiating physiological stress responses seem to be made at the level of limbic structures, which communicate information to subcortical sites positioned to interface with ongoing homeostatic feedback (Ulrich-Lai and Herman, 2009). The physical separation of autonomic and HPA stress effector circuits promotes some degree of independence of the two stress-modulatory cascades, allowing for appropriate tuning of neural and hormonal responses to specific demand characteristics of the actual or anticipated event. However, these two physiological systems also work together, both in terms of overlap in their underlying neural circuitry and in terms of their physiological functions. Charmandari et al., (2005) reviewed some of these connections. Reciprocal neural connections exist between the CRH and noradrenergic neurons of the

Immune effector cells

IL1 IL6 TNFα citoquines

cytokines

Locus

NA

mechanism in the evolution (see figure 2).

AVP

Pituitary

ACTH

GC

Fig. 2. Stress axis and main neuroimmunoendocrine interactions

TSH FSH ACTH PRL α-MSH β-End

Adrenal

central stress system, with CRH and noradrenalin stimulating each other primarily through CRH type 1 and *α*1-noradrenergic receptors, respectively. Autoregulatory negative feedback loops are also present in both the PVN, CRH and brainstem noradrenergic neurons, with collateral fibers inhibiting CRH and catecholamine secretion via presynaptic CRH and *α*2 noradrenergic receptors, respectively. Both the CRH and the noradrenergic neurons also receive stimulatory innervations from the serotoninergic and cholinergic systems, and inhibitory input from the *γ*-aminobutyric acid (GABA)-benzodiazepine (BZD) and opioid peptide neuronal systems of the brain, as well as from the end-product of the HPA axis, the GCs (Charmandari et al., 2005). It is likely that dysfunctions of information processing across these circuits, resulting from environmental adversity and/or genetic factors, generate alterations that can culminate in maladaption.

In terms of the bidirectional relationships between endocrine and immune systems many of the hormones assessed so far can have an influence on immune agents or mechanisms, and even more some of them play significant roles in the regulation of the immune response. This is the case of the hypothalamic CRH and the adrenal hormone cortisol. The other axis related to stress, the sympathetico-chromaffin axis also influences immune response through catecholamines. In the reverse direction, the majority of evidence indicates that either direct or indirect stimulation of hypothalamic CRH secretion is the primary means by which cytokines (IL-1, IL-6, and TNF-α) activate the HPA axis. Thus, the neural pathways which IL-1 have been proposed to influence the neuroendocrine hypothalamus in mammals are numerous and diverse, including the inhibitory effects of either inhibitors of prostaglandin synthesis or disruption of catecholaminergic input into the hypothalamus (Turnbull and Rivier, 1999)

Moreover, a reduced number of family molecules appear consistently in the interconnection pathways in all vertebrates. For example, immunocytes can release Proopiomelanocortin (POMC)-derived peptides which have been shown to be involved in the regulation of the immune system. The Growth Hormone can stimulate the activity of the immune system. Sympathetic neurotransmitters can modulate the respiratory burst activity of trout phagocytes. Melanotropins, Melanocyte Stimulating Hormone (MSH), and Melanocyte Concentrating Hormone, (MCH) exert relevant stimulatory effects of the immune system. In mammalian species POMC is produced not only by neuroendocrine tissues, but also in lymphoid cells, which reinforces the neuroimmunoendocrine connection. More work has to be done to ascertain the regulatory mechanisms, and in particular, the role of the receptors and the role of putative paracrine mechanisms that may explain at some extent the interconnection mechanisms between regulatory systems.

This may be the case of organs such the head kidney in fish, as it concentrates several secretory cells belonging to either immune or endocrine systems. It should be emphasized that in fish the head kidney or pronephros is an important centre of the endocrine response. In fact this organ becomes key player in the organization of the integrated response to stressors. Head kidney plays a substantial endocrine role in the secretion of the two main hormones of the hormonal axes, cortisol from interrenal cells, the major fish glucocorticoid and mineralocorticoid, and catecholamines, released by chromaffin cells which are components of the sympathetic nervous system releasing, and receiving specific connections from the central nervous system [30]. Moreover, not only the neural and the endocrine system meet in this organ, but also the immune and hematopoietic systems are found in the pronephros. Thus, this organ is the homologous to the bone marrow of higher vertebrates which involves that the production of lymphocytes, monocytes and neutrophils is localized

The Endocrine Response to Stress - A Comparative View 281

may be significantly conditioned by the coping strategy, not only in one species but

All living organisms have developed responses to face stress situations, and some at cellular level such as heat shock protein activation, have been well conserved along evolution. In vertebrates the physiological stress response is driven by the neuroendocrine axes which in turn affect many other physiological compartments until the homeostasis is regained. In this chapter, after revisiting the stress concept, we have reviewed the general character of the stress phenomenon and the cellular responses, and afterwards we summarize the physiological stress response in vertebrates: mammals, birds, reptiles, amphibians and fish. Finally, the consequences of the endocrine activation and the neuroimmunoendocrine integrated response to stressors have been reviewed in relation to the adaptive value of the

