Unraveling the periprandial changes in brain serotonergic activity and its correlation with food intake-related neuropeptides in rainbow trout Oncorhynchus mykiss.

This study explored changes in brain serotonin content and activity together with hypothalamic neuropeptide mRNA abundance around feeding time in rainbow trout, as well as the effect of one-day fasting. Groups of trout fed at two (ZT2) and six (ZT6) hours after lights on were sampled from 90 minutes before to 240 minutes after feeding, while additional groups of non-fed trout were also included in the study. Changes in brain amine and metabolite contents were measured in hindbrain, diencephalon and telencephalon, while in the diencephalon the mRNA abundance of tryptophan hydroxylase (tph1, tph2), serotonin receptors (5htr1a, 5htr1b and 5htr2c) and several neuropeptides (npy, agrp1, cartpt, pomca1, crfb) involved in the control of food intake were also assessed. The results showed changes in the hypothalamic neuropeptides that were consistent with the expected role for each in the regulation of food intake in rainbow trout. Serotonergic activity increased rapidly at the time of food intake in the diencephalon and hindbrain and remained high for much of the postprandial period. This increase in serotonin abundance was concomitant with elevated levels of pomca1 mRNA in the diencephalon, suggesting that serotonin might act on brain neuropeptides to promote a satiety profile. Furthermore, serotonin synthesis and neuronal activity appear to increase already before the time of feeding, suggesting additional functions for this amine before and during food intake. Exploration of serotonin receptors in the diencephalon revealed only small changes for gene expression of 5htr1b and 5htr2c receptors during the postprandial phase. Therefore, the results suggest that serotonin may play a relevant role in the regulation of feeding behavior in rainbow trout during periprandial time, but a better understanding of its interaction with brain centers involved in receiving and processing food-related signals is still needed.

Exploring the potential for an evolutionarily conserved role of the taste 1 receptor gene family in gut sensing mechanisms of fish.

In this study, we investigated the transcriptional spatio-temporal dynamics of the taste 1 receptor (T1R) gene family repertoire in seabream (Sparus aurata [sa]), during larval ontogeny and in adult tissues. In early larval development, saT1R expression arises heterochronously, i.e. the extraoral taste-related perception in the gastrointestinal tract (GIT) anticipates first exogenous feeding (at 9 days post hatching [dph]), followed by the buccal/intraoral perception from 14 dph onwards, supporting the hypothesis that the early onset of the molecular machinery underlying saT1R expression in the GIT is not induced by food but rather genetically hardwired. During adulthood, we characterized the expression patterns of sa T1R within specific tissues (n = 4) distributed in oropharingeal, GIT and brain regions substantiating their functional versatility as chemosensory signaling players to a variety of biological functions beyond oral taste sensation. Further, we provided for the first time direct evidences in fish for mRNA co-expression of a subset of saT1R genes (mostly sa T1R3, i.e. the common subunit of the heterodimeric T1R complexes for the detection of "sweet" and "umami" substances), with the selected gut peptides ghrelin (ghr), cholecystokinin (cck), hormone peptide yy (pyy) and proglucagon (pg). Each peptide defines the enteroendocrine cells (ECCs) identity, and establishes on morphological basis, a direct link for T1R chemosensing in the regulation of fish digestive processes. Finally, we analyzed the spatial gene expression patterns of 2 taste signaling components functionally homologous to the mammalian G(i)α subunit gustducin, namely sa G( i )α1 and sa G( i )α2, and demonstrated their co-localization with the saT1R3 in EECs, thus validating their direct involvement in taste-like transduction mechanisms of the fish GIT. In conclusion, data provide new insights in the evolutionary conservation of gut sensing in fish suggesting a conserved role for nutrient sensors modulating entero-endocrine secretion.

Insights into the Function and Evolution of Taste 1 Receptor Gene Family in the Carnivore Fish Gilthead Seabream (Sparus aurata).

