Role of melanocortin system in the locomotor activity rhythms and melatonin secretion as revealed by agouti-signalling protein (asip1) overexpression in zebrafish.

Temporal signals such as light and temperature cycles profoundly modulate animal physiology and behaviour. Via endogenous timing mechanisms which are regulated by these signals, organisms can anticipate cyclic environmental changes and thereby enhance their fitness. The pineal gland in fish, through the secretion of melatonin, appears to play a critical role in the circadian system, most likely acting as an element of the circadian clock system. An important output of this circadian clock is the locomotor activity circadian rhythm which is adapted to the photoperiod and thus determines whether animals are diurnal or nocturnal. By using a genetically modified zebrafish strain known as Tg (Xla.Eef1a1:Cau.asip1)iim04, which expresses a higher level of the agouti signalling protein 1 (Asip1), an endogenous antagonist of the melanocortin system, we observed a complete disruption of locomotor activity patterns, which correlates with the ablation of the melatonin daily rhythm. Consistent with this, in vitro experiments also demonstrated that Asip1 inhibits melatonin secretion from the zebrafish pineal gland, most likely through the melanocortin receptors expressed in this gland. Asip1 overexpression also disrupted the expression of core clock genes, including per1a and clock1a, thus blunting circadian oscillation. Collectively, these results implicate the melanocortin system as playing an important role in modulating pineal physiology and, therefore, circadian organisation in zebrafish.

Obesity Impairs Cognitive Function with No Effects on Anxiety-like Behaviour in Zebrafish.

Over the last decade, the zebrafish has emerged as an important model organism for behavioural studies and neurological disorders, as well as for the study of metabolic diseases. This makes zebrafish an alternative model for studying the effects of energy disruption and nutritional quality on a wide range of behavioural aspects. Here, we used the zebrafish model to study how obesity induced by overfeeding regulates emotional and cognitive processes. Two groups of fish (n = 24 per group) were fed at 2% (CTRL) and 8% (overfeeding-induced obesity, OIO) for 8 weeks and tested for anxiety-like behaviour using the novel tank diving test (NTDT). Fish were first tested using a short-term memory test (STM) and then trained for four days for a long-term memory test (LTM). At the end of the experiment, fish were euthanised for biometric sampling, total lipid content, and triglyceride analysis. In addition, brains (eight per treatment) were dissected for HPLC determination of monoamines. Overfeeding induced faster growth and obesity, as indicated by increased total lipid content. OIO had no effect on anxiety-like behaviour. Animals were then tested for cognitive function (learning and memory) using the aversive learning test in Zantiks AD units. Results show that both OIO and CTRL animals were able to associate the aversive stimulus with the conditioned stimulus (conditioned learning), but OIO impaired STM regardless of fish sex, revealing the effects of obesity on cognitive processes in zebrafish. Obese fish did not show a deficiency in monoaminergic transmission, as revealed by quantification of total brain levels of dopamine and serotonin and their metabolites. This provides a reliable protocol for assessing the effect of metabolic disease on cognitive and behavioural function, supporting zebrafish as a model for behavioural and cognitive neuroscience.

Role of the G protein-coupled receptors GPR84 and GPR119 in the central regulation of food intake in rainbow trout.

We evaluated the role of the G protein-coupled receptors GPR84 and GPR119 in food intake regulation in fish using rainbow trout (Oncorhynchus mykiss) as a model. In the first experiment, we assessed the effects on food intake of intracerebroventricular treatment with agonists of these receptors. In the second experiment, we assessed the impact of the same treatments on mRNA abundance in the hypothalamus and hindbrain of neuropeptides involved in the metabolic control of food intake (npy, agrp1, pomca1 and cartpt) as well as in changes in parameters related to signalling pathways and transcription factors involved in the integrative response leading to neuropeptide production. Treatment with both agonists elicited an anorectic response in rainbow trout attributable to changes observed in the mRNA abundance of the four neuropeptides. Changes in neuropeptides relate to changes observed in mRNA abundance and phosphorylation status of the transcription factor FOXO1. These changes occurred in parallel with changes in the phosphorylation status of AMPKα and Akt, the mRNA abundance of mTOR as well as signalling pathways related to PLCß and IP3. These results allow us to suggest that (1) at least part of the capacity of fish brain to sense medium-chain fatty acids such as octanoate depends on the function of GPR84, and (2) the capacity of fish brain to sense N-acylethanolamides or triglyceride-derived molecules occurs through the binding of these ligands to GPR119.

