Mineralocorticoid receptor activates postnatal adiposity in zebrafish lacking proopiomelanocortin.

The proopiomelanocortin (Pomc)-derived peptides, including adrenocorticotropic hormone and α-melanocyte stimulating hormone (α-Msh), play both a central and a peripheral role in modulating the stress response. The central role is predominantly associated with nutrient homeostasis, while peripherally they play an important role in the synthesis of glucocorticoids (GCs) in response to stress. Pomc mutations are a major risk factor in the development of early-onset childhood obesity in humans. This is attributed primarily to their central effects on melanocortin receptor dysfunction leading to hyperphagia and reduced energy expenditure, while the peripheral mechanism contributing to obesity has largely been unexplored. Here, we tested the hypothesis that Pomc mutation-mediated adrenal insufficiency and the associated changes in GC signaling contribute to postnatal adiposity using zebrafish as a model. We generated a ubiquitous Pomc knockout zebrafish that mimicked the mammalian mutant phenotype of adrenal insufficiency and enhanced adiposity. The loss of Pomc inhibited stress-induced cortisol production and reprogrammed GC signaling by reducing glucocorticoid receptor responsiveness, whereas the mineralocorticoid receptor (Mr) signaling was enhanced. Larval feeding led to enhanced growth and adipogenesis in the Pomc mutants, and this was inhibited by eplerenone, an Mr antagonist. Altogether, our results underscore a key role for Mr signaling in early developmental adipogenesis and a possible target for therapeutic intervention for early-onset childhood obesity due to Pomc dysfunction.

Exposure to Total Suspended Solids (TSS) Impacts Gill Structure and Function in Adult Zebrafish.

Total suspended solids (TSS) are a major contributor of anthropogenic impacts to aquatic systems. TSS exposure have been shown to affect the function of gills, but the mode of action is unclear. Zebrafish (Danio rerio) is emerging as an excellent model for mechanistic toxicology, and as there are no baseline studies on TSS effects in zebrafish gills, we tested the hypothesis that environmental concentrations of TSS damages gill structure and function in this species. Adult zebrafish were exposed to either 0, 10, 100, 500, 1000, or 2000 mg/L TSS for 4 days to assess the gill morphology. The minimal concentration that affected the gill structure was further tested for the distribution of key ion transporters, including Na+/K+- ATPase (NKA) and vacuolar-type H+-ATPase (VHA), using confocal microscopy. Our results reveal that TSS concentration as low as 100 mg/L alters the morphology of gills, including greater filament thickness, lamellae thickness, and epithelial lifting. This was also associated with a reduction in NKA immunoreactive (IR) cell count and intensity in the 100 mg/L TSS group, while there was neither a change in the VHA-IR cell count or expression nor the transcript abundance of atp6v1a and atp1a1a4 in the gills. Markers of stress response in these animals, including levels of cortisol, glucose, lactate, and glycogen were not altered after 4 days of TSS exposure. Overall, environmentally relevant concentrations of TSS can damage the gill structure and function in zebrafish and has the potential to enhance the toxicity of contaminants acting via the gills.

Valine administration in the hypothalamus alters the brain and plasma metabolome in rainbow trout.

Central administration of valine has been shown to cause hyperphagia in fish. Although mechanistic target of rapamycin (mTOR) is involved in this response, the contributions to feed intake of central and peripheral metabolite changes due to excess valine are unknown. Here, we investigated whether intracerebroventricular injection of valine modulates central and peripheral metabolite profiles and may provide insights into feeding response in fish. Juvenile rainbow trout (Oncorhynchus mykiss) were administered an intracerebroventricular injection of valine (10 µg·µL-1 at 1 µL·100·g-1 body wt), and the metabolite profile in plasma, hypothalamus, and rest of the brain (composing of telencephalon, optic tectum, cerebellum, and medulla oblongata) was carried out by liquid chromatography-mass spectrometry (LC/MS)-based metabolomics. Valine administration led to a spatially distinct metabolite profile at 1 h postinjection in the brain: enrichment of amino acid metabolism and energy production pathways in the rest of the brain but not in hypothalamus. This suggests a role for extrahypothalamic input in the regulation of feed intake. Also, there was enrichment of several amino acids, including tyrosine, proline, valine, phenylalanine, and methionine, in plasma in response to valine. Changes in liver transcript abundance and protein expression reflect an increased metabolic capacity, including energy production from glucose and fatty acids, and a lower protein kinase B (Akt) phosphorylation in the valine group. Altogether, valine intracerebroventricular administration affects central and peripheral metabolism in rainbow trout, and we propose a role for the altered metabolite profile in modulating the feeding response to this branched-chain amino acid.NEW & NOTEWORTHY Valine causes hyperphagia in fish when it is centrally administered; however, the exact mechanisms are far from clear. We tested how intracerebroventricular injection of valine in rainbow trout affected the brain and plasma metabolome. The metabolite changes in response to valine were more evident in the rest of the brain compared with the hypothalamus. Furthermore, we demonstrated for the first time that central valine administration affects peripheral metabolism in rainbow trout.