A plethora of molecular and functional studies in tetrapods has led to the discovery of multiple taste 1 receptor (T1R) genes encoding G-protein coupled receptors (GPCRs) responsible for sweet (T1R2 + T1R3) and umami (T1R1 + T1R3) taste. In fish, the T1R gene family repertoires greatly expanded because of several T1R2 gene duplications, and recent studies have shown T1R2 functional divergence from canonical mammalian sweet taste perceptions, putatively as an adaptive mechanism to develop distinct feeding strategies in highly diverse aquatic habitats. We addressed this question in the carnivore fish gilthead seabream (Sparus aurata), a model species of aquaculture interest, and found that the saT1R gene repertoire consists of eight members including saT1R1, saT1R3 and six saT1R2a-f gene duplicates, adding further evidence to the evolutionary complexity of fishT1Rs families. To analyze saT1R taste functions, we first developed a stable gene reporter system based on Ca2+-dependent calcineurin/NFAT signaling to examine specifically in vitro the responses of a subset of saT1R heterodimers to L-amino acids (L-AAs) and sweet ligands. We show that although differentially tuned in sensitivity and magnitude of responses, saT1R1/R3, saT1R2a/R3 and saT1R2b/R3 may equally serve to transduce amino acid taste sensations. Furthermore, we present preliminary information on the potential involvement of the Gi protein alpha subunits saGαi1 and saGαi2 in taste signal transduction.

Sensing Glucose in the Central Melanocortin Circuits of Rainbow Trout: A Morphological Study.

In mammals, glucosensing markers reside in brain areas known to play an important role in the control of food intake. The best characterized glucosensing mechanism is that dependent on glucokinase (GK) whose activation by increased levels of glucose leads in specific hypothalamic neurons to decreased or increased activity, ultimately leading to decreased food intake. In fish, evidence obtained in recent years suggested the presence of GK-like immunoreactive cells in different brain areas related to food intake control. However, it has not been established yet whether or not those neuronal populations having glucosensing capacity are the same that express the neuropeptides involved in the metabolic control of food intake. Therefore, we assessed through dual fluorescent in situ hybridization the possible expression of GK in the melanocortinergic neurons expressing proopiomelanocortin (POMC) or agouti-related protein (AGRP). POMC and AGRP expression localized exclusively in the rostral hypothalamus, in the ventral pole of the lateral tuberal nucleus, the homolog of the mammalian arcuate nucleus. Hypothalamic GK expression confined to the ependymal cells coating the ventral pole of the third ventricle but some expression level occurred in the AGRP neurons. GK expression seems to be absent in the hypothalamic POMC neurons. These results suggest that AGRP neurons might sense glucose directly through a mechanism involving GK. In contrast, POMC neurons would not directly respond to glucose through GK and would require presynaptic inputs to sense glucose. Ependymal cells could play a critical role relying glucose metabolic information to the central circuitry regulating food intake in fish, especially in POMC neurons.

Hypothalamic Integration of Metabolic, Endocrine, and Circadian Signals in Fish: Involvement in the Control of Food Intake.

The regulation of food intake in fish is a complex process carried out through several different mechanisms in the central nervous system (CNS) with hypothalamus being the main regulatory center. As in mammals, a complex hypothalamic circuit including two populations of neurons: one co-expressing neuropeptide Y (NPY) and Agouti-related peptide (AgRP) and the second one population co-expressing pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) is involved in the integration of information relating to food intake control. The production and release of these peptides control food intake, and the production results from the integration of information of different nature such as levels of nutrients and hormones as well as circadian signals. The present review summarizes the knowledge and recent findings about the presence and functioning of these mechanisms in fish and their differences vs. the known mammalian model.

Pigment patterns in adult fish result from superimposition of two largely independent pigmentation mechanisms.

Dorso-ventral pigment pattern differences are the most widespread pigmentary adaptations in vertebrates. In mammals, this pattern is controlled by regulating melanin chemistry in melanocytes using a protein, agouti-signalling peptide (ASIP). In fish, studies of pigment patterning have focused on stripe formation, identifying a core striping mechanism dependent upon interactions between different pigment cell types. In contrast, mechanisms driving the dorso-ventral countershading pattern have been overlooked. Here, we demonstrate that, in fact, zebrafish utilize two distinct adult pigment patterning mechanisms - an ancient dorso-ventral patterning mechanism, and a more recent striping mechanism based on cell-cell interactions; remarkably, the dorso-ventral patterning mechanism also utilizes ASIP. These two mechanisms function largely independently, with resultant patterns superimposed to give the full pattern.

Effect of different glycaemic conditions on gene expression of neuropeptides involved in control of food intake in rainbow trout; interaction with stress.