Central serotonin participates in the anorexigenic effect of GLP-1 in rainbow trout (Oncorhynchus mykiss).

The incretin, glucagon-like peptide-1 (GLP-1) is a major player in the gut-brain axis regulation of energy balance and in fish it seems to exert a negative influence on food intake. In this study, we investigated the role of the brain serotonergic system in the effects promoted by a peripheral GLP-1 injection on food intake in rainbow trout (Oncorhynchus mykiss). For this, in a first experiment the incretin was intraperitoneally injected (100 ng/g body weight) alone or in combination with a 5HT2C receptor antagonist (SB 242084, 1 µg/g body weight) and food intake was measured 30, 90, and 180 min later. In a second experiment, we studied the effect of these treatments on mRNA abundance of hypothalamic neuropeptides that control food intake. In addition, the effect of GLP-1 on serotonin metabolism was assessed in hindbrain and hypothalamus. Our results show that GLP-1 induced a significant food intake inhibition, which agreed with the increased expression of anorexigenic neuropeptides pomc and cart in the hypothalamus. Furthermore, GLP-1 stimulated the synthesis of serotonin in the hypothalamus, which might be indicative of a higher use of the neurotransmitter. The effects of GLP-1 on food intake were partially reversed when a serotonin receptor antagonist, SB 242084, was previously administered to trout. This antagonist also reversed the stimulatory effect of the hormone in hypothalamic pomca1 mRNA abundance. We conclude that hypothalamic serotonergic pathways are essential for mediating the effects of GLP-1 on food intake in rainbow trout. In addition, the 5HT2C receptor subtype seems to have a prominent role in the inhibition of food intake induced by GLP-1 in this species.

Viral Infection Drives the Regulation of Feeding Behavior Related Genes in Salmo salar.

The feeding behavior in fish is a complex activity that relies on the ability of the brain to integrate multiple signals to produce appropriate responses in terms of food intake, energy expenditure, and metabolic activity. Upon stress cues including viral infection or mediators such as the proinflammatory cytokines, prostaglandins, and cortisol, both Pomc and Npy/Agrp neurons from the hypothalamus are stimulated, thus triggering a response that controls both energy storage and expenditure. However, how appetite modulators or neuro-immune cues link pathogenesis and energy homeostasis in fish remains poorly understood. Here, we provide the first evidence of a molecular linkage between inflammation and food intake in Salmon salar. We show that in vivo viral challenge with infectious pancreatic necrosis virus (IPNV) impacts food consumption by activating anorexic genes such as mc4r, crf, and pomcb and 5-HT in the brain of S. salar. At the molecular level, viral infection induces an overall reduction in lipid content in the liver, favoring the production of AA and EPA associated with the increment of elovl2 gene. In addition, infection upregulates leptin signaling and inhibits insulin signaling. These changes are accompanied by a robust inflammatory response represented by the increment of Il-1b, Il-6, Tnfa, and Pge2 as well as an increased cortisol level in vivo. Thus, we propose a model in which hypothalamic neurons respond to inflammatory cytokines and stress-related molecules and interact with appetite induction/inhibition. These findings provide evidence of crosstalk between pathogenesis-driven inflammation and hypothalamic-pituitary-adrenocortical axes in stress-induced food intake behavior in fish.

The long-chain fatty acid receptors FFA1 and FFA4 are involved in food intake regulation in fish brain.