Zygotic Exposure to Venlafaxine Disrupts the Circadian Locomotor Activity Behaviour in Zebrafish Larvae.

The antidepressant venlafaxine, a selective serotonin and norepinephrine reuptake inhibitor, is commonly prescribed to treat major depressive disorder and is found at high concentrations in the aquatic environment. Concerns have been raised related to the health of aquatic organisms in response to this nontargeted pharmaceutical exposure. For instance, we previously demonstrated that exposure to venlafaxine perturbs neurodevelopment, leading to behavioural alterations in zebrafish (Danio rerio). We also observed disruption in serotonin expression in the pineal and raphe, regions critical in regulating circadian rhythms, leading us to hypothesize that zygotic exposure to venlafaxine disrupts the circadian locomotor rhythm in larval zebrafish. To test this, we microinjected zebrafish embryos with venlafaxine (1 or 10 ng) and recorded the locomotor activity in 5-day-old larvae over a 24-h period. Venlafaxine deposition reduced larval locomotor activity during the light phase, but not during the dark phase of the diurnal cycle. The melatonin levels were higher in the dark compared to during the light photoperiod and this was not affected by embryonic venlafaxine deposition. Venlafaxine exposure also did not affect the transcript abundance of clock genes, including clock1a, bmal2, cry1a and per2, which showed a clear day/night rhythmicity. A notable finding was that exposure to luzindole, a melatonin receptor antagonist, decreased the locomotor activity in the control group in light, whereas the activity was higher in larvae raised from the venlafaxine-deposited embryos. Overall, zygotic exposure to venlafaxine disrupts the locomotor activity of larval zebrafish fish during the day, demonstrating the capacity of antidepressants to disrupt the circadian rhythms in behaviour. Our results suggest that disruption in melatonin signalling may be playing a role in the venlafaxine impact on circadian behaviour, but further investigation is required to elucidate the possible mechanisms in larval zebrafish.

Chronic cortisol stimulation enhances hypothalamus-specific enrichment of metabolites in the rainbow trout brain.

The hypothalamus is a key integrating center that is involved in the initiation of the corticosteroid stress response, and in regulating nutrient homeostasis. Although cortisol, the principal glucocorticoid in humans and teleosts, plays a central role in feeding regulation, the mechanisms are far from clear. We tested the hypothesis that the metabolic changes to cortisol exposure signal an energy excess in the hypothalamus, leading to feeding suppression during stress in fish. Rainbow trout (Oncorhynchus mykiss) were administered a slow-release cortisol implant for 3 days, and the metabolite profiles in the plasma, hypothalamus, and the rest of the brain were assessed. Also, U-13C-glucose was injected into the hypothalamus by intracerebroventricular (ICV) route, and the metabolic fate of this energy substrate was followed in the brain regions by metabolomics. Chronic cortisol treatment reduced feed intake, and this corresponded with a downregulation of the orexigenic gene agrp, and an upregulation of the anorexigenic gene cart in the hypothalamus. The U-13C-glucose-mediated metabolite profiling indicated an enhancement of glycolytic flux and tricarboxylic acid intermediates in the rest of the brain compared with the hypothalamus. There was no effect of cortisol treatment on the phosphorylation status of AMPK or mechanistic target of rapamycin in the brain, whereas several endogenous metabolites, including leucine, citrate, and lactate were enriched in the hypothalamus, suggesting a tissue-specific metabolic shift in response to cortisol stimulation. Altogether, our results suggest that the hypothalamus-specific enrichment of leucine and the metabolic fate of this amino acid, including the generation of lipid intermediates, contribute to cortisol-mediated feeding suppression in fish.NEW & NOTEWORTHY Elevated cortisol levels during stress suppress feed intake in animals. We tested whether the feed suppression is associated with cortisol-mediated alteration in hypothalamus metabolism. The brain metabolome revealed a hypothalamus-specific metabolite profile suggesting nutrient excess. Specifically, we noted the enrichment of leucine and citrate in the hypothalamus, and the upregulation of pathways involved in leucine metabolism and fatty acid synthesis. This cortisol-mediated energy substrate repartitioning may modulate the feeding/satiety centers leading to the feeding suppression.