To assess mechanisms relating to food intake and glucosensing in fish, and their interaction with stress, we evaluated changes in the expression of orexigenic (NPY) and anorexigenic (POMC, CART and CRF) peptides in central glucosensing areas (hypothalamus and hindbrain) of rainbow trout subjected to normoglycaemic (control), hypoglycaemic (4 mg insulin kg(-1)) or hyperglycaemic (500 mg glucose kg(-1)) conditions for 6 h under normal stocking density (NSD; 10 kg fish mass m(-3)) or under stress conditions induced by high stocking density (HSD; 70 kg fish mass m(-3)). Hyperglycaemic NSD conditions resulted in decreased mRNA levels of NPY and increased levels of CART and POMC in the hypothalamus as well as increased mRNA levels of CART and CRF in the hindbrain compared with hypo- and normoglycaemic conditions. HSD conditions in normoglycaemic fish induced marked changes in the expression of all peptides assessed: mRNA levels of NPY and CRF increased and mRNA levels of POMC and CART decreased in the hypothalamus, whereas the expression of all four peptides (NPY, POMC, CART and CRF) decreased in the hindbrain. Furthermore, HSD conditions altered the response to changes in glycaemia of NPY and POMC expression in the hypothalamus and CART expression in the hypothalamus and the hindbrain. The results are discussed in the context of food intake regulation by glucosensor systems and their interaction with stress in fish.

Expression and role of Elovl4 elongases in biosynthesis of very long-chain fatty acids during zebrafish Danio rerio early embryonic development.

Elovl4 is a fatty acyl elongase that participates in the biosynthesis of very long-chain fatty acids (>/=C24), which are relatively abundant in skin (saturated chains), or retina, brain and testes (polyunsaturated chains) of mammals. In the present study we characterised two Elovl4 proteins, Elovl4a and Elovl4b, from zebrafish Danio rerio, and investigated their expression patterns during embryonic development. Heterologous expression in baker's yeast showed that both zebrafish Elovl4 proteins efficiently elongated saturated fatty acids up to C36, with 26:0 appearing the preferred substrate as reported for human ELOVL4. Interestingly, activity for the elongation of PUFA substrates was only shown by Elovl4b, which effectively converted eicosapentaenoic (20:5n-3) and arachidonic (20:4n-6) acids to elongated polyenoic products up to C36. Furthermore, zebrafish Elovl4b may be involved in the biosynthesis of docosahexaenoic acid (22:6n-3, DHA) as it had the capacity to elongate 22:5n-3 to 24:5n-3 which can be subsequently desaturated and chain shortened to DHA in peroxisomes. The distinct functional roles of zebrafish Elovl4 proteins were also reflected in their spatial-temporal expression patterns during ontogeny. Analyses by whole-mount in situ hybridisation in zebrafish embryos showed that elovl4a was expressed in neuronal tissues (wide-spread distribution in the head area), with elovl4b specifically expressed in epiphysis (pineal gland) and photoreceptor cells in the retina. Similarly, tissue distribution in adults revealed that elovl4a transcripts were found in most tissues analysed, whereas elovl4b expression was essentially restricted to eye and gonads. Overall, the results suggest that zebrafish elovl4b resembles other mammalian orthologues in terms of function and expression patterns, whereas elovl4a may represent an alternative elongase not previously described in vertebrates.

Expression of long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis genes during zebrafish Danio rerio early embryogenesis.

Long-chain polyunsaturated fatty acids (LC-PUFAs) are essential in important physiological processes, many of which are particularly vital during embryonic development. This study investigated the expression of genes encoding enzymes involved in LC-PUFA biosynthesis, namely fatty acyl desaturase (Fad) and Elovl5- and Elovl2-like elongases, during early embryonic development of zebrafish. First, zebrafish elovl2 cDNA was isolated and functionally characterised in yeast, showing high specificity towards C20- and C22-PUFAs, compared to C18 substrates. Second, spatial-temporal expression for elovl2 and the previously cloned fad and elovl5 were studied during zebrafish early embryonic development. Temporal expression shows that all three genes are expressed from the beginning of embryogenesis (zygote), suggesting maternal mRNA transfer to the embryo. However, a complete activation of the biosynthetic pathway seems to be delayed until 12 hpf, when noticeable increases of fad and elovl2 transcripts were observed, in parallel with high docosahexaenoic acid levels in the embryo. Spatial expression was studied by whole-mount in situ hybridisation in 24 hpf embryos, showing that fad and elovl2 are highly expressed in the head area where neuronal tissues are developing. Interestingly, elovl5 shows specific expression in the pronephric ducts, suggesting an as yet unknown role in fatty acid metabolism during zebrafish early embryonic development. The yolk syncytial layer also expressed all three genes, suggesting an important role in remodelling of yolk fatty acids during zebrafish early embryogenesis. Tissue distribution in zebrafish adults demonstrates that the target genes are expressed in all tissues analysed, with liver, intestine and brain showing the highest expression.