Dietary l-tryptophan leaves a lasting impression on the brain and the stress response.

Comparative models suggest that effects of dietary tryptophan (Trp) on brain serotonin (5-hydroxytryptamine; 5-HT) neurochemistry and stress responsiveness are present throughout the vertebrate lineage. Moreover, hypothalamic 5-HT seems to play a central role in control of the neuroendocrine stress axis in all vertebrates. Still, recent fish studies suggest long-term effects of dietary Trp on stress responsiveness, which are independent of hypothalamic 5-HT. Here, we investigated if dietary Trp treatment may result in long-lasting effects on stress responsiveness, including changes in plasma cortisol levels and 5-HT neurochemistry in the telencephalon and hypothalamus of Atlantic salmon. Fish were fed diets containing one, two or three times the Trp content in normal feed for 1 week. Subsequently, fish were reintroduced to control feed and were exposed to acute crowding stress for 1 h, 8 and 21 d post Trp treatment. Generally, acute crowding resulted in lower plasma cortisol levels in fish treated with 3×Trp compared with 1×Trp- and 2×Trp-treated fish. The same general pattern was reflected in telencephalic 5-HTergic turnover, for which 3×Trp-treated fish showed decreased values compared with 2×Trp-treated fish. These long-term effects on post-stress plasma cortisol levels and concomitant 5-HT turnover in the telencephalon lends further support to the fact that the extrahypothalamic control of the neuroendocrine stress response is conserved within the vertebrate lineage. Moreover, they indicate that trophic/structural effects in the brain underlie the effects of dietary Trp treatment on stress reactivity.

Changes in regional brain monoaminergic activity and temporary down-regulation in stress response from dietary supplementation with l-tryptophan in Atlantic cod (Gadus morhua).

The brain monoamines serotonin (5-hydroxytryptamine; 5-HT) and dopamine (DA) both play an integrative role in behavioural and neuroendocrine responses to challenges, and comparative models suggest common mechanisms for dietary modulation of transmission by these signal substances in vertebrates. Previous studies in teleosts demonstrate that 7 d of dietary administration with L-tryptophan (Trp), the direct precursor of 5-HT, suppresses the endocrine stress response. The present study investigated how long the suppressive effects of a Trp-enriched feed regimen, at doses corresponding to two, three or four times the Trp levels in commercial feed, last in juvenile Atlantic cod (Gadus morhua) when the fish are reintroduced to a diet with standard amino acid composition. We also wanted to determine whether Trp supplementation induced changes in brain monoaminergic neurochemistry in those forebrain structures innervated by DA and 5-HTergic neurons, by measuring regional activity of DA and 5-HT in the lateral pallial regions (Dl) of the telencephalon and nucleus lateralis tuberis (NLT) of the hypothalamus. Dietary Trp resulted in a dose-dependent suppression in plasma cortisol among fish exposed to confinement stress on the first day following experimental diet; however, such an effect was not observed at 2 or 6 d after Trp treatment. Feeding the fish with moderate Trp doses also evoked a general increase in DA and 5-HT-ergic activity, suggesting that these neural circuits within the NLT and Dl may be indirectly involved in regulating the acute stress response.

Stress effects on AVT and CRF systems in two strains of rainbow trout (Oncorhynchus mykiss) divergent in stress responsiveness.

The aim for this study was to examine whether the F4 generation of two strains of rainbow trout divergent in their plasma cortisol response to confinement stress (HR: high responder or LR: low responder) would also differ in stress-induced effects on forebrain concentrations of mRNA for corticotropin-releasing factor (CRF), arginine vasotocin (AVT), CRF receptor type 1 (CRF-R1), CRF receptor type 2 (CRF-R2) and AVT receptor (AVT-R). In addition, plasma cortisol concentrations, brainstem levels of monoamines and monoamine metabolites, and behaviour during confinement were monitored. The results confirm that HR and LR trout differ in their cortisol response to confinement and show that fish of these strains also differ in their behavioural response to confinement. The HR trout displayed significantly higher locomotor activity while in confinement than LR trout. Moreover, following 180 min of confinement HR fish showed significantly higher forebrain concentrations of CRF mRNA than LR fish. Also, when subjected to 30 min of confinement HR fish showed significantly lower CRF-R2 mRNA concentrations than LR fish, whereas there were no differences in CRF-R1, AVT or AVT-R mRNA expression between LR and HR fish either at 30 or 180 min of confinement. Differences in the expression of CRF and CRF-R2 mRNA may be related to the divergence in stress coping displayed by these rainbow trout strains.

Melanocortin peptides affect the motivation to feed in rainbow trout (Oncorhynchus mykiss).