We hypothesized that the free fatty acid receptors FFA1 and FFA4 might be involved in the anorectic response observed in fish after rising levels of long-chain fatty acids (LCFAs) such as oleate. In one experiment we demonstrated that intracerebroventricular (i.c.v.) treatment of rainbow trout with FFA1 and FFA4 agonists elicited an anorectic response 2, 6 and 24 h after treatment. In a second experiment, the same i.c.v. treatment resulted after 2 h in an enhancement in the mRNA abundance of anorexigenic neuropeptides pomca1 and cartpt and a decrease in the values of orexigenic peptides npy and agrp1 These changes occurred in parallel with those observed in the mRNA abundance and/or protein levels of the transcription factors Creb, Bsx and FoxO1, protein levels and phosphorylation status of Ampkα and Akt, and mRNA abundance of plcb1 and itrp3 Finally, we assessed in a third experiment the response of all these parameters after 2 h of i.c.v. treatment with oleate (the endogenous ligand of both free fatty acid receptors) alone or in the presence of FFA1 and FFA4 antagonists. Most effects of oleate disappeared in the presence of FFA1 and FFA4 antagonists. The evidence obtained supports the involvement of FFA1 and FFA4 in fatty acid sensing in fish brain, and thus involvement in food intake regulation through mechanisms not exactly comparable (differential response of neuropeptides and cellular signalling) to those known in mammals.

SIRT1 mediates the effect of stress on hypothalamic clock genes and food intake regulators in rainbow trout, Oncorhynchus mykiss.

Stress negatively affects a wide range of physiological and behavioural functions (circadian physiology and food intake, among others), thus compromising animal welfare. Cortisol mediates the effect of stress on food intake, but other mediators (such as sirtuins) may participate in that related to circadian physiology. We evaluated 1) the effect of stress on the day-night variation of hypothalamic clock genes and food intake regulators, 2) changes of mRNA abundance in cortisol biosynthesis at the head kidney, and 3) changes of glucocorticoid receptors in both tissues of rainbow trout, together with the involvement of SIRT1 in such effect. Trout receiving or not SIRT1 inhibitor (EX527) and subjected or not to stress by high stocking density (72 h), were sampled at day- (ZT10) and night-time (ZT18). Our results indicate that SIRT1 mediates the effect of stress on mRNA abundance of clock genes in trout hypothalamus, but it also influences those changes occurring on food intake-related peptides. High stocking density inhibits clock genes expression, but enhances that of food intake-related peptides. EX527 treatment prevents stress-related changes observed in clock genes, thus evidencing a key role played by SIRT1 in mediating this effect on trout circadian oscillators. On the other hand, EX527 treatment partially prevents changes of food intake-related peptides, indicating that an interaction between SIRT1 and other mediators (such as cortisol) exists during response to stress. In support of that, our results reveal that SIRT1 influences cortisol biosynthesis during stress. Whatever the case is, further research will help understanding the underlying mechanisms involved.

Evidence for the presence in rainbow trout brain of amino acid-sensing systems involved in the control of food intake.

To assess the hypothesis of central amino acid-sensing systems involved in the control of food intake in fish, we carried out two experiments in rainbow trout. In the first one, we injected intracerebroventricularly two different branched-chain amino acids (BCAAs), leucine and valine, and assessed food intake up to 48 h later. Leucine decreased and valine increased food intake. In a second experiment, 6 h after similar intracerebroventricular treatment we determined changes in parameters related to putative amino acid-sensing systems. Different areas of rainbow trout brain present amino acid-sensing systems responding to leucine (hypothalamus and telencephalon) and valine (telencephalon), while other areas (midbrain and hindbrain) do not respond to these treatments. The decreased food intake observed in fish treated intracerebroventricularly with leucine could relate to changes in mRNA abundance of hypothalamic neuropeptides [proopiomelanocortin (POMC), cocaine- and amphetamine-related transcript (CART), neuropeptide Y (NPY), and agouti-related peptide (AgRP)]. These in turn could relate to amino acid-sensing systems present in the same area, related to BCAA and glutamine metabolism, as well as mechanistic target of rapamycin (mTOR), taste receptors, and general control nonderepressible 2 (GCN2) kinase signaling. The treatment with valine did not affect amino acid-sensing parameters in the hypothalamus. These responses are comparable to those characterized in mammals. However, clear differences arise when comparing rainbow trout and mammals, in particular with respect to the clear orexigenic effect of valine, which could relate to the finding that valine partially stimulated two amino acid-sensing systems in the telencephalon. Another novel result is the clear effect of leucine on telencephalon, in which amino acid-sensing systems, but not neuropeptides, were activated as in the hypothalamus.

Response of rainbow trout's (Oncorhynchus mykiss) hypothalamus to glucose and oleate assessed through transcription factors BSX, ChREBP, CREB, and FoxO1.

