Stress in Atlantic salmon: response to unpredictable chronic stress.

Combinations of stressors occur regularly throughout an animal's life, especially in agriculture and aquaculture settings. If an animal fails to acclimate to these stressors, stress becomes chronic, and a condition of allostatic overload arises with negative results for animal welfare. In the current study, we describe effects of exposing Atlantic salmon parr to an unpredictable chronic stressor (UCS) paradigm for 3 weeks. The paradigm involves exposure of fish to seven unpredictable stressors three times a day. At the end of the trial, experimental and control fish were challenged with yet another novel stressor and sampled before and 1 h after that challenge. Plasma cortisol decreased steadily over time in stressed fish, indicative of exhaustion of the endocrine stress axis. This was confirmed by a lower cortisol response to the novel stressor at the end of the stress period in chronically stressed fish compared with the control group. In the preoptic area (POA) and pituitary gland, chronic stress resulted in decreased gene expression of 11ßhsd2, gr1 and gr2 in the POA and increased expression of those genes in the pituitary gland. POA crf expression and pituitary expression of pomcs and mr increased, whereas interrenal gene expression was unaffected. Exposure to the novel stressor had no effect on POA and interrenal gene expression. In the pituitary, crfr1, pomcs, 11ßhsd2, grs and mr were down-regulated. In summary, our results provide a novel overview of the dynamic changes that occur at every level of the hypothalamic-pituitary gland-interrenal gland (HPI) axis as a result of chronic stress in Atlantic salmon.

Melanocortin receptor subtypes in interrenal cells and corticotropic activity of α-melanocyte-stimulating hormones in barfin flounder, Verasper moseri.

The aim of this study was to characterize the pituitary-interrenal axis in barfin flounder, a flatfish. Adrenocorticotropic hormone (ACTH) and melanocortin 2 receptor (MC2R) have been shown to be indispensable substances in pituitary and interrenal cells for cortisol release, respectively. We previously identified ACTH in the pars distalis of the barfin flounder pituitary gland, and detected transcripts of Mc1r, Mc4r, and Mc5r in the head kidney wherein interrenal cells are located. We have now demonstrated the presence of MC2R, which is a specific receptor for ACTH, in interrenal cells by molecular cloning of Mc2r cDNA and in situ hybridization, and confirmation of the in vitro cortisol-releasing activity of ACTH. These results show the presence of a classical pituitary-interrenal axis in this fish. We also evaluated the role of α-melanocyte-stimulating hormone (α-MSH) and its related peptides. In situ hybridization was used to demonstrate the expression of Mc5r in interrenal cells; both desacetyl-α-MSH and diacetyl-α-MSH showed in vitro cortisol-releasing activities, while the activity of α-MSH was negligible. These findings indicate the presence of an additional pituitary-interrenal axis consisting of α-MSH-like peptides secreted from the neurointermediate lobe of the pituitary and MC5R in the interrenal cells. The cortisol-releasing activity of desacetyl-α-MSH and diacetyl-α-MSH, compared with the low activity of α-MSH, suggest a unique and specific functional role of these forms of MSH peptides. The interrenal co-expression of two subtypes of Mcrs may play a role in this specialization.

Melanocortins regulate the electric waveforms of gymnotiform electric fish.

The hypothalamic-pituitary-adrenal/interrenal axis couples serotonergic activity in the brain to the peripheral regulators of energy balance and response to stress. The regulation of peripheral systems occurs largely through the release of peptide hormones, especially the melanocortins (adrenocorticotropic hormone [ACTH] and alpha melanocyte stimulating hormone [alpha-MSH]), and beta-endorphin. Once in circulation, these peptides regulate a wide range of processes; alpha-MSH in particular regulates behaviors and physiologies with sexual and social functions. We investigated the role of the HPI and melanocortin peptides in regulation of electric social signals in the gymnotiform electric fish, Brachyhypopomus pinnicaudatus. We found that corticotropin releasing factor, thyrotropin-releasing hormone, and alpha-MSH, three peptide hormones of the HPI/HPA, increased electric signal waveform amplitude and duration when injected into free-swimming fish. A fourth peptide, a synthetic cyclic-alpha-MSH analog attenuated the normal circadian and socially-induced EOD enhancements in vivo. When applied to the electrogenic cells (electrocytes) in vitro, only alpha-MSH increased the amplitude and duration of the electrocyte discharge similar to the waveform enhancements seen in vivo. The cyclic-alpha-MSH analog had no effect on its own, but blocked or attenuated alpha-MSH-induced enhancements in the single-cell discharge parameters, demonstrating that this compound functions as a silent antagonist at the electrocyte. Overall, these results strongly suggest that the HPI regulates the EOD communication signal, and demonstrate that circulating melanocortin peptides enhance the electrocyte discharge waveform.