In this study, we investigated the effects of one melanocortin receptor (MCR) agonist and two antagonists on food intake in juvenile rainbow trout. Baseline food intake was established prior to 1 microl intracerebroventricular injection (ICV) of the non-specific agonist MTII, the MC4R antagonist HS024 and the MC3/4R antagonist SHU9119 at concentrations of 0.3, 1 or 3 nM. Saline-injected fish and untreated fish served as controls. Changes in food intake were observed 1h after the ICV injections. Our results showed that treatment with MTII significantly decreased food intake at 3 nM compared to control, HS024 significantly increased food intake at 3 nM compared to control and saline-treated fish, and SHU9119 significantly increased food intake at 3 nM compared to saline-treated fish. In conclusion, our study provides further evidence, and hence strengthens the hypothesis, that MC4R participates in the control of energy balance in fish in the same manner as in mammals. Our findings that HS024 is more potent than SHU9119 in increasing food intake suggest that the effects of melanocortin on energy balance in rainbow trout are mainly regulated by activation of MC4R. Hence, HS024 seems an excellent tool as a MC4R antagonist in rainbow trout.

Genetically determined variation in stress responsiveness in rainbow trout: behavior and neurobiology.

It is becoming increasingly recognized that the diversity in stressors, their intensity, predictability and the context in which they are experienced, will result in behavioral and physiological responses just as diverse. In addition, stress responses are characterized by individual variations where the physiological and behavioral reactions are associated in such a manner that distinct stress coping styles encompassing suites of correlated traits can be identified. These are often referred to as proactive and reactive stress coping styles. Proactive coping is characterized by more aggression, higher general activity and higher sympathetic activation, whereas reactive coping is characterized by immobility, lack of initiative and a higher parasympathetic/hypothalamic activation. Stable coping styles appear to coexist within populations, and these strategies appear to be largely innate. Moreover, the physiological and behavioral traits of coping styles appear to be heritable. These stress coping styles have proven to play a major role in competitive ability and subsequent social position in different species of vertebrates. However, there are also studies showing that social position can affect parameters encompassing the stress coping style of individuals. In this regard it is important, but not always easy, to distinguish between causes and effects of behavioral and physiological responses to stressors. The question raised is to what extent and rigidness stress coping styles are guided by genetic factors.

The toxicity of copper to crucian carp (Carassius carassius) in soft water.

Crucian carp (Carassius carassius) were exposed to a Cu rich medium (pH 6.6, conductivity 25 microS/cm, 2.91 mg Ca(2+)/l, approximately 300 microg Cu(2+)/l). Untreated department water (pH 6.6, conductivity 25 microS/cm, 2.91 mg Ca(2+)/l) acted as control. Mortality in crucian carp was first observed after 13 days of exposure to the Cu rich medium. There were, however, significant changes in haematocrit, plasma chloride, plasma sodium and water content in muscle in fish exposed to the Cu rich medium after two days. After 14 days of exposure to copper, haematocrit increased to 52+/-2% (control: between 37 and 40%), plasma chloride decreased to 45+/-5 mmol/l (control: 99-106 mmol/l), plasma sodium decreased to 81+/-6 mmol/l (control: 116-137 mmol/l), and water content in muscle increased to 83.0+/-0.3% (control: 78.7-79.9%). No apparent changes in blood ethanol, and minor changes in plasma lactate were observed in copper exposed fish. Analyses of the gills revealed an increasing concentration of copper on the gills from fish exposed to Cu rich water. After 14 days, the concentration of copper accumulated in the gill was 12.8+/-4.1 microg Cu/g wet weight (control: 0.91-1.19 microg Cu/g wet weight). A reduction of the respiratory area in fish exposed to copper was observed, in terms of both lamellar and filamental fusion. The normoxic O(2) uptake did not change, but the critical oxygen tension was elevated to 6.12+/-1.04 mg O(2)/l after a 6 day exposure to copper (control: 1.03+/-0.05 mg O(2)/l). This study shows that crucian carp has a higher tolerance to copper compared to other freshwater fish species. Our results suggest that this tolerance is based on the ability of crucian carp to avoid becoming hypoxic as well as an extreme tolerance to severe loss of plasma ions.

The high tolerance to aluminium in crucian carp (Carassius carassius) is associated with its ability to avoid hypoxia.

