The influence of repeated stress on the release of melanin\x=req-\ concentrating hormone in the rainbow trout J. A. Green and B. I. Baker Department of Biological Sciences, University of Bath, Claverton Down, Bath ba2 7AY received
11
May
1990
ABSTRACT
Melanin-concentrating hormone (MCH) is a neurohypophysial peptide that induces pigmentary pallor in teleosts and which is released when the fish are placed on a white background. An additional effect of the peptide is the depression of ACTH and hence cortisol secretion during moderate stress. The present work on rainbow trout shows that plasma MCH concentrations, while unaffected by a single stress, are raised by repeated stress (1 ml saline injected i.p. without anaesthesia) and remain high for several hours thereafter. The response to stress is observed only in white\x=req-\ adapted fish and not in fish kept in black-coloured
tanks, when MCH release is normally low. Plasma concentrations of MCH vary diurnally but stress induces an equivalent incremental rise in plasma MCH, whether administered in the middle or towards the end of the photophase. The stress-induced rise in MCH concentrations is prevented by treatment with dexamethasone. The results support the suggestion that the modulatory effect of MCH on the hypothalamopituitary-interrenal axis of fish might be enhanced under conditions of stress. Journal of Endocrinology (1991) 128, 261\p=n-\266
INTRODUCTION
secreted, have lower plasma ACTH and cortisol levels than trout maintained on a dark-coloured background, in which MCH secretion is depressed (Baker & Ranee, 1981 ; Gilham & Baker, 1985). Subsequent work showed that the in-vitro release of bioactive ACTH by incu¬ bated pituitary glands is depressed if the donor fish have previously received chronic treatment with MCH via minipumps (Baker, Bird & Buckingham, 1986). Also, the in-vitro release of bioactive ACTH in response to CRF is suppressed in a dose-dependent manner ifMCH is added to the pituitary incubation medium (Baker, Bird & Buckingham, 1985). If MCH does indeed play a physiological role in modulating the response of fish to stress, it could be envisaged that the release of MCH might itself be affected by stress and feedback on the HPI axis to con¬ trol the intensity of cortisol secretion. The aim of the work reported here was to investigate this possibility.
The response of vertebrates, including fish, to stressful conditions has been extensively studied. Persistent or excessive stress can have very deleterious effects result¬ ing, for example, in impaired growth and depression of immune and inflammatory responses. It may be antici¬ pated that endocrine systems which respond to stress, such as the hypothalamo-pituitary-interrenal (HPI) axis, are under multiple controls to regulate their activity under different circumstances. Several hypothalamic factors stimulate adrenocorticotrophin (ACTH) release in fish including, as in mammals, corticotrophin-releasing factor (CRF) and vasotocin, the homologue of vasopressin (Fryer, Lederis & Rivier, 1985). Another hormone which seems to exert an inhibitory effect on ACTH release is melanin-concentrating hormone (MCH). This is a neurohypophysial peptide which serves as a colour change hormone in many teleosts and whose plasma concentration varies as fish move between pale- and dark-coloured backgrounds (Baker, 1988; Kishida, Baker & Eberle, 1989). Although discovered because of its pigmentary effects, it was subsequently observed that MCH has effects on the responsiveness offish to stress. Thus it was found that trout adapted to a palecoloured background, in which MCH is being actively
MATERIALS AND METHODS
Fish
Rainbow trout aged 1-2 years (150-250 g) were obtained from a local fish farm (Alderley Trout Ltd, Wotton-Under-Edge, Glos.). In the aquarium they were kept in white- or black-coloured tanks (250
litres) in slow-flowing tap water (100 ml/min) at 11 °C, under a photoperiod of 18h light: 6h darkness. All fish were adapted to aquarium conditions for at least 2 weeks before the experiment, during which time they were
not fed.
Administration of stress
=
Control fish remained undisturbed throughout the Stress was caused by injecting 1 ml iso¬ tonic saline (0-8% (w/v) NaCl) into the peritoneal cavity without anaesthesia. Trout were usually given one injection per day for one or several days (see indi¬ vidual experimental details) and killed 1 h after the final injection.
experiment.
