**4. Consequences of the neuroendocrine activation**

Response involving the sympatico-chromaffin axis, involves the activation of chromaffin tissue by specific neurons, resulting in the release of catecholamines, adrenalin and noradrenalin, which in turn induce the generation of large amounts of energy to meet the energetic needs of this active reaction. This is a very rapid response among all vertebrates and mainly involves the cardiovascular system. Regarding the HPA axis, a longer time is needed, since an endocrine cascade is activated and therefore the effects will be produced when the final hormone in the cascade, a corticosteroid has been released. Looking at the knowledge on comparative animal physiology, it can be sustained that the proteins, gene structures, and signalling pathways of the HPA axis are present in the earliest vertebrates and have been maintained by natural selection because of their critical adaptive roles. In all vertebrates so far studied, the HPA axis is activated as a response to stressors and is controlled centrally by peptides of the CRH family. It can be assumed that, irrespective of the behavioral or physiological outcomes, acute and chronic elevations of corticosteroid secretion initiate metabolic alterations and biochemical processes. Because of the dichotomy in the effects of acute and chronic GC responses, studies generally focus on either acute or chronic elevations of GCs (elevated basal values). When basal GC concentrations are

2009) and a range of other immune system responses (Maule and VanDerKooi, 1999), and appears to attenuate the cellular heat shock protein response to thermal insult (Ackerman et al., 2000; Basu et al., 2001 in Pankhurst 2010). Corticosteroid hormones also play a key role in osmoregulation (reviewed in McCormick, 1995), and are critical for controlling metabolism, hydromineral balance, and the overall stress response. Among corticosteroid hormones that have been well characterized in most vertebrate groups, the identity of one of the earliest vertebrate corticosteroid hormones, 11-deoxycortisol has been recently found in lampreys, a member of the agnathans that evolved more than 500 million years ago. This corticosteroid is regulated by the hypothalamus–pituitary axis and responds to acute stress (Close et al., 2010). This indicates that a complex and highly specific corticosteroid signalling pathway evolved at least 500 million years ago with the arrival of the earliest vertebrate, although it is assumed that this molecule may be derived also from an early ancestral

Regarding the nuclear glucocorticoid receptor (GR), found in all vertebrates, it is known that in fish regulates cell growth, bone density, metabolism and modulates the cardiovascular system. Besides cortisol, also significant levels of 11-deoxycortisol and 11 deoxycorticosterone occur and these ligands bind to GRs and Mineralocorticoid receptors (MRs). It has been shown in fish that teleosts may have one or two different GR genes (Acerete et al., 2007; Bury et al. 2003). This discrepancy likely results from the fish specific genome duplication event. One of these two GR genes has two different transcripts that are generated by alternative splicing (Bury et al. 2003; Greenwood et al. 2003). Most interestingly, the three different GR forms in fish are differentially expressed in vivo and show different transactivational capacities, but only slightly different affinity for their ligand. As both genes and different splice variants are transcriptionally active, it is suggested that they both play an important and probably different role in the fish physiology (Bury et al. 2003, Greenwood et al. 2003). This multiple corticoid receptors in fish and a more complex signalling mechanism by related steroids provides an interesting model

Response involving the sympatico-chromaffin axis, involves the activation of chromaffin tissue by specific neurons, resulting in the release of catecholamines, adrenalin and noradrenalin, which in turn induce the generation of large amounts of energy to meet the energetic needs of this active reaction. This is a very rapid response among all vertebrates and mainly involves the cardiovascular system. Regarding the HPA axis, a longer time is needed, since an endocrine cascade is activated and therefore the effects will be produced when the final hormone in the cascade, a corticosteroid has been released. Looking at the knowledge on comparative animal physiology, it can be sustained that the proteins, gene structures, and signalling pathways of the HPA axis are present in the earliest vertebrates and have been maintained by natural selection because of their critical adaptive roles. In all vertebrates so far studied, the HPA axis is activated as a response to stressors and is controlled centrally by peptides of the CRH family. It can be assumed that, irrespective of the behavioral or physiological outcomes, acute and chronic elevations of corticosteroid secretion initiate metabolic alterations and biochemical processes. Because of the dichotomy in the effects of acute and chronic GC responses, studies generally focus on either acute or chronic elevations of GCs (elevated basal values). When basal GC concentrations are

corticosteroid receptor molecule (Thornton and Carroll, 2011).

for comparative GR function (Stolte et al., 2006).