It is well known that aluminium is the principle toxicant killing fish in acidified freshwater systems, and it has been shown that crucian carp (Carassius carassius) can survive exposures to aqueous aluminium levels toxic to most other freshwater fish species. The crucian carp has a remarkable ability to survive anoxic conditions, and the aim of the present study was to reveal if the tolerance to aluminium can be associated with the ability to survive prolonged anoxia. Crucian carps were exposed to either acidic Al-rich water (pH 5.8; 960 µg Al/l), acidic Al-poor water (pH 5.8; 50 µg Al/l) or untreated control water (pH 6.5; 50 µg Al/l). Blood, muscle and gill samples were collected from exposed fish, and closed respirometry was performed to measure critical O2-tension an normoxic O2-consumption. The results show an increased gill surface area in Al-exposed fish, while the critical O2-tension did not change. The normoxic O2-consumption was lower in Al-exposed fish and might be due to a reduced metabolic rate. The results suggest that crucian carp exposed to aluminium do not become hypoxic, since haematocrit, plasma lactate and blood ethanol did not differ from that of control fish after 14 days of exposure. We also observed an initial loss of plasma chloride and sodium, followed by a stabilisation of these ions at a lower level than in control fish. The decrease in plasma ions caused a transient increase in haematocrit and water content in muscle tissue, returning to control levels when the ion concentrations stabilised, suggesting that the water balance was restored. We conclude that the high tolerance to aluminium in crucian carp is associated with its ability to avoid hypoxia as well as an ability to counteract a continuous loss of plasma ions.

Avoidance behavior and brain monoamines in fish.

The crucian carp performs a typical avoidance behavior when exposed to olfactory cues from injured skin of conspecifics. They swim rapidly to the bottom and hide in available material. This work examines the effects of skin extract exposure and availability of hiding material on this behavior, and concomitant changes in brain monoaminergic activity in crucian carp. Individual fish were exposed to skin extract in aquaria with or without hiding material. Exposure to skin extract resulted in the expected avoidance behavior consisting of rapid movement towards the bottom of the aquarium. This lasted for 1-2 min. Activity then decreased below the level observed before exposure, suggesting a "freezing" type of avoidance behavior. This behavior was independent of availability of hiding material. Brain dopaminergic activity increased in telencephalon and decreased in the brain stem following skin extract exposure, again independent of availability of hiding material. However, fish kept in aquaria without hiding material showed an elevation of serotonergic activity in the brain stem and the optic tectum compared to fish with available hiding material. Absence of hiding material increased serotonergic activity also without exposure to skin extract. In aquaria with hiding material, the fish stirred up a cloud of fine sediments and showed a more pronounced decrease in locomotor activity in agreement with this being a more efficient freezing or immobile avoidance behavior. These results show that basic components of avoidance behavior and related brain changes are present in the fish brain, in accordance with the common phylogenetic roots of avoidance behavior in all vertebrates.

Does individual variation in stress responses and agonistic behavior reflect divergent stress coping strategies in juvenile rainbow trout?

Individual rainbow trout were transferred to visual isolation in experimental aquaria. As a measure of the speed of acclimation, individual food intake was quantified during the first 6 d following transfer. Following acclimation, aggression was quantified by subjecting the fish to three resident-intruder tests, with 30 d of recovery between the tests. Moreover, between the resident-intruder tests (i.e., two times) the fish were exposed to an unfamiliar environment and their cortisol response was measured. The results of this study show that individuals of juvenile rainbow trout differ distinctly in their response to changes in their environment, and that this diversity in behavior is reflected by consistent behavioral traits displayed by individual fish. These traits have proven to be consistent not only over time but also across situations, revealing two distinct behavioral profiles, in the same manner as shown in studies on proactive and reactive mammals. Our results also show that the reactivity of the hypothalamic-pituitary-interrenal (HPI) axis, when exposed to a stressor, is a consistent physiological trait in juvenile rainbow trout. We found that difference in HPI axis reactivity is linked to the different behavioral profiles. However, HPI axis reactivity could not be linked directly to the singular behavioral traits measured. In other words, we did not find that the consistent behavioral traits shown by the fish were associated with a difference in HPI axis reactivity in the same manner as the reactivity of the hypothalamic-pituitary-adrenocortical axis does in mammals. Taken together, our results show that stress coping strategies akin to what has been described as reactive and proactive stress coping in mammals appear to exist in juvenile rainbow trout.

Divergence in behavioural responses to stress in two strains of rainbow trout (Oncorhynchus mykiss) with contrasting stress responsiveness.

The aim of this study was to establish whether two lines of rainbow trout divergent for their plasma cortisol response to a standardized stressor would show consistent differences in their behavioural response to a range of challenging situations. Our results show that the high- and low-responding (HR and LR) lines of rainbow trout did not differ in the aggression shown towards an intruder or in their response to the introduction of a novel object to their home environment. However, there was a difference in behaviour between the two selection lines when they were exposed to two unfamiliar environments. These results suggest that the behaviour of the HR and LR fish differs when they are challenged in unfamiliar environments, while their behaviour does not differ when they are challenged in their home environment. These observations are in agreement with studies on mammals that show that individuals with reactive coping styles perform similarly to proactive animals when they are challenged in a familiar environment, while they show different behaviour when they are challenged in unfamiliar environments. Thus, these results provide further evidence that the HR and LR selection lines of rainbow trout exemplify the two different coping styles described in mammals.