Sarah Alderman

Ontogeny of the corticotropin-releasing factor system in zebrafish.

The corticotropin-releasing factor (CRF) system in fish functions to maintain homeostasis during stress in part by regulating cortisol production via the hypothalamus-pituitary-interrenal (HPI) axis. Towards understanding the role of the CRF system in vertebrate development, we describe the ontogeny of the CRF system, cortisol, and the stress response in the zebrafish, Danio rerio. Early embryonic expression of mRNA encoding CRF, urotensin I (UI), CRF-binding protein (CRF-BP), and two CRF receptors (CRF-R1 and CRF-R2) suggest a function in the early organization of the developing embryo. The expression patterns of CRF, UI, and CRF-BP in the larval brain are consistent with the adult distribution patterns for these genes and support HPI-axis independent functions. The relative amounts of CRF and UI mRNA in the heads and tails of developing and adult zebrafish suggest that CRF functions primarily in the brain while UI also plays an important role in the caudal neurosecretory system. The amount of cortisol in developing zebrafish is low and relatively constant through the first 6 days of development. The commencement of feeding after 4 dpf, however, significantly increases basal cortisol production. Finally, we show that zebrafish larvae are able to respond to an osmotic stressor as early as 3 dpf. Overall, results from this study establish the zebrafish as a model species for research on stress during ontogeny and offer new insights into an HPI-axis independent function for the CRF system during embryogenesis.

Heads or tails? Stressor-specific expression of corticotropin-releasing factor and urotensin I in the preoptic area and caudal neurosecretory system of rainbow trout

Corticotropin-releasing factor (CRF)- and urotensin I (UI)-expressing cells of the preoptic area (POA) and caudal neurosecretory system (CNSS) are considered key contributors to the regulation of the stress response in fish; however, the expression pattern of these neurons to environmental and social challenges have not been compared in a single study. Therefore, we characterized in rainbow trout (Oncorhynchus mykiss) the central distribution of CRF and UI expression and quantified the POA and CNSS mRNA levels of both transcripts in response to hyperammonemia, hypoxia, isolation, or subordination. The tissue distribution demonstrated that the POA and the CNSS are dominant sites of CRF and UI expression. Comparison of the plasma cortisol levels in response to the diverse treatments showed that subordination was the most severe stressor followed by hyperammonemia, isolation, and hypoxia. In the POA, with the exception of subordination that had no effect on UI expression, all stressors resulted in increase in CRF and UI mRNA levels. In the CNSS, while hyperammonemia was associated with increase in CRF and UI mRNA levels, and hypoxia induced an increase in CRF expression, isolation caused a decrease in the expression of both transcripts, and subordination had no effect. Independent of the stressor, we found strong positive correlations between CRF and UI expression in the POA and the CNSS, and no correlation in the expression of either gene between regions. Overall, the results demonstrate that the contribution of POA and CNSS CRF and UI neurons to the stress response in rainbow trout is stressor-, time-, and region-specific.

Localization of corticotropin-releasing factor, urotensin I, and CRF-binding protein gene expression in the brain of the zebrafish, Danio rerio

Our current understanding of the corticotropin‐releasing factor (CRF) system distribution in the teleost brain is restricted by limited immunohistochemical studies and a lack of complete transcriptional distribution maps. The present study used in situ hybridization to localize and compare CRF, urotensin I (UI), and CRF‐binding protein (CRF‐BP) expression in the brain of adult zebrafish (Danio rerio). All three peptides were localized in the preoptic area, periventricular hypothalamic and tectal regions, and dorsal part of the trigeminal motor nucleus. CRF and UI were both expressed in several nuclei of the dorsal telencephalon, whereas CRF and CRF‐BP were both expressed in the ventral nucleus of the ventral telencephalon. Sole expression of CRF and CRF‐BP was apparent in the olfactory bulbs and superior raphe nucleus, respectively, whereas only UI was observed in the corpus mamillare, nucleus of the medial longitudinal fascicle, dorsal tegmental nucleus, nucleus lateralis valvulae, and nucleus interpeduncularis. A major finding of this study was the general regional overlapping of CRF‐BP with its ligands and a tendency to be expressed in tandem with CRF rather than UI. Overall, the mRNA expression patterns outlined in this study support the stress‐related neuroendocrine, autonomic, and behavioral functions generally ascribed to the vertebrate CRF system and suggest some unique functional roles for CRF and UI in the teleost brain. J. Comp. Neurol. 502:783–793, 2007.