Effects of subchronic exposure to waterborne cadmium on H-P-I axis hormones and related genes in rare minnows (Gobiocypris rarus).

The H (hypothalamic)-P (pituitary)-I (interrenal) axis is critical in the stress response and other activities of fish. To further investigate cadmium (Cd) toxicity on the H-P-I axis and to identify its potential regulatory genes in fish, the adult female rare minnows (Gobiocypris rarus) were exposed to subchronic (5weeks) levels of waterborne Cd in the present study. This kind of treatment caused dose-dependent decline in fish growth, with significance in the high dose group (100µg/L). Correspondingly, low dose (5-50µg/L) waterborne Cd disrupted the endocrine system of H-P-I axis just at the secretion level, while high dose Cd disrupted both the secretion and synthesis of cortisol and its downstream signals in rare minnows, revealed by the significantly upregulation and positive correlation of corticosteroidogenic genes including MC2R, StAR, CYP11A1, and CYP11B1 in the kidney (including the interrenal tissue) (P<0.05), and the significant alteration of Glcci1, Hsp90AA and Hsp90AB in the hepatopancreas, gill and intestine as well (P<0.05). The expression of Glcci1 was significantly decreased in hepatopancreas, gill and intestine of tested fish following treatment, and its positive correlation with GR (Glucocorticoid receptor) suggested its potential regulation on the cortisol and/or H-P-I axis in fish. The expression of FKBP5 in the intestine was positively and significantly correlated with that of Hsp90AA (P<0.05), and the Hsp90AB transcript in the hepatopancreas was positively correlated with that of Hsp90AA (P<0.05), which indicated that Hsp90AA and Hsp90AB were more likely to serve as cofactors of GR and FKBP5 in response to Cd exposure.

A novel neuropeptide cellular mechanism in amphibian interrenal steroidogenesis.

Interrenals of female Rana esculenta were incubated with gonadotropin-releasing hormone (GnRH), 9-ketoreductase inhibitor (palmitic acid), acetyl salicyclic acid, prostaglandin F2 alpha (PGF2 alpha), forskolin, isobutylmethyl xanthine (IBMX), dibutyril cyclic adenosine monophosphate (dbcAMP). Prostaglandin E2 (PGE2), PGF2 alpha, testosterone and 17 beta-estradiol were assessed on the incubation media. In addition, in the same interrenals, 9-ketoreductase and aromatase activities were evaluated. GnRH increased PGF2 alpha, 17 beta-estradiol, 9-ketoreductase and aromatase, and decreased PGE2 and testosterone. PGF2 alpha increased 17 beta-estradiol and aromatase, and decreased testosterone. Palmitic acid counteracted GnRH effects, while forskolin, IBMX and dbcAMP showed the same PGF2 alpha effects. These results suggest that GnRH stimulates 9-ketoreductase enhancing PGF2 alpha which in turn activates aromatase through cAMP mediation in the interrenal of Rana esculenta.

Metabolic response in liver and Brockmann bodies of rainbow trout to inhibition of lipolysis; possible involvement of the hypothalamus-pituitary-interrenal (HPI) axis.