Excess feeding increases adipogenesis but lowers leptin transcript abundance in zebrafish larvae.

Although fish exposed to municipal wastewater effluents (MWWE) show higher lipid accumulation, whether this is due to adipogenesis is unclear. The objective here was to identify molecular markers of adipogenesis in zebrafish (Danio rerio) larvae for use as high throughput screening tools for environmental contaminants, including obesogens in MWWE. Zebrafish larvae were fed a commercial diet at a maintenance level (5 % body mass) or in excess (25 or 50 % body mass) from day 6 to 30 days post-fertilization (dpf) to stimulate adipogenesis. We monitored fat accumulation and markers of lipid metabolism, including peroxisome proliferator-activated receptor γ (ppar γ), fatty acid synthase (fas), ELOVL fatty acid elongase 2 (elovl2), diacylglycerol O-acyltransferase 2 (dgat2), leptin (lepa and lepb), leptin receptor (lepr), and lipoprotein lipase (lpl). Excess feeding led to a higher growth rate, protein content and an increase in igf1 transcript abundance. Also, these larvae had higher triglyceride levels and accumulated lipids droplets in the abdominal cavity and viscera. The molecular markers of adipogenesis, including fas, elovl2, and dgat2, were upregulated, while the transcript abundance of lpl, a lipolytic gene, was transiently lower due to excess feeding. The increased adiposity seen at 30 dpf due to excess feeding coincided with a lower lep but not lepr transcript abundance in zebrafish. Our results demonstrate that excess feeding alters the developmental programming of key genes involved in lipid homeostasis, leading to excess lipid accumulation in zebrafish larvae. Overall, fas, elovl2, lpl, and dgat2, but not lep or ppar γ, have the potential to be biomarkers of adipogenesis in zebrafish larvae.

Negative feedback regulation in the hypothalamic-pituitary-interrenal axis of rainbow trout subjected to chronic social stress.

The formation of dominance hierarchies in pairs of juvenile rainbow trout (Oncorhynchus mykiss) results in subordinate individuals exhibiting chronically elevated plasma cortisol concentrations. Cortisol levels reflect a balance between cortisol production, which is coordinated by the hypothalamic-pituitary-interrenal (HPI) axis in teleost fish, and negative feedback regulation and hormone clearance, which act to lower cortisol levels. However, the mechanisms contributing to the longer-term elevation of cortisol levels during chronic stress are not well established in fishes. The current study aimed to determine how subordinate fish maintain elevated cortisol levels, by testing the prediction that negative feedback and clearance mechanisms are impaired by chronic social stress. Plasma cortisol clearance was unchanged by social stress based on a cortisol challenge trial, hepatic abundance of the cortisol-inactivating enzyme 11-beta hydroxysteroid dehydrogenase type 2 (11ßHSD2), and tissue fate of labelled cortisol. The capacity for negative feedback regulation in terms of transcript and protein abundances of corticosteroid receptors in the preoptic area (POA) and pituitary appeared stable. However, changes in 11ßHSD2 and mineralocorticoid receptor (MR) expression suggest subtle regulatory changes in the pituitary that may alter negative feedback. The chronic cortisol elevation observed during social subordination likely is driven by HPI axis activation and compounded by dysregulated negative feedback.

Cortisol Rapidly Facilitates Glucocorticoid Receptor Translocation to the Plasma Membrane in Primary Trout Hepatocytes.