Central and peripheral glucocorticoid receptors are involved in the plasma cortisol response to an acute stressor in rainbow trout.

Cortisol, the primary circulating corticosteroid in teleosts, is elevated during stress following activation of the hypothalamus-pituitary-interrenal (HPI) axis. Cortisol exerts genomic effects on target tissues in part by activating glucocorticoid receptors (GR). Despite a well-established negative feedback loop involved in plasma cortisol regulation, the role of GR in the functioning of the HPI axis during stress in fish is still unclear. We used mifepristone (a GR antagonist) to suppress GR signaling in rainbow trout (Oncorhynchus mykiss) and assessed the resultant changes to HPI axis activity. We show for the first time that mifepristone caused a functional knockdown of GR by depleting protein expression 40-75%. The lower GR protein expression corresponded with a compensatory up-regulation of GR mRNA levels across tissues. Mifepristone treatment completely abolished the stressor-induced elevation in plasma cortisol and glucose levels seen in the control fish. A reduction in corticotropin-releasing factor (CRF) mRNA abundance in the hypothalamic preoptic area was also observed, suggesting that GR signaling is involved in maintaining basal CRF levels. We further characterized the effect of mifepristone treatment on the steroidogenic capacity of interrenal tissue in vitro. A marked reduction in cortisol production following adrenocorticotropic hormone stimulation of head kidney pieces was observed from mifepristone treated fish. This coincided with the suppression of steroidogenic acute regulatory protein, but not P450 side chain cleavage mRNA abundances. Overall, our results underscore a critical role for central and peripheral GR signaling in the regulation of plasma cortisol levels during stress in fish.

11β-Hydroxysteroid dehydrogenase type 2 in zebrafish brain: a functional role in hypothalamus-pituitary-interrenal axis regulation.

The type 2, 11β-hydroxysteroid dehydrogenase (Hsd11b2) converts active glucocorticoids to their inactive derivatives (e.g. cortisol to cortisone). In most vertebrates, Hsd11b2 is essential for conferring aldosterone-specific actions in mineralocorticoid target tissues and for protecting glucocorticoid-sensitive tissues during stress. However, teleosts do not synthesize aldosterone, and the function of Hsd11b2 is poorly defined. The distribution of Hsd11b2 in nonmammalian brain is also largely unexplored. We tested the hypothesis that modulation of brain Hsd11b2 activity is involved in stressor-mediated cortisol regulation in zebrafish (Danio rerio). In adult zebrafish, the stress effect on Hsd11b2 expression in the brain was tested using acute air exposure followed by recovery over a 24-h period. hsd11b2 transcripts were found in nearly all peripheral tissues examined, and a spatial map of its mRNA abundance in unstressed zebrafish brain revealed extensive distribution. Stressor exposure increased the conversion of (3)H-cortisol to (3)H-cortisone indicating enhanced Hsd11b2 activity in zebrafish brain. Promoter analysis of zebrafish hsd11b2 gene revealed putative sites for cortisol-mediated transcriptional regulation of this gene. Furthermore, inhibition of Hsd11b2 activity by 18β-glycyrrhetinic acid resulted in elevated whole-body cortisol levels and preoptic area mRNA abundance of corticotropin-releasing factor and mineralocorticoid receptor. Taken together, our results underscore an important role for brain Hsd11b2 involvement in the negative feedback regulation of cortisol poststress in zebrafish.