We previously demonstrated in rainbow trout that the decrease in circulating levels of fatty acid (FA) induced by treating fish with SDZ WAG 994 (SDZ) induced a counter-regulatory response in which the activation of the hypothalamus-pituitary-interrenal (HPI, equivalent to mammalian hypothalamus-pituitary-adrenal) axis was likely involved. This activation, probably not related to the control of food intake through FA sensor systems but to the modulation of lipolysis in peripheral tissues, liver and Brockmann bodies (BB, the main site of pancreatic endocrine cells in fish), would target the restoration of FA levels in plasma. To assess this hypothesis, we lowered circulating FA levels by treating fish with SDZ alone, or SDZ in the presence of metyrapone (an inhibitor of cortisol synthesis). In liver, the changes observed were not compatible with a direct FA-sensing response but with a stress response, which allows us to suggest that the detection of a FA decrease in the hypothalamus elicits a counter-regulatory response in liver, resulting in an activation of lipolysis to restore FA levels in plasma. The activation of these metabolic changes in liver could be attributable to the activation of the HPI axis and/or to the action of sympathetic pathways. In contrast, in BB, changes in circulating FA levels induce changes in several parameters compatible with the function of FA-sensing systems informing about the decrease in circulating FA levels.

The hypothalamic-pituitary-interrenal axis and the control of food intake in teleost fish.

Although environmental, social and physical stressors have been shown to inhibit food intake and feeding behavior in fish, little is known about the mechanisms that mediate the appetite-suppressing effects of stress. Since the hypothalamic-pituitary-interrenal (HPI) axis is activated in response to most forms of stress in fish, components of this axis may be involved in mediating the food intake reductions elicited by stress. Recent investigations into the brain regulation of food intake in fish have identified several signals with orexigenic and anorexigenic properties. Among these appetite-regulating signals are related neuropeptides that can activate the HPI axis, namely corticotropin-releasing factor (CRF) and urotensin I (UI). Central injections of CRF or UI, or treatments that result in an increase in hypothalamic CRF and UI gene expression, can elicit dose-dependent decreases in food intake that can be reversed by pre-treatment with a CRF-receptor antagonist. Evidence also suggests that cortisol, the end product of HPI activation in most fishes (i.e. Osteichthyes), may be involved in the regulation of food intake. Overall, while elements of the HPI axis may mediate some of the appetite-suppressing effects of stress, it is undetermined how either CRF-related peptides, cortisol, or other elements of the stress response interact with the complex circuitry of the hypothalamic feeding center.

Responsiveness of the hypothalamo-pituitary-interrenal axis in an amphibian (Bufo terrestris) exposed to coal combustion wastes.

To assess the responsiveness of the interrenal axis to stress, we injected toads exposed to coal combustion wastes and toads from an unpolluted reference site with adrenocorticotropic hormone (ACTH), as well as the vehicle alone (saline). Initial circulating levels of corticosterone in toads captured at the polluted area were significantly higher than levels in toads from the reference site. Corticosterone levels in toads from the polluted site remained high even after 2 weeks of laboratory acclimation and injection with saline. The results may suggest disruption of hepatic enzymes responsible for the metabolic clearance of steroid hormones. Injection of toads from the polluted site with ACTH had no effect on plasma corticosterone levels, whereas a similar treatment of toads from the reference site stimulated a marked increase in corticosterone. Our study provides evidence that toads exposed to coal combustion wastes may be less efficient at responding to additional environmental stressors.

Interrenal responses to high ambient temperature in soft-shelled turtle, Lissemys punctata punctata.

An exposure to ambient temperature of 25 degrees C had no perceptible effect on interrenal function but further increase of temperature to 35 degrees C caused nuclear hypertrophy with increase of nuclear diameter, RNA concentration, acid phosphatase and alkaline phosphatase activities, accompanied by quantitative depletions of cholesterol (free, esterified and total) and ascorbic acid levels in the interrenal gland of the soft-shelled turtle Lissemys p. punctata. Similar manifestations of stimulation, except in the nucleus, were marked after exposure to 38 degrees C, but the degree of response in respect of esterified and free cholesterol levels was higher at 38 degrees C than at 35 degrees C. Moreover, withdrawal of 38 degrees C temperature and subsequently maintaining at 25 degrees C for 15 days showed reverse manifestations to those of 35 degrees C/38 degrees C, leading to a tendency towards normalcy. It is suggested that high a ambient temperature of 35 degrees C significantly stimulates interrenal function of Lissemys turtles, but further increase of 38 degrees C does not cause further overall stimulation, and withdrawal of higher temperature (38 degrees C) shows a tendency towards normalcy. It is also suggested that (a) high ambient temperature causes thermal stress, (b) it is reversible and (c) it acts on interrenal activity presumably via CRF-ACTH-axis in turtles.