**4. Consequences of the neuroendocrine activation** 

elevated, a common consequence is a weakening of the further acute GC response, probably through negative feedback mechanisms in the hypothalamic–pituitary-adrenal axis. This is an endocrine vicious circle: a chronically high baseline can provoke pathologies, and a weak acute response is ineffective at handling short-term stressors (Creel, 2001).

Since the energetic cost of the stress situation is high, it is clear that other hormone axes, mostly devoted to the metabolic support will have an influence on the immune function. Thus, the growth hormone and the somatotropic axis have also been shown to affect immune processes, and opioids and thyroid hormones have also been shown to modulate immune responses. The activation of catecholamines and corticosteroids induce a wide number of changes, in particular because these molecules have receptors in most tissues. Therefore, many if not all of the hormones involved in stress responses possess, in addition to their direct effects, induce pleiotropic or collateral consequences that may or may not reinforce direct or primary effect. These other effects can mediate the mechanisms that might affect other unrelated adaptive needs, as for example to modulate the responses of the cardiovascular system, osmotic equilibrium, disease resistance mechanisms and immunocompetence, energetic metabolism and reproduction (see Figure 1).

Fig. 1. Schematic diagrams of the endocrine interactions between the stress axis and growth, reproduction, energetics and metabolism

In terms of energetics and growth effects, stress generally shows depressive effects, inhibiting feeding behavior in all vertebrates. The effects of CRH on food intake are evolutionarily ancient, as this peptide inhibits feeding already in fishes, and the same effect is observed in birds and mammals. The effects of melanocortins on food intake have not been as extensively studied, but available evidence suggests that the anorexic role of neuronal melanocortins has been conserved. Data from mammals suggest an important role for hypothalamic neuropeptides, in particular the melanocortins and CRH-like peptides, in mediating stress-induced inhibition of feeding. Although there is evidence that CRH and the melanocortins influence hypothalamic circuitry controlling food intake, these peptides may have a more primitive role in modulating visuomotor pathways involved in the recognition

The Endocrine Response to Stress - A Comparative View 277

reproductive system can become resistant to inhibition by GCs in some reproductive contexts. For example, if GCs allocate resources away from reproduction, and thereby reduce individual fitness by impairing successful production of offspring, the benefit of the reproductive system ignoring the GC signal may outweigh the cost of not responding to the stressor. In salmon species and several marsupials, death occurs shortly after breeding. The proximate cause of death is the extremely high levels of GCs that catabolize essential proteins (Wingfield & Romero, 2001). Reproduction in these animals clearly continues despite elevated GCs. Furthermore, GCs do not inhibit reproduction in many short-lived species and in older individuals, and in dominant individuals in some species where the dominant individual has a limited period with access to mates (Wingfield & Sapolsky, 2003). Consequently, susceptibility to GC-induced inhibition of reproduction is highly specific depending on the importance of continuing reproduction in the presence of stress, which may vary depending upon age, sex or stage of the breeding cycle and of course, the species

In the regulation of the stress response by steroid hormones it has been assumed that the production and regulation of steroid hormones has been viewed as a multi-organ process involving glucocorticoids and sex steroids. However, active steroids can also be synthesized locally in target tissues, either from circulating inactive precursors or de novo from cholesterol. This may be the case in the brain for neurosteroids and in the immune system. Furthermore, recent evidence suggests that other steroid hormones LH, GnRH, ACTH and CRH are expressed locally in target tissues, potentially providing a mechanism for local regulation of neurosteroid and immunosteroid synthesis. The balance between systemic and local steroid signals depends critically on life history stage, species adaptations, and the costs of systemic signals. Thus, individual tissues and organs may become capable of autonomously synthesizing and modulating local steroid signals, perhaps interacting with

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 HPG and HPA axes and the overall response to stress (Schmidt et al., 2008).

(Romero and Butler, 2007).