Glucocorticoids (GCs) stimulate rapid cell signalling by activating the membrane-anchored intracellular glucocorticoid receptor (GR). However, the recruitment of the GR to the plasma membrane to facilitate nongenomic signalling is far from clear. As cytosolic free calcium ([Ca2+]i) is involved in intracellular protein dynamics, we tested the hypothesis that acute elevation in cortisol levels rapidly stimulates GR translocation to the plasma membrane via a calcium-dependent process in rainbow trout (Oncorhynchus mykiss) hepatocytes. To test this, we monitored temporal changes in intracellular GR distribution in response to cortisol exposure. Immunofluorescence labelling showed that the GR was present in cytosolic and nuclear compartments in trout hepatocytes. However, upon cortisol exposure, the GR rapidly (within 5 min) formed punctate and colocalized with caveolin-1, suggesting plasma membrane localization of the receptor. This redistribution of the GR to the plasma membrane was transient and lasted for 30 min and was evident even upon exposure to cortisol-BSA, a membrane-impermeable analogue of the steroid. The rapid cortisol-mediated GR translocation to the plasma membrane involved F-actin polymerization and was completely abolished in the presence of either EGTA or Cpd5J-4, a calcium release-activated calcium (CRAC) channel blocker. Additionally, the modulation of the biophysical properties of the plasma membrane by cholesterol or methyl ß-cyclodextrin, which led to changes in ([Ca2+]i) levels, modified GR translocation to the plasma membrane. Altogether, acute cortisol-mediated rise in ([Ca2+]i) levels rapidly stimulated the translocation of intracellular GR to the plasma membrane, and we propose this as a mechanism promoting the nongenomic action of the GR for hepatocyte stress resistance.

Chronic cortisol elevation restricts glucose uptake but not insulin responsiveness in zebrafish skeletal muscle.

Although teleosts show an elevated insulin response to hyperglycemia, the circulating glucose levels are not normalized as rapidly as in mammals. While this may suggest a lack of target tissue insulin responsiveness, the underlying mechanisms are unclear. We investigated whether changes in skeletal muscle insulin sensitivity and glucose uptake underlie the cortisol-mediated elevated blood glucose levels. Adult zebrafish (Danio rerio) were exposed to water-borne cortisol for 3 days followed by an intraperitoneal injection of glucose with or without insulin. Cortisol treatment resulted in a temporal delay in the reduction in blood glucose levels, and this corresponded with a reduced glucose uptake capacity and lower glycogen content in the skeletal muscle. The transcript abundance of slc2a1b (which encodes for GLUT1b) and a suite of genes encoding enzymes involved in muscle glycogenesis and glycolysis were upregulated in the cortisol group. Both the control and cortisol groups showed higher whole body insulin expression in response to blood glucose elevation, which also resulted in enhanced insulin-stimulated phosphorylation of AKT in the skeletal muscle. The insulin-mediated phosphorylation of S6 kinase was lower in the cortisol group. Altogether, chronic cortisol stimulation restricts glucose uptake and enhances the glycolytic capacity without affecting insulin responsiveness in zebrafish skeletal muscle.

Divergent Pathways of Ammonia and Urea Production and Excretion during the Life Cycle of the Sea Lamprey.