Gonadotropins and sex hormones modulate interrenal function in soft-shelled turtle.

The aim of the current work was to investigate the role of gonadotropins and female sex hormones on interrenal activity in soft-shelled turtles, Lissemys punctata punctata. 1) FSH treatment (3 microg/100 g body wt daily for 10 days) caused interrenal hypertrophy with increased nuclear diameter, raises of acid phosphatase and alkaline phosphatase concentrations, and depletions of cholesterol (except the free fraction) and ascorbic acid levels from the interrenal gland. However, LH treatment (3 microg/100 g body wt daily for 10 days) failed to produce any perceptible change in the interrenal activity. The combined treatments of FSH and LH (3 microg each/100 gm body wt daily for 10 days) produced no further change beyond that of FSH alone. 2) Estrogen treatment with the low dose (25 microg/100 g body wt daily for 10 days) had no effect, but with higher doses (50 microg or 100 microg/100 gm body wt daily for 10 days) is caused interrenal stimulation by inducing the same manifestations to those of FSH. The degree of manifestations was higher with the higher dose (100 microg daily) than that of the moderate dose (50 microg daily). Progesterone treatment with the low dose (25 microg /100 g body wt daily for 10 days) had no significant effect, but with the moderate (50 microg daily) and higher (100 microg daily) doses suppressed interrenal activity by showing the reverse changes to those of estrogen. The degree of manifestations was higher with the higher dose than that of the moderate one. The combined treatments of estrogen and progesterone (100 microg each/100 g body wt daily for 10 days) caused interrenal stimulation but to a lesser extent than that of estrogen alone. The findings are briefly discussed.

[Ultrastructure of the interrenal gland in young beluga in fresh water and during adaptation to salt water]. / Ul'trastruktura interrenalovoi zhelezy molodi belugi v presnoi vode i v protsesse solevoi adaptatsii.

Two groups of cells (light and dark) were found in the interrenal gland of the young beluga from the fresh-water. Ultrastructural differencies are presumably associated with different functional activity of cells. 12 hours after the transfer of fishes to the sea water, the interrenal cells appeared to be most active with significant elevation of volumetric density of mitochondria and endoplasmic reticulum. After 8 days in the sea water, dark cells were found seldom; a great number of small lyposomes and large myelin-like figures were observed in the cytoplasm of the light cells. The volumetric density of mitochondria and of the reticulum was seen decreased. The functional activity of interrenal gland of the young beluga was lower after 8 days than after 12 hours in the sea water, but higher than in the fresh-water.

[The direct activating effect of a nonapeptide neurohormone (vasotocin) on the thyroid and interrenal glands of sturgeons in in-vitro experiments]. / Neposredstvennoe aktiviruiushchee vliianie nonapeptidnogo neirogormona (vazototsina) na shchitovidnuiu i interrenalovuiu zhelezy osetrovykh ryb v opytakh in vitro.

An in-vitro effect of nonapeptide neurohormone vasotocin on thyroid and interrenal glands was studied in hybrid of Siberian and Lena sturgeons [correction of salmons] at light microscopy level using morphometric method. At a concentration of 0.1 and 1 nmol/l vasotocin was shown to exert undirectional stimulating effect on the thyroid and interrenal gland functions. In the presence of vasotocin at a concentration of 1 nmol/l in culture media the activity of glands is even more pronounced than under the influence of adenohypophyseal hormones, adrenocorticotropic (8 x 10 ng/ml) and thyrotropic (5 ng/ml).

[Morphofunctional research on the interrenal gland of the frog Rana temporaria following arginine vasotocin administration]. / Morfofunktsional'noe issledovanie interrenalovoi zhelezy liagushki Rana temporaria posle vvedeniia arginin-vazototsina.

Studies have been made of the effect of injections of hypothalamic nonapeptide neurohormone, arginine vasotocin, on functional condition of the interrenal gland in mature frogs. In unoperated, sham-operated and in animals 10 days after hypophysectomy, single and especially three subsequent injections of arginine vasotocin (5 x 10(-9) M per 1 kg of the body weight) result in evident activation of glandular cells of the interrenal gland which is manifested in the increase of the volume of their nuclei and cytoplasmic area, as well as in the dilatation of the blood vessels. Activation of the interrenal gland in hypophysectomized frogs, which lack endogenous ACTH, indicate the direct para-adenohypophyseal influences of nonapeptide hypothalamic hormones on the activity of glandular cells in the peripheral endocrine glands, in particular, the interrenal gland of the grass frog.