We aimed to obtain information regarding mechanisms that link glucose- and fatty acid-sensing systems to expression of neuropeptides that regulate food intake in the fish brain. We assessed the relative expression and protein levels of the transcription factors BSX, ChREBP, FoxO1, and CREB in the hypothalamus of rainbow trout (Oncorhynchus mykiss) treated for 6 h with either glucose or oleate in vivo (intra-cerebroventricular treatment with 1 µl 100 g- 1 body weight of 40 µg glucose or 1 µmol oleate) or in vitro (incubation with 4-8 mM glucose or 100-500 µM oleate). BSX levels decreased after oleate treatment for mRNA (10% in vitro and 47% in vivo) and protein (25%), while minor changes occurred after glucose treatment. CREB values generally decreased after glucose or oleate treatment for mRNA (50% in vivo) as well as the phosphorylation status of protein (80%). Foxo1 mRNA levels increased in vivo with glucose (129%) and decreased in vivo with oleate (60%), and protein phosphorylation status increased with glucose (in vivo) and oleate. mRNA values of chrebpα decreased in response to glucose and oleate, while protein levels decreased with oleate and increased with glucose. The results support the association of several transcription factors with metabolic control of food intake in fish.

Changes in the levels and phosphorylation status of Akt, AMPK, CREB and FoxO1 in hypothalamus of rainbow trout under conditions of enhanced glucosensing activity.

There is no available information about mechanisms linking glucosensing activation in fish and changes in the expression of brain neuropeptides controlling food intake. Therefore, we assessed in rainbow trout hypothalamus the effects of raised levels of glucose on the levels and phosphorylation status of two transcription factors, FoxO1 and CREB, possibly involved in linking these processes. We also aimed to assess the changes in the levels and phosphorylation status of two proteins possibly involved in the modulation of these transcription factors: Akt and AMPK. Therefore, in pooled preparations of hypothalamus incubated for 3 and 6 h in the presence of 2, 4 or 8 mmol l-1 d-glucose, we evaluated the response of parameters related to glucosensing mechanisms, neuropeptide expression and levels and phosphorylation status of the proteins of interest. The activation of hypothalamic glucosensing systems and the concomitant enhanced anorectic potential occurred in parallel with activation of Akt and inhibition of AMPK. The changes in these proteins relate to neuropeptide expression through changes in the level and phosphorylation status of transcription factors under their control, such as CREB and FoxO1, which displayed inhibitory (CREB) or activatory (FoxO1) responses to increased glucose.

Ceramide counteracts the effects of ghrelin on the metabolic control of food intake in rainbow trout.

In mammals, ceramides are involved in the modulation of the orexigenic effects of ghrelin (GHRL). We previously demonstrated in rainbow trout that intracerebroventricular (ICV) treatment with ceramide (2.5 µg/100 g fish) resulted in an anorexigenic response, i.e. a response opposed to that described in mammals, where ceramide treatment is orexigenic. Therefore, we hypothesized that the putative interaction between GHRL and ceramide must be different in fish. Accordingly, in a first experiment, we observed that ceramide levels in the hypothalamus of rainbow trout did not change after ICV treatment with GHRL. In a second experiment, we assessed whether the effects of GHRL treatment on the regulation of food intake in rainbow trout changed in the presence of ceramide. Thus, we injected ICV GHRL and ceramide alone or in combination to evaluate in hypothalamus and hindbrain changes in parameters related to the metabolic control of food intake. The presence of ceramide generally counteracted the effects elicited by GHRL on fatty acid-sensing systems, the capacity of integrative sensors (AMPK, mTOR and SIRT-1), proteins involved in cellular signalling pathways (Akt and FoxO1) and neuropeptides involved in the regulation of food intake (AgRP, NPY, POMC and CART). The results are discussed in the context of regulation of food intake by metabolic and endocrine inputs.

Ceramides are involved in the regulation of food intake in rainbow trout (Oncorhynchus mykiss).