Little is known about nitrogenous waste (N waste) handling and excretion (JN waste) during the complex life cycle of the sea lamprey (Petromyzon marinus), an extant jawless fish that undergoes a complete metamorphosis from a filter-feeding larva (ammocoete) into a parasitic juvenile that feeds on the blood of larger, jawed fishes. Here, we investigate the ammonia- and urea-handling profiles of sea lampreys before, during, and after metamorphosis. The rates of ammonia excretion (Jamm) and urea excretion (Jurea) significantly decreased after the onset of metamorphosis, with the lowest rates observed during midmetamorphosis. Near the completion of metamorphosis, rates of JN waste (JN waste=Jamm+Jurea) significantly increased as sea lampreys entered the juvenile period. Feeding juvenile lampreys had greater than 10- to 15-fold higher Jamm and fivefold higher Jurea compared to nonfed juveniles, which corresponded to higher postprandial (postfeeding) concentrations of plasma ammonia and urea. The routes of Jamm and Jurea completely diverged following metamorphosis. In larvae, Jamm was equally split between branchial (gills) and extrabranchial (skin plus renal) pathways, but following metamorphosis, >80% of ammonia was excreted via the gills in nonfeeding juvenile lampreys, and >95% of ammonia was excreted via the gills in adult sea lampreys. Urea, on the other hand, was predominantly excreted via extrabranchial routes and, to a lesser extent, the gills in larvae and in nonfeeding juveniles. In adults, however, virtually all urea was excreted via urine. Reverse transcription polymerase chain reaction and in silico analyses also indicated that a urea transporter encoded by a slc4a2-like gene is present in lampreys. The branchial expression of this transporter is modulated throughout sea lamprey life history, as it is higher in the larvae and steadily decreases until the adult stage. We conclude that the divergent pathways of Jamm and Jurea during the sea lamprey life cycle reflect changes in their habitat, lifestyle, and diet. Further, the near-complete reliance on renal routes for Jurea in adult sea lampreys is unique among fishes and may reflect the ancestral condition of how this N waste product was handled and excreted by the earliest vertebrates.

Glucocorticoid receptor activation reduces food intake independent of hyperglycemia in zebrafish.

Chronic cortisol exposure suppresses food intake in fish, but the central mechanism(s) involved in appetite regulation are unclear. Stress and the associated increase in cortisol levels increase hepatic gluconeogenesis, leading to hyperglycemia. As hyperglycemia causes a reduction in food intake, we tested the hypothesis that cortisol-induced hyperglycemia suppresses feeding in zebrafish (Danio rerio). We first established that stress-independent hyperglycemia suppressed food intake, and this corresponded with a reduction in the phosphorylation of the nutrient sensor, AMP-activated protein kinase (AMPK) in the brain. Chronic cortisol exposure also led to hyperglycemia and reduced food intake, but the mechanisms were distinct. In cortisol-exposed fish, there were no changes in brain glucose uptake or AMPK phosphorylation. Also, the phosphorylation of Akt and mTOR was reduced along with an increase in redd1, suggesting an enhanced capacity for proteolysis. Loss of the glucocorticoid receptor did not rescue cortisol-mediated feeding suppression but did increase glucose uptake and abolished the changes seen in mTOR phosphorylation and redd1 transcript abundance. Taken together, our results indicate that GR activation enhances brain proteolysis, and the associated amino acids levels, and not hyperglycemia, maybe a key mediator of the feeding suppression in response to chronic cortisol stimulation in zebrafish.

The Mineralocorticoid Receptor Functions as a Key Glucose Regulator in the Skeletal Muscle of Zebrafish.

Glucocorticoids (GCs) are essential for maintaining energy homeostasis as part of the adaptive stress response. Most work to date has characterized the metabolic role of GCs via the activation of the glucocorticoid receptor (nr3c1; GR), which is activated under high GC conditions. However, GCs also bind to the mineralocorticoid receptor (nr3c2; MR), a high-affinity corticosteroid receptor active under basal GC conditions. Despite the expression of MR in skeletal muscles, almost nothing is known about its physiological role. Here we tested the hypothesis that the MR promotes anabolic processes during resting cortisol levels and curtails the catabolic actions of the GR during high (stressed) levels of cortisol. To determine the effect of MR, a zebrafish line with a ubiquitous MR knockout (MRca402/ca402) was utilized. The GR was activated in the same group by chronically treating fish with exogenous cortisol. In the muscle, MR primarily promoted nutrient storage, and restricted energy substrate mobilization under resting conditions, whereas GR activation resulted in increased nutrient utilization. Interestingly, MR loss improved GR-driven metabolic flexibility, suggesting that the activation state of these receptors is a key determinant of skeletal muscle ability to switch fuel sources. To determine if the anabolism-promoting nature of MR was due to an interaction with insulin, fish were co-injected with insulin and the fluorescent glucose analogue 2-NBDG. A loss of MR abolished insulin-stimulated glucose uptake in the skeletal muscle. Taken together, we postulate that MR acts as a key modulator of glucose metabolism in the musculature during basal and stress conditions.