[The direct stimulating effect of arginine vasotocin on the functional activity of the interrenal gland in the frog Rana temporaria]. / Priamoe stimuliruiushchee vliianie arginin-vazototsina na funktsional'nuiu aktivnost' interrenalovoi zhelezy liagushki Rana temporaria.

Using radioimmunoassay it has been detected that both nonoperated and hypophysectomized, lacking endogenous ACTH, frogs injected one or three times with arginine vasotocin (5.10(-9) M/kg b. w.) show a statistically significant increase of plasma corticosterone level as compared with that in control animals and frogs injected with Ringer solution. The level of 11-hydroxycorticosteroids (fluorometric determination) in the interrenal gland decreases significantly only in animals three times injected with arginine vasotocin. It is assumed that arginine vasotocin produces a direct stimulatory effect on corticosteroid-producing cells of the frog interrenal gland.

[The effect of injections of arginine vasotocin and mesotocin on the function of the interrenal gland in the frog Rana temporaria 2 months after the removal of the anterior lobe of the hypophysis]. / Vliianie in''ektsii arginin-vazototsina i mezototsina na sostoianie interrenalovoi zhelezy liagushki Rana temporaria spustia dva mesiatsa posle udaleniia perednei doli gipofiza.

Morphometric and biochemical studies have been made on the activity of interrenal gland in the frog Rana temporaria after removal of the anterior lobe of pituitary and injections of arginine vasotocin and mesotocin. It was demonstrated that the activity of this gland after the operation decreases increasing after arginine vasotocin injections.

Intra-adrenal interactions in fish: catecholamine stimulated cortisol release in sea bass (Dicentrarchus labrax L.).

The effect of the catecholamines, adrenaline and noradrenaline, on sea bass (Dicentrarchus labrax) and sea bream (Sparus auratus) interrenal cortisol production was studied in vitro using a dynamic superfusion system technique. Increasing concentrations of catecholamines (10(-6), 10(-8) and 10(-10) M) stimulated cortisol production in a dose-dependent manner, in sea bass only. The increase in cortisol production stimulated by adrenaline (10(-6) M) and noradrenaline (10(-6) M) was inhibited by sotalol (2 x 10(-5) M), but not by prazosin suggesting that catecholamines stimulate cortisol release through the beta-receptor subtype. To evaluate catecholamine-induced signal transduction in head kidney cells, measurements of cAMP production and [H3]myo-inositol incorporation were determined in head kidney cell suspensions. Adrenaline and noradrenaline (10(-6) M) increased cAMP production, but had no effect on total inositol phosphate accumulation. These results indicate that catecholamines released from the chromaffin cells within the interrenal tissue may act as a paracrine factor to stimulate interrenal steroidogenesis in the sea bass.

The corticotropin-releasing factor system as a mediator of the appetite-suppressing effects of stress in fish.

A characteristic feature of the behavioural response to intensely acute or chronic stressors is a reduction in appetite. In fish, as in other vertebrates, the corticotropin-releasing factor (CRF) system plays a key role in coordinating the neuroendocrine, autonomic, and behavioural responses to stress. The following review documents the evidence implicating the CRF system as a mediator of the appetite-suppressing effects of stress in fish. Central injections of CRF or the related peptide, urotensin I (UI), or pharmacological treatments or stressors that result in an increase in forebrain CRF and UI gene expression, can elicit dose-dependent reductions in food intake that are at least partially reversed by pre-treatment with a CRF receptor antagonist. In addition, the appetite suppressing effects of various environmental, pathological, physical, and social stressors are associated with elevated levels of forebrain CRF and UI gene expression and with an activation of the hypothalamic-pituitary-interrenal (HPI) stress axis. In contrast, although stressors can also be associated with an increase in caudal neurosecretory system CRF and UI gene expression and an endocrine role for CRF-related peptides has been suggested, the physiological effects of peripheral CRF-related peptides on the gastrointestinal system and in the regulation of appetite have not been investigated. Overall, while CRF and UI appear to participate in the stress-induced changes in feeding behaviour in fish, the role of other know components of the CRF system is not known. Moreover, the extent to which the anorexigenic effects of CRF-related peptides are mediated through the hypothalamic feeding center, the HPI axis and cortisol, or via actions on descending autonomic pathways remains to be investigated.