Collection of plasma
samples At the end ofthe experiment, fish were caught in a single
sweep of a net and anaesthetized as a group in a tank of phenoxyethanol (diluted 1 : 1667, v/v). Blood was
collected from the severed caudal peduncle in cold poly¬ propylene tubes containing 50 pi 6% (w/v) EDTA disodium salt as anticoagulant and 2500 KIU aprotinin (Sigma Chemical Co. Ltd, Poole, Dorset, U.K.) as enzyme inhibitor. Blood samples were collected within 5 min of catching the fish. After centrifugation at 2000 g for 15 min, aliquots of plasma were stored at 20 °C. —
Radioimmunoassays
Statistics All statistical comparisons of means were made using the pooled Student's t-test; the appropriate trans¬ formations (square root or logarithmic) were per¬ formed on all data to give homogeneity of variance. Results are expressed as means ± s.e.m. RESULTS
Acute stress and MCH release
single injection of isotonic saline did not signifi¬ cantly affect the plasma concentration of MCH in white-adapted fish at 1 or 2 h after injection (plasma MCH concentration in pmol/1: 1-h control, 44-5 ± 12-0, 1-h injected, 61-2 + 8 ; 2-h control, 48-1 + 8-5, 2-h injected, 60T + 10-0; w 8). By contrast, cortisol concentrations were significantly (P
11
May
1990
ABSTRACT
Melanin-concentrating hormone (MCH) is a neurohypophysial peptide that induces pigmentary pallor in teleosts and which is released when the fish are placed on a white background. An additional effect of the peptide is the depression of ACTH and hence cortisol secretion during moderate stress. The present work on rainbow trout shows that plasma MCH concentrations, while unaffected by a single stress, are raised by repeated stress (1 ml saline injected i.p. without anaesthesia) and remain high for several hours thereafter. The response to stress is observed only in white\x=req-\ adapted fish and not in fish kept in black-coloured
tanks, when MCH release is normally low. Plasma concentrations of MCH vary diurnally but stress induces an equivalent incremental rise in plasma MCH, whether administered in the middle or towards the end of the photophase. The stress-induced rise in MCH concentrations is prevented by treatment with dexamethasone. The results support the suggestion that the modulatory effect of MCH on the hypothalamopituitary-interrenal axis of fish might be enhanced under conditions of stress. Journal of Endocrinology (1991) 128, 261\p=n-\266
INTRODUCTION
secreted, have lower plasma ACTH and cortisol levels than trout maintained on a dark-coloured background, in which MCH secretion is depressed (Baker & Ranee, 1981 ; Gilham & Baker, 1985). Subsequent work showed that the in-vitro release of bioactive ACTH by incu¬ bated pituitary glands is depressed if the donor fish have previously received chronic treatment with MCH via minipumps (Baker, Bird & Buckingham, 1986). Also, the in-vitro release of bioactive ACTH in response to CRF is suppressed in a dose-dependent manner ifMCH is added to the pituitary incubation medium (Baker, Bird & Buckingham, 1985). If MCH does indeed play a physiological role in modulating the response of fish to stress, it could be envisaged that the release of MCH might itself be affected by stress and feedback on the HPI axis to con¬ trol the intensity of cortisol secretion. The aim of the work reported here was to investigate this possibility.
The response of vertebrates, including fish, to stressful conditions has been extensively studied. Persistent or excessive stress can have very deleterious effects result¬ ing, for example, in impaired growth and depression of immune and inflammatory responses. It may be antici¬ pated that endocrine systems which respond to stress, such as the hypothalamo-pituitary-interrenal (HPI) axis, are under multiple controls to regulate their activity under different circumstances. Several hypothalamic factors stimulate adrenocorticotrophin (ACTH) release in fish including, as in mammals, corticotrophin-releasing factor (CRF) and vasotocin, the homologue of vasopressin (Fryer, Lederis & Rivier, 1985). Another hormone which seems to exert an inhibitory effect on ACTH release is melanin-concentrating hormone (MCH). This is a neurohypophysial peptide which serves as a colour change hormone in many teleosts and whose plasma concentration varies as fish move between pale- and dark-coloured backgrounds (Baker, 1988; Kishida, Baker & Eberle, 1989). Although discovered because of its pigmentary effects, it was subsequently observed that MCH has effects on the responsiveness offish to stress. Thus it was found that trout adapted to a palecoloured background, in which MCH is being actively
MATERIALS AND METHODS
Fish
Rainbow trout aged 1-2 years (150-250 g) were obtained from a local fish farm (Alderley Trout Ltd, Wotton-Under-Edge, Glos.). In the aquarium they were kept in white- or black-coloured tanks (250
litres) in slow-flowing tap water (100 ml/min) at 11 °C, under a photoperiod of 18h light: 6h darkness. All fish were adapted to aquarium conditions for at least 2 weeks before the experiment, during which time they were
not fed.
Administration of stress
=
Control fish remained undisturbed throughout the Stress was caused by injecting 1 ml iso¬ tonic saline (0-8% (w/v) NaCl) into the peritoneal cavity without anaesthesia. Trout were usually given one injection per day for one or several days (see indi¬ vidual experimental details) and killed 1 h after the final injection.
experiment.
Collection of plasma
samples At the end ofthe experiment, fish were caught in a single
sweep of a net and anaesthetized as a group in a tank of phenoxyethanol (diluted 1 : 1667, v/v). Blood was
collected from the severed caudal peduncle in cold poly¬ propylene tubes containing 50 pi 6% (w/v) EDTA disodium salt as anticoagulant and 2500 KIU aprotinin (Sigma Chemical Co. Ltd, Poole, Dorset, U.K.) as enzyme inhibitor. Blood samples were collected within 5 min of catching the fish. After centrifugation at 2000 g for 15 min, aliquots of plasma were stored at 20 °C. —
Radioimmunoassays
Statistics All statistical comparisons of means were made using the pooled Student's t-test; the appropriate trans¬ formations (square root or logarithmic) were per¬ formed on all data to give homogeneity of variance. Results are expressed as means ± s.e.m. RESULTS
Acute stress and MCH release
single injection of isotonic saline did not signifi¬ cantly affect the plasma concentration of MCH in white-adapted fish at 1 or 2 h after injection (plasma MCH concentration in pmol/1: 1-h control, 44-5 ± 12-0, 1-h injected, 61-2 + 8 ; 2-h control, 48-1 + 8-5, 2-h injected, 60T + 10-0; w 8). By contrast, cortisol concentrations were significantly (P