Antidepressants as Endocrine Disrupting Compounds in Fish.

As antidepressant usage by the global population continues to increase, their persistent detection in aquatic habitats from municipal wastewater effluent release has led to concerns of possible impacts on non-target organisms, including fish. These pharmaceuticals have been marketed as mood-altering drugs, specifically targeting the monoaminergic signaling in the brain of humans. However, the monoaminergic systems are highly conserved and involved in the modulation of a multitude of endocrine functions in vertebrates. While most studies exploring possible impact of antidepressants on fish have focused on behavioural perturbations, a smaller spotlight has been placed on the endocrine functions, especially related to reproduction, growth, and the stress response. The purpose of this review is to highlight the possible role of antidepressants as endocrine disruptors in fish. While studies linking the effects of environmentally relevant levels of antidepressant on the endocrine system in fish are sparse, the emerging evidence suggests that early-life exposure to these compounds have the potential to alter the developmental programming of the endocrine system, which could persist as long-term and multigenerational effects in teleosts.

Increased gut serotonin production in response to bisphenol A structural analogs may contribute to their obesogenic effects.

Obesogens are synthetic, environmental chemicals that can disrupt endocrine control of metabolism and contribute to the risk of obesity and metabolic disease. Bisphenol A (BPA) is one of the most studied obesogens. There is considerable evidence that BPA exposure is associated with weight gain, increased adiposity, poor blood glucose control, and nonalcoholic fatty liver disease in animal models and human populations. Increased usage of structural analogs of BPA has occurred in response to legislation banning their use in some commercial products. However, BPA analogs may also cause some of the same metabolic impairments because of common mechanisms of action. One key effector that is altered by BPA and its analogs is serotonin, however, it is unknown if BPA-induced changes in peripheral serotonin pathways underlie metabolic perturbations seen with BPA exposure. Upon ingestion, BPA and its analogs act as endocrine-disrupting chemicals in the gastrointestinal tract to influence serotonin production by the gut, where over 95% of serotonin is produced. The purpose of this review is to evaluate how BPA and its analogs alter gut serotonin regulation and then discuss how disruption of serotonergic networks influences host metabolism. We also provide evidence that BPA and its analogs enhance serotonin production in gut enterochromaffin cells. Taken together, we propose that BPA and many BPA analogs represent endocrine-disrupting chemicals that can influence host metabolism through the endogenous production of gut-derived factors, such as serotonin.

Sex-Specific and Long-Term Impacts of Early-Life Venlafaxine Exposure in Zebrafish.

Venlafaxine, a selective serotonin and norepinephrine reuptake inhibitor, is a widely prescribed antidepressant that is detected in municipal wastewater effluents at µg/L concentrations. It has been shown to impact the early life stages of fish, including neurodevelopment and behaviour in larvae, but whether such early exposures have longer-term consequences are far from clear. Here, we sought to determine whether zygotic deposition of venlafaxine, mimicking a maternal transfer scenario, disturbs the metabolic rate and behavioural performance using zebrafish (Danio rerio). This was tested using freshly fertilized embryos (1-4 cell stage) microinjected with either 0, 1 or 10 ng of venlafaxine and raised to either juvenile (60 days post-fertilization) or adult (10-12 months post-fertilization). Zygotic venlafaxine exposure led to a reduction in the active metabolic rate and aerobic scope, but this was only observed in female fish. On the other hand, the total distance travelled in an open field assessment was greater at the highest concentration of venlafaxine only in the adult males. At the juvenile stage, behavioural assessments demonstrated that venlafaxine exposure may increase boldness-including hyperactivity, lower thigmotaxis, and a reduction in the distance to a novel object. Taken together, these results demonstrate that zygotic venlafaxine exposure may impact developmental programming in a sex-specific manner in fish.

Coordinated Action of Corticotropin-Releasing Hormone and Cortisol Shapes the Acute Stress-Induced Behavioural Response in Zebrafish.