We hypothesize that ceramides are involved in the regulation of food intake in fish. Therefore, we assessed in rainbow trout (Oncorhynchus mykiss) the effects of intracerebroventricular treatment with C6:0 ceramide on food intake. In a second experiment, we assessed the effects in brain areas of ceramide treatment on neuropeptide expression, fatty acid-sensing systems, and cellular signaling pathways. Ceramide treatment induced a decrease in food intake, a response opposed to the orexigenic effect described in mammals, which can be related to enhanced mRNA abundance of cocaine and amphetamine-related transcript and proopiomelanocortin and decreased mRNA abundance of Agouti-related protein and neuropeptide Y. Fatty acid-sensing systems appear to be inactivated by ceramide treatment. The mRNA abundance of integrative sensors AMPK and sirtuin 1, and the phosphorylation status of cellular signaling pathways dependent on protein kinase B, AMPK, mammalian target of rapamycin (mTOR), and forkhead box protein O1 (FoxO1) are generally activated by ceramide treatment. However, there are differences between hypothalamus and hindbrain in the phosphorylation status of AMPK (decreased in hypothalamus and increased in hindbrain), mTOR (decreased in hypothalamus and increased in hindbrain), and FoxO1 (increased in hypothalamus and decreased in hindbrain) to ceramide treatment. The results suggest that ceramides are involved in the regulation of food intake in rainbow trout through mechanisms comparable to those characterized previously in mammals in some cases.

Behind melanocortin antagonist overexpression in the zebrafish brain: A behavioral and transcriptomic approach.

Melanocortin signaling is regulated by the binding of naturally occurring antagonists, agouti-signaling protein (ASIP) and agouti-related protein (AGRP) that compete with melanocortin peptides by binding to melanocortin receptors to regulate energy balance and growth. Using a transgenic model overexpressing ASIP, we studied the involvement of melanocortin system in the feeding behaviour, growth and stress response of zebrafish. Our data demonstrate that ASIP overexpression results in enhanced growth but not obesity. The differential growth is explained by increased food intake and feeding efficiency mediated by a differential sensitivity of the satiety system that seems to involve the cocaine- and amphetamine- related transcript (CART). Stress response was similar in both genotypes. Brain transcriptome of transgenic (ASIP) vs wild type (WT) fish was compared using microarrays. WT females and males exhibited 255 genes differentially expressed (DEG) but this difference was reduced to 31 after ASIP overexpression. Statistical analysis revealed 1122 DEG when considering only fish genotype but 1066 and 981 DEG when comparing ASIP males or females with their WT counterparts, respectively. Interaction between genotype and sex significantly affected the expression of 97 genes. Several neuronal systems involved in the control of food intake were identified which displayed a differential expression according to the genotype of the fish that unravelling the flow of melanocortinergic information through the central pathways that controls the energy balance. The information provided herein will help to elucidate new central systems involved in control of obesity and should be of invaluable use for sustaining fish production systems.

In vitro evidence supports the presence of glucokinase-independent glucosensing mechanisms in hypothalamus and hindbrain of rainbow trout.

We previously obtained evidence in rainbow trout for the presence and response to changes in circulating levels of glucose (induced by intraperitoneal hypoglycaemic and hyperglycaemic treatments) of glucosensing mechanisms based on liver X receptor (LXR), mitochondrial production of reactive oxygen species (ROS) leading to increased expression of uncoupling protein 2 (UCP2), and sweet taste receptor in the hypothalamus, and on sodium/glucose co-transporter 1 (SGLT-1) in hindbrain. However, these effects of glucose might be indirect. Therefore, we evaluated the response of parameters related to these glucosensing mechanisms in a first experiment using pooled sections of hypothalamus and hindbrain incubated for 6 h at 15°C in modified Hanks' medium containing 2, 4 or 8 mmol l(-1) d-glucose. The responses observed in some cases were consistent with glucosensing capacity. In a second experiment, pooled sections of hypothalamus and hindbrain were incubated for 6 h at 15°C in modified Hanks' medium with 8 mmol l(-1) d-glucose alone (control) or containing 1 mmol l(-1) phloridzin (SGLT-1 antagonist), 20 µmol l(-1) genipin (UCP2 inhibitor), 1 µmol l(-1) trolox (ROS scavenger), 100 µmol l(-1) bezafibrate (T1R3 inhibitor) and 50 µmol l(-1) geranyl-geranyl pyrophosphate (LXR inhibitor). The response observed in the presence of these specific inhibitors/antagonists further supports the proposal that critical components of the different glucosensing mechanisms are functioning in rainbow trout hypothalamus and hindbrain.

