Nicholas J. Bernier

Brain regulation of feeding behavior and food intake in fish.

In mammals, the orexigenic and anorexigenic neuronal systems are morphologically and functionally connected, forming an interconnected network in the hypothalamus to govern food intake and body weight. However, there are relatively few studies on the brain control of feeding behavior in fish. Recent studies using mammalian neuropeptides or fish homologs of mammalian neuropeptides indicate that brain orexigenic signal molecules include neuropeptide Y, orexins, galanin and beta-endorphin, whereas brain anorexigenic signal molecules include cholecystokinin, bombesin, corticotropin-releasing factor, cocaine- and amphetamine-regulated transcript, and serotonin. Tachykinins may also have an anorectic action in fish. The brain hypothalamic area is associated with regulation of food intake, while sites outside the hypothalamus are also involved in this function. There is correlation between short-term changes in serum growth hormone levels and feeding behavior, although possible mechanisms integrating these functions remain to be defined.

Stress and Reproduction

Publisher Summary This chapter focuses specifically on the interactions between the hypothalamic–pituitary–interrenal (HPI) stress axis and the hypothalamic–pituitary–gonadal (HPG) reproductive axis and the consequences of these interactions for reproductive processes. It also examines that how stressors and the mediators of the stress response affect the key effectors of the HPG axis, including the hypothalamic gonadotropin–releasing hormones, the pituitary gonadotropins, the gonadal sex steroids, and hepatic vitellogeni. The chapter reviews how stressors and stress hormones impact the development of the reproductive system and reproductive functions at the embryonic, larval, pubertal, and adult life stages. The influence of stress on reproduction depends on the type of stressor, its intensity and duration, the sex of the fish, its developmental stage, its nutritional status, and its reproductive strategy. The effects of gender and reproduction on the activity of the HPI axis are discussed.

Two divergent leptin paralogues in zebrafish (Danio rerio) that originate early in teleostean evolution.

We describe duplicate leptin genes in zebrafish (Danio rerio) that share merely 24% amino acid identity with each other and only 18% with human leptin. We were also able to retrieve a second leptin gene in medaka (Oryzias latipes). The presence of duplicate leptin genes in these two distantly related teleosts suggests that duplicate leptin genes are a common feature of teleostean fishes. Despite low primary sequence conservation, we are confident in assigning orthology between mammalian and zebrafish leptins for several reasons. First, both zebrafish leptins share their characteristic gene structure and display key features of conserved synteny with mammalian leptin genes. Secondly, the cysteine residues that make up leptins single disulphide bridge are equally spaced in mammalian and zebrafish leptins and are unique among all members of the class-I helical cytokine family. Thirdly, the zebrafish leptins cluster with other fish leptins and mammalian leptins in phylogenetic analysis, supported by high bootstrap values. Within the leptin cluster, leptin-b forms a separate clade with the leptin-b orthologue from medaka. Finally, our prediction of the tertiary structures shows that both leptins conform to the typical four alpha-helix bundle structure of the class-I alpha-helical cytokines. The zebrafish leptins are differentially expressed; the liver shows high leptin-a expression (in concordance with what we observed for carp leptins), while leptin-b is expressed at much lower levels, which are downregulated further upon fasting. The finding of duplicate leptin genes in teleosts adds to our understanding of the evolution of leptin physiology in the early vertebrate lineage.