INTRODUCTION: The stress response mediated by the hypothalamus-pituitary-adrenal (HPA) axis activation is highly conserved in vertebrates. Hyperactivity is one such established acute stress response, and corticotropin-releasing hormone (CRH), the primary step in HPA activation, signalling has been implicated in this stressor-mediated behaviour. However, whether CRH mediates the acute behavioural effects either alone or in conjunction with glucocorticoids (GCs) are far from clear. We hypothesized that the CRH receptor 1 (CRHR1)-mediated rise in GCs post-stress is necessary for the initiation and maintenance of the acute stress-related behaviour. METHODS: We first generated zebrafish (Danio rerio) with a mutation in the CRHR1 gene (CRHR1-KO) to assess the function of CRH. The behavioural readout utilized for this study was the locomotor activity of larval zebrafish in response to an acute light exposure, a protocol that freezes the larvae in response to the light stimulus. To test whether cortisol signalling is involved in the stress-mediated hyperactivity, we treated wildtype fish with metyrapone (MET), an inhibitor of 11ß-hydroxylase, to suppress cortisol production. The temporal role for cortisol signalling in the stress-related hyperactivity was tested using the glucocorticoid receptor knockout (GRKO) and mineralocorticoid receptor knockout (MRKO) zebrafish mutants. RESULTS: CRHR1-KO larvae did not increase cortisol, the principal GC in teleosts, post-stress, confirming a functional knockout. An acute stress resulted in the hyperactivity of the larvae in light at 15, 60, and 240 min post-stress, and this was absent in CRHR1-KO larvae. Addition of MET effectively blocked the attendant rise in cortisol post-stress; however, the stress-mediated hyperactivity was inhibited only at 60 and 240 min but not at 15 min post-stress. Addition of human CRH peptide caused hyperactivity at 15 min, and this response was also abolished in the CRHR1-KO mutants. The stress-induced hyperactivity was absent in the MRKO fish, while GRKO mutants showed transient effects. CONCLUSIONS: The results suggest that the stress-induced hyperactivity is induced by the CRH/CRHR1 system, while the temporal activation of cortisol production and the associated GR/MR signalling is essential for prolonging the stressor-induced hyperactivity. This study underscores the importance of systems-level analysis to assess stress responsivity.

The antidepressant venlafaxine perturbs cardiac development and function in larval zebrafish.

Venlafaxine, a selective serotonin and norepinephrine reuptake inhibitor, is a highly prescribed antidepressant and is detected at µg/L concentrations in waterways receiving municipal wastewater effluents. We previously showed that early-life venlafaxine exposure disrupted the normal development of the nervous system and reduces larval activity in zebrafish (Danio rerio). However, it is unclear whether the reduced swimming activity may be associated with impaired cardiac function. Here we tested the hypothesis that zygotic exposure to venlafaxine impacts the development and function of the larval zebrafish heart. Venlafaxine (0, 1 or 10 ng) was administered by microinjection into freshly fertilized zebrafish embryos (1-4 cell stage) to assess heart development and function during early-life stages. Venlafaxine deposition in the zygote led to precocious development of the embryo heart, including the timing of the first heartbeat, increased heart size, and a higher heart rate at 24- and 48-hours post-fertilization (hpf). Also, waterborne exposure to environmental levels of this antidepressant during early development increased the heart rate at 48 hpf of zebrafish larvae mimicking the zygotic deposition. The venlafaxine-induced higher heart rate in the embryos was abolished in the presence of NAN-190, an antagonist of the 5HT1A receptor. Also, heart rate dropped below control levels in the 10 ng, but not 1 ng venlafaxine group at 72 and 96 hpf. An acute stressor reduced the venlafaxine-induced heart rate at 48 hpf but did not affect the already reduced heart rate at 72 and 96 hpf in the 10 ng venlafaxine group. Our results suggest that the higher heart rate in the venlafaxine group may be due to an enhanced serotonin stimulation of the 5HT1A receptor. Taken together, early-life venlafaxine exposure disrupts cardiac development and has the potential to compromise the cardiovascular performance of larval zebrafish.

Venlafaxine deposition in the zygote disrupts the endocrine control of growth in juvenile zebrafish.