Distribution and Regional Stressor-Induced Regulation of Corticotrophin-Releasing Factor Binding Protein in Rainbow Trout (Oncorhynchus mykiss)

The corticotrophin‐releasing factor (CRF) system plays a key role in the co‐ordination of the physiological response to stress in vertebrates. Although the binding protein (BP) for CRF‐related peptides, CRF‐BP, is an important player in the many functions of the CRF system, the distribution of CRF‐BP and the impact of stressors on its expression in fish are poorly understood. In the present study, we describe the distribution of CRF‐BP in the brain and peripheral tissues of rainbow trout (Oncorhynchus mykiss) using a combination of real‐time reverse transcriptase‐polymerase chain reaction, in situ hybridisation and immunohistochemistry. Our results indicate a widespread and highly localised distribution of CRF‐BP in the central nervous system, but do not support a significant peripheral production of the protein. Major expression sites in the brain include the area ventralis telencephali, nucleus preopticus, anterior and lateral tuberal nuclei, and the posterior region of the pituitary pars distalis. We further characterise changes in CRF‐BP gene expression in three discrete brain regions after exposure to 8 h and 24 h of social stress or hypoxia. The plasma cortisol concentration in subordinate fish was much higher than in dominant fish and controls, and was indicative of a relatively severe stressor. By contrast, the increase in plasma cortisol concentration in fish exposed to hypoxia was characteristic of the response to a mild stressor. Changes in CRF‐BP gene expression were only observed after 24 h of either stressor, and were region‐specific. CRF‐BP mRNA in the telencephalon increased in both subordinate fish and fish exposed to hypoxia, but CRF‐BP in the preoptic area only increased after 24 h of hypoxia exposure. In the hypothalamus, CRF‐BP mRNA levels decreased in dominant fish relative to controls after 24 h. Taken together, our results support a diverse role for CRF‐BP in the central actions of the fish CRF system, but a negligible role in the peripheral functions of circulating CRF‐related peptides. Furthermore, the differential changes in forebrain CRF‐BP mRNA appear to occur independently of the hypothalamic‐pituitary‐inter‐renal axis.

Hypoxemia-induced leptin secretion: a mechanism for the control of food intake in diseased fish

Leptin is a potent anorexigen, but little is known about the physiological conditions under which this cytokine regulates food intake in fish. In this study, we characterized the relationships between food intake, O2-carrying capacity, liver leptin-A1 (lep-a1) gene expression, and plasma leptin-A1 in rainbow trout infected with a pathogenic hemoflagellate, Cryptobia salmositica. As lep gene expression is hypoxia-sensitive and Cryptobia-infected fish are anemic, we hypothesized that Cryptobia-induced anorexia is mediated by leptin. A 14-week time course experiment revealed that Cryptobia-infected fish experience a transient 75% reduction in food intake, a sharp initial drop in hematocrit and hemoglobin levels followed by a partial recovery, a transient 17-fold increase in lep-a1 gene expression, and a sustained increase in plasma leptin-A1 levels. In the hypothalamus, peak anorexia was associated with decreases in mRNA levels of neuropeptide Y (npy) and cocaine- and amphetamine-regulated transcript (cart), and increases in agouti-related protein (agrp) and pro-opiomelanocortin A2 (pomc). In contrast, in non-infected fish pair-fed to infected animals, lep-a1 gene expression and plasma levels did not differ from those of non-infected satiated fish. Pair-fed fish were also characterized by increases in hypothalamic npy and agrp, no changes in pomc-a2, and a reduction in cart mRNA expression. Finally, peak infection was characterized by a significant positive correlation between O2-carrying capacity and food intake. These findings show that hypoxemia, and not feed restriction, stimulates leptin-A1 secretion in Cryptobia-infected rainbow trout and suggest that leptin contributes to anorexia by inhibiting hypothalamic npy and stimulating pomc-a2.