Intracerebroventricular ghrelin treatment affects lipid metabolism in liver of rainbow trout (Oncorhynchus mykiss).

We aimed to elucidate in rainbow trout (Oncorhynchus mykiss) the effects of central ghrelin (GHRL) treatment on the regulation of liver lipid metabolism, and the possible modulatory effect of central GHRL treatment on the simultaneous effects of raised levels of oleate. Thus, we injected intracerebroventricularly (ICV) rainbow trout GHRL in the presence or absence of oleate and evaluated in liver variables related to lipid metabolism. Oleate treatment elicited in liver of rainbow trout decreased lipogenesis and increased oxidative capacity in agreement with previous studies. Moreover, as demonstrated for the first time in fish in the present study, GHRL also acts centrally modulating lipid metabolism in liver, resulting in increased potential for lipogenesis and decreased potential for fatty acid oxidation, i.e. the converse effects to those elicited by central oleate treatment. The simultaneous treatment of GHRL and oleate confirmed these counteractive effects. Thus, the nutrient sensing mechanisms present in hypothalamus, particularly those involved in sensing of fatty acid, are involved in the control of liver energy metabolism in fish, and this control is modulated by the central action of GHRL. These results give support to the notion of hypothalamus as an integrative place for the regulation of peripheral energy metabolism in fish.

Is gill cortisol concentration a good acute stress indicator in fish? A study in rainbow trout and zebrafish.

Cortisol is the main biomarker of physiological stress in fish. It is usually measured in plasma, which requires blood collection. Though cortisol is produced in the anterior kidney, it can diffuse easily through cell membranes due to its lipophilic nature. Taking advantage of that, some non-invasive techniques have been developed to measure cortisol directly in the water from fish-holding tanks, in skin mucus or in scales. In this study, we explored the possibility to analyze fish cortisol from gill filaments as a reliable acute stress marker. Our results show that gill cortisol levels correlate well with plasma cortisol levels in both rainbow trout and zebrafish exposed or not to an acute stress protocol. Measuring cortisol in gill filaments increases the available possibilities for stress assessment in fish. Although this approach should yet be tested for its use with other stressors, it has several advantages: In relatively large fish (i.e. above 30 g) gill cortisol levels could be measured in vivo. Sampling of gill biopsies is very fast and easy, and the procedure does not induce stress if properly performed, making it an ideal option for in vivo stress assessment. In small fish, the use of gill tissue to measure cortisol has important technical advantages with respect to the current methods using whole-body homogenates. Gill homogenates could be used directly for ELISA cortisol analysis, avoiding the need of tedious and expensive cortisol extraction protocols, and, since no organic solvent is required, contributing for a more environmentally friendly analysis.

Stress inhibition of melatonin synthesis in the pineal organ of rainbow trout (Oncorhynchus mykiss) is mediated by cortisol.

Cortisol has been suggested to mediate the effect of stress on pineal melatonin synthesis in fish. Therefore, we aimed to determine how pineal melatonin synthesis is affected by exposing rainbow trout to different stressors, such as hypoxia, chasing and high stocking density. In addition, to test the hypothesis that cortisol is a mediator of such stress-induced effects, a set of animals were intraperitoneally implanted with coconut oil alone or containing cortisol (50 mg kg(-1) body mass) and sampled 5 or 48 h post-injection at midday and midnight. The specificity of such effect was also assessed in cultured pineal organs exposed to cortisol alone or with the general glucocorticoid receptor antagonist, mifepristone (RU486). Stress (in particular chasing and high stocking density) affected the patterns of plasma and pineal organ melatonin content during both day and night, with the greatest reduction occurring at night. The decrease in nocturnal melatonin levels in the pineal organ of stressed fish was accompanied by increased serotonin content and decreased AANAT2 enzymatic activity and mRNA abundance. Similar effects on pineal melatonin synthesis to those elicited by stress were observed in trout implanted with cortisol for either 5 or 48 h. These data indicate that stress negatively influences the synthesis of melatonin in the pineal organ, thus attenuating the day-night variations of circulating melatonin. The effect might be mediated by increased cortisol, which binds to trout pineal organ-specific glucocorticoid receptors to modulate melatonin rhythms. Our results in cultured pineal organs support this. Considering the role of melatonin in the synchronization of daily and annual rhythms, the results suggest that stress-induced alterations in melatonin synthesis could affect the availability of fish to integrate rhythmic environmental information.