**5. Integration and regulation** 

and acquisition of food. (Carr, 2002). Stress rapidly reduces visually guided prey-catching behavior in toads, an effect that can be mimicked by administration of CRH, while corticosterone and isoproterenol are without effect. Melanocortins also reduce prey oriented turning movements in amphibians and, in addition, facilitate the acquisition of habituation to a moving prey. The effects of these neuropeptides are rapid, occurring within 30 min after administration. By modulating visual and motor processing circuits these neuropeptides may help animals make appropriate behavioral decisions during stress episodes.

Many studies in the last decade have shown that stress can affect immune system both in mammals and lower vertebrates (Verburg-Van Kemenade et al., 2009; Dhabhar et al., 2009), although most of the reports are related to the effects of corticosteroids, and describe generally suppressive effects. Studies with human, murine and rat immune cells showed the immunosuppressive effects of elevated GC levels following stressful circumstances. GCs suppress Th1 cellular immunity and mediate a Th2 shift by suppressing production of T helper cells type 1/(Th1; tumor necrosis factor-a, interferon-g, interleukin-2 and interleukin -12 cytokines and inducing production of T helper cells type 1 (Elenkov 2004). Suppressive effects have been shown also in fish, where administration of cortisol induce a reduction of cytokine expression in cultured macrophages (Mackenzie et al., 2006; Castillo et al, 2009). Husbandry stressors applied to different species of fish result in a reduction of immunocompetence, showing decreased activity of immune response mechanisms (Montero et al., 1999), and reduction in efficiency after a combination of stress and pathogen treatment (Mauri et al., 2011).

Nevertheless, there are also episodes in which enhancement of immune function is observed, for instance during the immediate reaction, and this may depend on several considerations. One is the time-course, since acute or short-term stress often enhances innate and adaptive immune responses whereas chronic or long-term stress normally suppress or dysregulate immune function (Dhabhar et al., 2009; Tort, 2011). Another is the body compartment at which we are looking. Thus, skin is enriched with immune cells during acute stress, showing immuno-enhancement, while circulating blood may show depletion of leukocytes. A third factor may be the energetic situation of the animal since the demand of resources after stress are increased and they may be insufficient for other needs than facing the coping with stress itself. Whether there is an excess of demands and a shortage of energy resources, the immune system can be suppressed. Nevertheless, these arguments based on conservation of energy have been invoked to explain potential adaptive benefits of stress-induced immuno-suppression, but in one hand, some mechanisms for immunosuppression expend, rather than conserve, energy. On the other hand, it can be also observed that while some immune responses are depressed or delayed, others are present without any decrease in efficiency, for instance, reducing the number of lymphocytes but increasing granulocytes . Therefore, the hypothesis is rather a temporal reorganization of immune resources than a pure suppression response. Maladaptive implications are present when stress becomes chronic; a situation that is unusual in nature and that evolution has yet to resolve (Dhabhar et al., 2009).

Stressors have been shown to reduce reproductive performance and, in lower vertebrates, even impair completely the reproductive process (Pottinger, 1999; Pankhurst 2009; Tort, 2011). In fact, the Hypothalamic-Pituitary-Gonadal axis and the Hypothalamic-Pituitary-Adrenal/Interrenal axis share a number of mediators, mainly steroid hormones. Therefore, stress and the activation of HPA axis affects the production of steroids. Interestingly, the

and acquisition of food. (Carr, 2002). Stress rapidly reduces visually guided prey-catching behavior in toads, an effect that can be mimicked by administration of CRH, while corticosterone and isoproterenol are without effect. Melanocortins also reduce prey oriented turning movements in amphibians and, in addition, facilitate the acquisition of habituation to a moving prey. The effects of these neuropeptides are rapid, occurring within 30 min after administration. By modulating visual and motor processing circuits these neuropeptides may help animals make appropriate behavioral decisions during