Effect of cold acclimation on troponin I isoform expression in striated muscle of rainbow trout

In vertebrates each of the three striated muscle types (fast skeletal, slow skeletal, and cardiac) contain distinct isoforms of a number of different contractile proteins including troponin I (TnI). The functional characteristics of these proteins have a significant influence on muscle function and contractility. The purpose of this study was to characterize which TnI gene and protein isoforms are expressed in the different muscle types of rainbow trout (Oncorhynchus mykiss) and to determine whether isoform expression changes in response to cold acclimation (4°C). Semiquantitative real-time PCR was used to characterize the expression of seven different TnI genes. The sequence of these genes, cloned from Atlantic salmon (Salmo salar) and rainbow trout, were obtained from the National Center for Biotechnology Information databases. One-dimensional gel electrophoresis and tandem mass spectrometry were used to identify the TnI protein isoforms expressed in each muscle type. Interestingly, the results indicate that each muscle type expresses the gene transcripts of up to seven TnI isoforms. There are significant differences, however, in the expression pattern of these genes between muscle types. In addition, cold acclimation was found to increase the expression of specific gene transcripts in each muscle type. The proteomics analysis demonstrates that fast skeletal and cardiac muscle contain three TnI isoforms, whereas slow skeletal muscle contains four. No other vertebrate muscle to date has been found to express as many TnI protein isoforms. Overall this study underscores the complex molecular composition of teleost striated muscle and suggests there is an adaptive value to the unique TnI profiles of each muscle type.

Effects of diluted bitumen exposure on juvenile sockeye salmon: From cells to performance

Diluted bitumen (dilbit; the product of oil sands extraction) is transported through freshwater ecosystems critical to Pacific salmon. This is concerning, because crude oil disrupts cardiac development, morphology, and function in embryonic fish, and cardiac impairment in salmon can have major consequences on migratory success and fitness. The sensitivity of early life-stage salmon to dilbit and its specific cardiotoxic effects are unknown. Sockeye salmon parr were exposed to environmentally relevant concentrations of the water-soluble fraction (WSF) of dilbit for 1 wk and 4 wk, followed by an examination of molecular, morphological, and organismal endpoints related to cardiotoxicity. We show that parr are sensitive to WSF of dilbit, with total polycyclic aromatic hydrocarbon (PAH) concentrations of 3.5 µg/L sufficient to induce a liver biomarker of PAH exposure, and total PAH of 16.4 µg/L and 66.7 µg/L inducing PAH biomarkers in the heart. Furthermore, WSF of dilbit induces concentration-dependent cardiac remodeling coincident with performance effects: fish exposed to 66.7 µg/L total PAH have relatively fewer myocytes and more collagen in the compact myocardium and impaired swimming performance at 4 wk, whereas the opposite changes occur in fish exposed to 3.5 µg/L total PAH. The results demonstrate cardiac sensitivity to dilbit exposure that could directly impact sockeye migratory success. Environ Toxicol Chem 2017;36:354-360.

Evidence for a plasma-accessible carbonic anhydrase in the lumen of salmon heart that may enhance oxygen delivery to the myocardium

ABSTRACT Oxygen supply to the heart of most teleosts, including salmonids, relies in part or in whole on oxygen-depleted venous blood. Given that plasma-accessible carbonic anhydrase (CA) in red muscle of rainbow trout has recently been shown to facilitate oxygen unloading from arterial blood under certain physiological conditions, we tested the hypothesis that plasma-accessible CA is present in the lumen of coho salmon (Oncorhynchus kisutch) hearts, and may therefore assist in the luminal oxygen supply to the spongy myocardium, which has no coronary circulation. We demonstrate a widespread distribution of CA throughout the heart chambers, including lumen-facing cells in the atrium, and confirm that the membrane-bound isoform ca4 is expressed in the atrium and ventricle of the heart. Further, we confirm that CA catalytic activity is available to blood in the atrial lumen using a modified electrometric ΔpH assay in intact atria in combination with either a membrane-impermeable CA inhibitor or specific cleavage of the Ca4 membrane anchor. Combined, these results support our hypothesis of the presence of an enhanced oxygen delivery system in the lumen of a salmonid heart, which could help support oxygen delivery when the oxygen content of venous blood becomes greatly reduced, such as after burst exercise and during environmental hypoxia. Highlighted Article: Results from research in coho salmon support the presence of an enhanced oxygen delivery system in the salmonid heart, which could help support cardiac function when oxygen supply to this vital organ becomes limiting.