The authors want to thank the support of the Spanish Ministry of Science and Innovation (BFU2009-07354) and Generalitat de Catalunya (SGR2009-0554). LT and MT are members of

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Ackerman, P. A., R. B. Forsyth, Mazur C.F.and Iwama G.K. (2000). Stress hormones and the cellular stress response in salmonids*. Fish Physiology Biochemistry* 23: 327-336. Aluru, N. and M. M. Vijayan (2009). Stress transcriptomics in fish: A role for genomic cortisol

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Barton, B. A. (2002). Stress in fishes: A diversity of responses with particular reference to changes in circulating corticosteroids. *Integrative Comparative Biology* 42: 517-525. Basu, N., T. Nakano, Grau, E. G. Iwama, G. K. (2001). The effects of cortisol on heat shock protein 70 levels in two fish species. *General Comparative Endocrinology* 124: 97-105. Baumeister, R., Schaffitzel, E., Hertweck, M. (2006). Endocrine signalling in *Caenorhabditis elegans* controls stress response and longevity *Journal of Endocrinology* 190: 191-202 Binkley, S.A. (1995). *Endocrinology*. HarperCollins College Publishers. New York, pp. 283-327. Blas J., Bortolotti G. R., Tella J. L., Baos R., and Marchant T. A. (2007). Stress response during

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MacKenzie, S. and Tort, L. (2007). Cloning of glucocorticoid receptor (GR) in gilthead seabream (*Sparus aurata*). Differential expresion of GR and immune genes after an

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*York Academy of Sciences* 104: 8880–8884

**7. Conclusion** 

stress reaction.

**9. References** 

**8. Acknowledgements** 

in this organ. It is concluded, then, that the head kidney deals with a number of key physiological responses in fish species. Therefore, fish represent an interesting comparative model for the study of the stress effects on the nervous, endocrine and immune systems, due to the structure and composition of the head kidney (Tort, 2010). In summary, the concept of neuroimmunoendocrine connection identifying specific tissues with specific systems (lymphoid tissue/ immune system, gland/ hormones or neurones/ nervous system) appears to be no longer appropriate or complete as well in lower vertebrates [28]. Clearly in this case, the head kidney in fish represents a tissue in which all three regulatory systems are integrally connected forming a centralized network to coordinate endocrine, neural, and immune, responses after stress.

## **6. Stress and adaptation in vertebrates**

The adaptive value of responsiveness to stressors in animals in nature may provide invaluable information regarding the dynamics and flexibility of neuroendocrine responses. Absolute levels of transmitters or hormones may not matter in the induction of responses and survival. Relative elevation or inhibition related to previous experience may adjust specific neural centers to produce relevant output specifically related to the appropriate environmental context. The neural mechanisms for transduction of relevant information are necessarily very plastic, with many transmitters, neuromodulators and peripheral hormone systems interacting between them. These systems influence behavioral and physiological stress responses, but are also influenced by that output (Greenberg and Summers, 2002).

Taking the whole amount of data on the research in stress, it can be said that it generally focuses on the use of negative stimuli. However, positive stimuli such as immediate reaction or novel rewards can cause comparable physiological stress responses (Ulrich-Lai and Herman, 2009). It has been proposed that acute rises in corticosteroids following perturbations of the environment may actually avoid chronic stress as they may work primarily as ''anti-stress'' hormones. Free-living populations may have elevated circulating levels of corticosteroids under emergency stages. However, this situation may not always be advantageous and there is accumulating evidence from birds that the adrenocortical responses to perturbating factors are modulated both on seasonal and individual bases. These data suggest that corticosteroid secretions allow flexibility so that the response is integrated in relation to time of year, time of the day, as well as for individual differences owing to body condition, disease and social status (Wingfield and Kitaysky 2002).

Although GC responses are viewed as a major evolutionary mechanism to maximize fitness through stress management, phenotypic variability exists within animal populations, and it remains unclear whether inter-individual differences in stress physiology can explain variance in unequivocal components of fitness. For instance, it has been shown that the magnitude of the adrenocortical response to a standardized perturbation during development is negatively related to survival and recruitment in a wild population of birds, providing empirical evidence for a link between stress response, not exposure to stressors, and fitness in a vertebrate under natural conditions. Recent studies suggest that variability in the adrenocortical response to stress may be maintained if high and low responders represent alternative coping strategies, with differential adaptive value depending on environmental conditions (Blas et al., 2007). In fish it has been shown that such a coping strategy is an important determinant of the physiological response, either in behavior, physiology and even gene expression, and therefore much of the phenotypic expression may be significantly conditioned by the coping strategy, not only in one species but specifically in groups of individuals (Mackenzie et al., 2009).