Many studies in the last decade have shown that stress can affect immune system both in mammals and lower vertebrates (Verburg-Van Kemenade et al., 2009; Dhabhar et al., 2009), although most of the reports are related to the effects of corticosteroids, and describe generally suppressive effects. Studies with human, murine and rat immune cells showed the immunosuppressive effects of elevated GC levels following stressful circumstances. GCs suppress Th1 cellular immunity and mediate a Th2 shift by suppressing production of T helper cells type 1/(Th1; tumor necrosis factor-a, interferon-g, interleukin-2 and interleukin -12 cytokines and inducing production of T helper cells type 1 (Elenkov 2004). Suppressive effects have been shown also in fish, where administration of cortisol induce a reduction of cytokine expression in cultured macrophages (Mackenzie et al., 2006; Castillo et al, 2009). Husbandry stressors applied to different species of fish result in a reduction of immunocompetence, showing decreased activity of immune response mechanisms (Montero et al., 1999), and reduction in efficiency after a combination of stress and pathogen

Nevertheless, there are also episodes in which enhancement of immune function is observed, for instance during the immediate reaction, and this may depend on several considerations. One is the time-course, since acute or short-term stress often enhances innate and adaptive immune responses whereas chronic or long-term stress normally suppress or dysregulate immune function (Dhabhar et al., 2009; Tort, 2011). Another is the body compartment at which we are looking. Thus, skin is enriched with immune cells during acute stress, showing immuno-enhancement, while circulating blood may show depletion of leukocytes. A third factor may be the energetic situation of the animal since the demand of resources after stress are increased and they may be insufficient for other needs than facing the coping with stress itself. Whether there is an excess of demands and a shortage of energy resources, the immune system can be suppressed. Nevertheless, these arguments based on conservation of energy have been invoked to explain potential adaptive benefits of stress-induced immuno-suppression, but in one hand, some mechanisms for immunosuppression expend, rather than conserve, energy. On the other hand, it can be also observed that while some immune responses are depressed or delayed, others are present without any decrease in efficiency, for instance, reducing the number of lymphocytes but increasing granulocytes . Therefore, the hypothesis is rather a temporal reorganization of immune resources than a pure suppression response. Maladaptive implications are present when stress becomes chronic; a situation that is unusual in nature and that evolution has yet

Stressors have been shown to reduce reproductive performance and, in lower vertebrates, even impair completely the reproductive process (Pottinger, 1999; Pankhurst 2009; Tort, 2011). In fact, the Hypothalamic-Pituitary-Gonadal axis and the Hypothalamic-Pituitary-Adrenal/Interrenal axis share a number of mediators, mainly steroid hormones. Therefore, stress and the activation of HPA axis affects the production of steroids. Interestingly, the

stress episodes.

treatment (Mauri et al., 2011).

to resolve (Dhabhar et al., 2009).

reproductive system can become resistant to inhibition by GCs in some reproductive contexts. For example, if GCs allocate resources away from reproduction, and thereby reduce individual fitness by impairing successful production of offspring, the benefit of the reproductive system ignoring the GC signal may outweigh the cost of not responding to the stressor. In salmon species and several marsupials, death occurs shortly after breeding. The proximate cause of death is the extremely high levels of GCs that catabolize essential proteins (Wingfield & Romero, 2001). Reproduction in these animals clearly continues despite elevated GCs. Furthermore, GCs do not inhibit reproduction in many short-lived species and in older individuals, and in dominant individuals in some species where the dominant individual has a limited period with access to mates (Wingfield & Sapolsky, 2003). Consequently, susceptibility to GC-induced inhibition of reproduction is highly specific depending on the importance of continuing reproduction in the presence of stress, which may vary depending upon age, sex or stage of the breeding cycle and of course, the species (Romero and Butler, 2007).

In the regulation of the stress response by steroid hormones it has been assumed that the production and regulation of steroid hormones has been viewed as a multi-organ process involving glucocorticoids and sex steroids. However, active steroids can also be synthesized locally in target tissues, either from circulating inactive precursors or de novo from cholesterol. This may be the case in the brain for neurosteroids and in the immune system. Furthermore, recent evidence suggests that other steroid hormones LH, GnRH, ACTH and CRH are expressed locally in target tissues, potentially providing a mechanism for local regulation of neurosteroid and immunosteroid synthesis. The balance between systemic and local steroid signals depends critically on life history stage, species adaptations, and the costs of systemic signals. Thus, individual tissues and organs may become capable of autonomously synthesizing and modulating local steroid signals, perhaps interacting with the HPG and HPA axes and the overall response to stress (Schmidt et al., 2008).