The hypothalamic-pituitary-interrenal axis and the control of food intake in teleost fish

Although environmental, social and physical stressors have been shown to inhibit food intake and feeding behavior in fish, little is known about the mechanisms that mediate the appetite-suppressing effects of stress. Since the hypothalamic-pituitary-interrenal (HPI) axis is activated in response to most forms of stress in fish, components of this axis may be involved in mediating the food intake reductions elicited by stress. Recent investigations into the brain regulation of food intake in fish have identified several signals with orexigenic and anorexigenic properties. Among these appetite-regulating signals are related neuropeptides that can activate the HPI axis, namely corticotropin-releasing factor (CRF) and urotensin I (UI). Central injections of CRF or UI, or treatments that result in an increase in hypothalamic CRF and UI gene expression, can elicit dose-dependent decreases in food intake that can be reversed by pre-treatment with a CRF-receptor antagonist. Evidence also suggests that cortisol, the end product of HPI activation in most fishes (i.e. Osteichthyes), may be involved in the regulation of food intake. Overall, while elements of the HPI axis may mediate some of the appetite-suppressing effects of stress, it is undetermined how either CRF-related peptides, cortisol, or other elements of the stress response interact with the complex circuitry of the hypothalamic feeding center.

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.

Appetite-Suppressing Effects of Urotensin I and Corticotropin-Releasing Hormone in Goldfish (Carassius auratus)

Fish urotensin I (UI), a member of the corticotropin-releasing hormone (CRH) family of peptides, is a potent inhibitor of food intake in mammals, yet the role of UI in the control of food intake in fish is not known. Therefore, to determine the acute effects of UI on appetite relative to those of CRH, goldfish were given intracerebroventricular (i.c.v.) injections of carp/goldfish UI and rat/human CRH (0.2–200 ng/g) and food intake was assessed for a 2-hour period after the injection. UI and CRH both suppressed food intake in a dose-related manner and UI (ED50 = 3.8 ng/g) was significantly more potent than CRH (ED50 = 43.1 ng/g). Pretreatment with the CRH receptor antagonist, α-helical CRH(9–41), reversed the reduction in food intake induced by i.c.v. UI and CRH. To assess whether endogenous UI and CRH modulate fish appetite, goldfish were given intraperitoneal implants of the glucocorticoid receptor antagonist, RU-486 (50 and 100 µg/g), or the cortisol synthesis inhibitor, metyrapone (100 and 200 µg/g), and food intake was monitored over the following 72 h. Fish treated with either RU-486 or metyrapone were characterized by a sustained and dose-dependent reduction in food intake. Pretreatment with i.c.v. implants of α-helical CRH(9–41) partially reversed the appetite-suppressing effects of RU-486 and metyrapone. In a parallel experiment, the effects of RU-486 (100 µg/g) and metyrapone (200 µg/g) intraperitoneal implants on brain UI and CRH gene expression were assessed. Relative to sham-implanted controls, fish treated with RU-486 or metyrapone had elevated UI mRNA levels in the hypothalamus and CRH mRNA levels in the telencephalon-preoptic brain region. Together, these results suggest that UI is a potent anorectic peptide in the brain of goldfish and that endogenous CRH-related peptides can play a physiological role in the control of fish appetite.

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.

Chapter 6 Regulation And Contribution Of The Corticotropic, Melanotropic And Thyrotropic Axes To The Stress Response In Fishes

Publisher Summary This chapter discusses the regulatory pathways, the targets, the functions, and the interactions among the corticotropic, melanotropic, and thyrotropic axes, and the evidence that implicates each axis in the stress response. Multiple hypothalamic factors are involved in the regulation of the secretions from the corticotrope, melanotrope, and thyrotrope pituitary cells in fishes. Among these factors, corticotropin‐releasing factor (CRF) and thyrotropin‐releasing hormone (TRH) stimulate, and dopamine generally inhibits the secretions from all three hypothalamo–pituitary axes. The CRF system also plays a master role in the regulation of the endocrine response to stressors. In general, the contributions of the corticotropic, melanotropic, and thyrotropic axes to the stress response are species‐specific and depend on the challenge imposed on the system, its duration, and the homeostatic resilience of the fish. Multiple interactions and feedback effects have been identified among these endocrine axes. In the chapter it has been postulated that the extensive multidirectional communication as well as the cross‐talk among the corticotropic, melanotropic, and thyrotropic axes forms a “stress web” that exerts well‐concerted actions on energy metabolism as its prime task.