Is plasma cortisol response to stress in rainbow trout regulated by catecholamine-induced hyperglycemia?

Based on previous studies we hypothesize that under stress conditions catecholamine-induced hyperglycemia contributes to enhance cortisol production in head kidney of rainbow trout. Therefore, treatment with propranolol (ß-adrenoceptor blocker) should reduce the hyperglycemia elicited by stress and, therefore, we expected reduced glucosensing response and cortisol production in head kidney. Propranolol treatment was effective in blocking most of the effects of catecholamines in liver energy metabolism resulting in a lower glycemia in stressed fish. The decreased glycemia of stressed fish treated with propranolol was observed along with reduced transcription of genes involved in the cortisol synthetic pathway, which supports our hypothesis. However, changes in putative glucosensing parameters assessed in head kidney were scarce and in general did not follow changes noted in glucose levels in plasma. Furthermore, circulating cortisol levels did not change in parallel with changes in glycemia. As a whole, the present results suggest that glycemia could participate in the regulation of cortisol synthetic pathways but other factors are also likely involved. Propranolol effects on trout stress response were different depending on time passed after stress onset; the direct or indirect involvement of catecholaminergic response in the regulation of cortisol production and release deserves further investigation.

Short-term time course of liver metabolic response to acute handling stress in rainbow trout, Oncorhynchus mykiss.

To elucidate the short-term time-course of liver metabolic response in rainbow trout to acute handling stress we subjected rainbow trout to 5min chasing and obtained samples 0 to 480min post-stress. Levels of cortisol, glucose and lactate were measured in plasma, whereas metabolite levels, enzyme activities, mRNA abundance of parameters related to energy metabolism, and glucocorticoid receptors were assessed in liver. Acute stress affected many parameters related to energy metabolism, with most of them turning back to normal levels after 480min. In general, the present results support the existence of two stages in the short-term time-course of metabolic response to handling stress. A first stage occurring few minutes post-stress (15-45min), was characterized by increased mobilization of liver glycogen resulting in increased production of endogenous glucose, reduced use of exogenous glucose and reduced lipogenic potential. A second stage, occurring 60-120min post-stress onwards was characterized by the recovery of liver glycogen levels, the increased capacity of liver for releasing glucose, and the recovery of lipogenic capacity whereas no changes were noted in gluconeogenic potential, which probably needs longer time periods to become enhanced.

The response of brain serotonergic and dopaminergic systems to an acute stressor in rainbow trout: a time course study.

The brain monoaminergic neurotransmitter systems are known to be involved in the integrated response to stress in vertebrates. However, present knowledge about the timing of their actions as well as their specific roles in the regulation of the endocrine axes that drive the stress response is incomplete. This is partly because of the complexity of the reciprocal interactions among the monoaminergic systems and other biochemical effectors of the stress response such as corticotropin-releasing factor (CRF), arginine vasotocin (AVT), adrenocorticotropic hormone (ACTH) and corticosteroids. In this study, we show for the first time in teleost fish (rainbow trout) the short- and mid-term time course of the response of the forebrain serotonergic and dopaminergic activities after exposure to an acute stressor. Other stress markers like the plasma levels of cortisol, glucose and lactate were also monitored, providing a context in which to precisely locate the monoaminergic activation within the fish acute stress response. Our results show that acute stress induced a rapid increase in forebrain serotonergic activity, which became elevated after only 15 s of chasing. Several hours after stress, serotonergic activity recovered its basal levels, in parallel with the recovery of other stress markers such as plasma catecholamines and cortisol. Dopaminergic activity was also increased after stress, but only in the telencephalon and only after 20 min. The increase in serotonergic activity happened before the elevation of plasma catecholamines, suggesting that this monoamine system could have a key role in triggering the initial steps of the activation of not only the hypothalamus-pituitary-inter-renal axis but also the brain-sympathetic-chromaffin axis in fish.