Are hsps suitable for indicating stressed states in fish?

In response to most stressors, fish will elicit a generalized physiological stress response, which involves the activation of the hypothalamic-pituitary-interrenal axis (HPI). As in other vertebrates, this generalized stress response comprises physiological responses that are common to a wide range of environmental, physical and biological stressors. Recently, several families of heat shock proteins (hsps) have been proposed as indicators of a generalized stress response at the cellular level. Recent findings that hsp levels, in various fish tissues, respond to a wide range of stressors have supported the use of these proteins as indicators of stressed states in fish. However, the cellular stress response can vary, for example, according to tissue, hsp family and type of stressor. This brief overview of these responses in fish asks the question of whether changes in levels and families of hsps can be used as a suitable indicator of stressed states in fish. By casting this question in the context of the well-established generalized physiological stress response in fish, we argue that the use of hsps as indicators of stressed states in fish in general is premature.

Differences in behaviour between rainbow trout selected for high- and low-stress responsiveness.

Two F1 lines of rainbow trout Oncorhynchus mykiss, divergent for plasma cortisol responsiveness, were generated by individual selection for post-stress cortisol values within the F0 generation. Adult females of the F1 generation were transferred to rearing in social isolation in observation tanks. After 6 days, locomotor activity in high-responding (HR) and low-responding (LR) individuals was quantified as time spent moving during a 20 min observation period. Behavioural observations were repeated the next day with a smaller conspecific intruder present in each observation tank. Differential hypothalamus-pituitary-interrenal axis activity in the two lines was subsequently confirmed by a standardised confinement stress test, which resulted in significantly higher plasma cortisol concentrations in HR than LR fish. HR fish displayed higher levels of locomotor activity than LR fish in the presence of an intruder, but not when in isolation. Aggressive behaviour towards the intruder was not seen, suggesting either a state-dependent lack of territorial aggression, or chronic stress in the experimental fish. A significantly higher incidence of feed intake was seen in LR trout when held in observation tanks (40% versus 0% of the fish took food when in isolation), suggesting that these fish acclimated more successfully to the experimental conditions than HR fish did. These results suggest that selection for stress responsiveness in salmonid fish leads to behavioural alterations, which are of potential importance to the performance of these fish in aquaculture rearing operations.

The influence of social rank on adenohypophysial cell activity in Salmo irideus. With special reference to basophil ACTH-cells of the proximal pars distalis.

In nine cell types of the adenohypophysis in untreated adult rainbow trout, histologically different activity phases, seasonal changes in activity, and the relation between certain cell types and the interrenal gland, thyroid or gonads were investigated by light and, occasionally, by electron microscopy. Special attention was given to the effect of social rank on the synthetic activity in adenohypophysial cells of trout kept in small groups in which a social hierarchy with one (light) dominant and several (dark) submissives is established. Cell types in the rostral pars distalis were azocarminophil (I) or amphiphil (II). Proximal pars distalis cell types were slightly basophil (IV), orangeophil (V), strongly basophil (VI) or chromophobe (VII). In the pars intermedia, cell types were amphiphil (VIII) or very slightly basophil (IX). Type III was a non-secretory supporting (?) cell. Histologically different activity phases abounded in type IV cells, which mainly occurred in the proximal pars distalis but were also found dispersed in the rostral pars distalis, the pars intermedia and the neurohypophysis. Influences of social rank were pronounced in type IV cells. Phases with a high synthetic activity were exclusively found in submissive animals, phases with a low synthetic activity occurred in dominants. As a positive relation existed between type IV cell activity and the social rank dependent activity of the interrenal gland, it was suggested that type IV cells produce ACTH. In (dominant) male trout treated with DOCG or ACTH, colloid-containing type IV cell phases, reflecting accumulation of the secretory product, were found. This supported the earlier suggestion that ACTH in the trout is produced in the basophil type IV cells and not, as reported in the literature, in cells comparable to type II.