The antidepressant venlafaxine can be found at levels nearing µg/L in waterways receiving municipal wastewater effluent, exposing non-target organisms, such as fish, to this chemical. We showed previously that zygotic exposure to venlafaxine alters neurodevelopment and behaviour in zebrafish (Danio rerio) larvae. Here, we tested the hypothesis that the zygotic deposition of venlafaxine disrupts endocrine pathways related to growth in zebrafish. This was carried out by microinjecting embryos (1-4 cell stage) with either 0, 1, or 10 ng venlafaxine. Zygotic venlafaxine deposition reduced the growth of fish after 30 days post-fertilization. Specific growth rate was particularly impacted by 1 ng venlafaxine. This growth retardation corresponded with the disruption of endocrine pathways involved in growth and metabolism. Venlafaxine exposed embryos displayed reduced transcript abundance of key genes involved in anabolic hormone action. Early-life venlafaxine exposure also reduced whole-body insulin and glucose content in juveniles. Target-tissue glucose uptake measurements indicated that high venlafaxine deposition preferentially increased glucose uptake to the brain. Zygotic venlafaxine did not affect feed intake nor altered the transcript abundance of key feeding-related peptides. Taken together, zygotic venlafaxine deposition compromises zebrafish growth by disrupting multiple endocrine pathways, and this study has identified key markers for potential use in risk assessment.

Wild longnose dace downstream of wastewater treatment plants display an obese phenotype.

Wild fish living downstream of wastewater treatment plants (WWTPs) often have increased body condition factors or body mass indices compared to upstream fish. This observation has been largely attributed to increased nutrient loading and food availability around wastewater effluent outflows. While a higher condition factor in fish is generally considered a predictor of healthy ecosystems, the metabolic status and capacity of the animals downstream of WWTPs may be a better predictor of fitness and potential population level effects. To address this, we sampled wild longnose dace (Rhinichthys cataractae), a native species in North American waterways, from sites upstream and downstream of WWTPs. Downstream fish had higher body mass indices, which corresponded with higher nutrient (lipid, protein, and glycogen) storage in somatic tissues compared to upstream fish. Liver transcriptome analysis revealed metabolic reprogramming favoring lipid synthesis, including higher hepatic triglyceride levels and transcript abundance of targeted lipogenic genes. This suggests that effluent exposure-mediated obesity in dace is a result of changes at the transcriptional level. To determine potential ecological consequences, we subjected these fish to an acute stressor in situ to determine their stress performance. Downstream fish failed to mobilize metabolites post-stress, and showed a reduction in liver aerobic and anaerobic metabolic capacity. Taken together, fish living downstream of WWTPs exhibit a greater lipid accumulation that results in metabolic disruption and may compromise the ability of these fish to cope with subsequent environmental and/or anthropogenic stressors.

Cortisol modulates calcium release-activated calcium channel gating in fish hepatocytes.

Glucocorticoids (GCs) are rapidly released in response to stress and play an important role in the physiological adjustments to re-establish homeostasis. The mode of action of GCs for stress coping is mediated largely by the steroid binding to the glucocorticoid receptor (GR), a ligand-bound transcription factor, and modulating the expression of target genes. However, GCs also exert rapid actions that are independent of transcriptional regulation by modulating second messenger signaling. However, a membrane-specific protein that transduces rapid GCs signal is yet to be characterized. Here, using freshly isolated hepatocytes from rainbow trout (Oncorhynchus mykiss) and fura2 fluorescence microscopy, we report that stressed levels of cortisol rapidly stimulate the rise in cytosolic free calcium ([Ca2+]i). Pharmacological manipulations using specific extra- and intra-cellular calcium chelators, plasma membrane and endoplasmic reticulum channel blockers and receptors, indicated extracellular Ca2+ entry is required for the cortisol-mediated rise in ([Ca2+]i). Particularly, the calcium release-activated calcium (CRAC) channel gating appears to be a key target for the rapid action of cortisol in the ([Ca2+]i) rise in trout hepatocytes. To test this further, we carried out in silico molecular docking studies using the Drosophila CRAC channel modulator 1 (ORAI1) protein, the pore forming subunit of CRAC channel that is highly conserved. The result predicts a putative binding site on CRAC for cortisol to modulate channel gating, suggesting a direct, as well as an indirect regulation (by other membrane receptors) of CRAC channel gating by cortisol. Altogether, CRAC channel may be a novel cortisol-gated Ca2+ channel transducing rapid nongenomic signalling in hepatocytes during acute stress.