A.L. Alonso-Gómez

Role of corticotropin-releasing factor (CRF) as a food intake regulator in goldfish

The effect of intraperitoneal or intracerebroventricular corticotropin-releasing factor (CRF) administration on food intake has been studied in goldfish after 24 h of food deprivation. Food intake was evaluated at different time periods after injection, 0-2, 2-8, and 0-8 h. The 1 and 2 micrograms doses of CRF intracerebroventricularly administered induced a reduction in food intake during the first 2 h, although an increased feeding was observed in the next 6 h. The higher dose of CRF used in this study (3.3 micrograms) increased cumulative food intake at 8 h postinjection. However, intraperitoneal injection of 1 microgram CRF did not modify food intake in any of the studied intervals. These results suggest that CRF may play a role in central regulation of feeding behavior intake in goldfish, and show that CRF effects are time- and dose-dependent.

Circadian Clock Genes of Goldfish, Carassius auratus: cDNA Cloning and Rhythmic Expression of Period and Cryptochrome Transcripts in Retina, Liver, and Gut:

Clock genes are known to be the molecular core of biological clocks of vertebrates. They are expressed not only in those tissues considered central pacemakers, but also in peripheral tissues. In the present study, partial cDNAs for 6 of the principal clock genes (Period 1-3 and Cryptochrome 1-3) were cloned from a teleost fish, the goldfish (Carassius auratus ). These genes showed high homology (approximately 90%) with the respective cDNAs of zebrafish (Danio rerio), the only other teleost from which clock genes have been cloned. The daily expression pattern of each gene in retina, gut, and liver of goldfish was investigated using quantitative RT-PCR and cosinor analysis. All clock genes analyzed in the retina showed circadian rhythmicity; however, only Per 2-3 and Cry 2-3 were rhythmic in goldfish liver and gut. The amplitude and phase of the expression in liver and gut were different from those found in goldfish retina. Such differences suggest that other cues, such as feeding time, may contribute to the entrainment of oscillators in goldfish liver and gut. Our results support the use of goldfish as a teleost model to investigate the location and functioning of the circadian oscillators.

Corticotropin-releasing factor stimulates metamorphosis and increases thyroid hormone concentration in prometamorphic Rana perezi larvae

Attempts to identify a hypothalamic molecule that stimulates thyrotropin (TSH) secretion from amphibian pituitary have been unsuccessful to date. The effects of mammalian (ovine and human) corticotropin-releasing factor (CRF) on the thyroid function of prometamorphic (Taylor & Kollros stages XI-XVII) (Taylor and Kollros, 1946) Rana perezi larvae were studied. Chronic treatments with both ovine and human CRF (oCRF, hCRF) stimulated metamorphosis while delaying larval growth. Chronic hCRF (1 microgram) administration induced 3.2- and 5.3-fold increases in whole body concentration of thyroxine (T4) and triiodothyronine (T3), respectively. In contrast, the 0.5-microgram dose of hCRF stimulated a significant (3.4-fold) increase in whole body concentration of T4 but not of T3. Histological studies of the thyroid gland revealed a 22% increase in the number of follicles per section as a result of the chronic treatment with oCRF (1 microgram). Acute oCRF (2 micrograms) treatment induced a significant increase in T4 concentration at 4 hr (1.3-fold) and 8 hr (2.3-fold) postinjection. T3 concentration was not altered. These results support previous reports and lead us to conclude that a CRF-like peptide, and not TRH, is involved in the regulation of thyroid activity in anuran amphibians during metamorphosis.

Effect of α-Helical-CRF[9-41] on feeding in goldfish : Involvement of cortisol and catecholamines

The anoretic effect of corticotropin -releasing factor (CRF) was not dependent on adrenal activation in goldfish (Carassius auratus). Moreover, an interaction between CRF and the hypothalamic catecholaminergic system in the central regulation of food intake was observed. The intracerebroventricular (icv) administration of CRF increased cortisol levels and reduced food intake and hypothalamic norepinephrine and dopamine content at 2 hr postinjection, with these effects reversed by a-helical CRF[9_41] pretreatment. The anoretic effect of CRF was independent of the circulating cortisol increase, because it was only evoked after icv injections but not after intraperitoneal (ip) administration. Furthermore, the increase in plasma cortisol levels induced by ip administration of this steroid did not modify feeding.

Food intake inhibition by melatonin in goldfish (Carassius auratus).

Feeding regulation by monoamines, neuropeptides and certain hormones has been studied in fish, but a possible role of melatonin is unknown. The purpose of the present study was to investigate the effects of melatonin on food intake in goldfish. Fishes were housed in 12L:12D and injected with different doses of either melatonin or 2-iodomelatonin. Two routes of administration, intracerebroventricular and intraperitoneal injections, and two times of the daily photocycle, midday and midnight, were tested. Food intake was measured at 2, 5 and 8 h postinjection. Melatonin and its analog, 2-iodomelatonin intracerebroventricularly injected had no effect on food intake at any time. However, intraperitoneal injections of both indoleamines significantly reduced food intake at different postinjection times. The inhibitory effect of melatonin was blocked by intraperitoneal administration of its antagonist, luzindole. These results demonstrate the in vivo efficiency of luzindole as melatonin antagonist, and thus provide a useful experimental tool to investigate melatonin functions. In conclusion, both melatonin and its agonist 2-iodomelatonin administered peripherally, inhibit food intake in goldfish, and this inhibitory effect appears to be mediated via luzindole-sensitive melatonin receptors. Our results strongly suggest that melatonin is involved in the peripheral satiety mechanisms in goldfish.

Seasonal variation of gonadal development, sexual steroids, and lipid reserves in a population of the lizard Psammodromus algirus

We studied the seasonal variations of reproductive characteristics in a wild population of the lacertid lizard Psammodromus algirus by measuring gonadal weight, germinal epithelium height, sexual steroids (testosterone, estradiol, and progesterone), and lipid reserves (in fat bodies and liver) in three phases of the reproductive cycle: early breeding (late April), late breeding (mid-June), and post-breeding season (late July). In males, testis size, germinal epithelium, and testosterone plasma levels were high in the breeding season and decreased dramatically in July. However, spermatogenesis (maximum in June) was slightly delayed with respect to testosterone secretion (peak in April), suggesting that a previous increase in testosterone levels could be necessary to stimulate the initial stages of spermatogenesis

Melatonin-synthesizing enzymes in pineal, retina, liver, and gut of the goldfish (Carassius): mRNA expression pattern and regulation of daily rhythms by lighting conditions

It has been suggested that melatonin is synthesized in nonphotosensitive organs of vertebrates in addition to the well-known sites of the pineal gland and retina. However, very few studies have demonstrated the gene expression of melatonin-synthesizing enzymes in extrapineal and extraretinal locations. This study focuses on the circadian expression of the two key enzymes of the melatoninergic pathway, arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT), in central and peripheral locations of a teleost fish, the goldfish (Carassius auratus). First, the full-length cDNA sequences corresponding to the goldfish AANAT-2 (gAanat-2) and HIOMT-2 (gHiomt-2) were cloned, showing high similarity with other teleost sequences. Two forms of AANAT exist in teleosts. Here, for the first time, two isoforms of HIOMT are deduced from phylogenetic analysis. Moreover, both HIOMT and AANAT were detected in several peripheral locations, including liver and gut, the present results being the first to find HIOMT in nonphotosensitive structures of a fish species. Second, quantitative real-time polymerase chain reaction (PCR) studies were performed to investigate regulation of gAanat-2 in pineal and peripheral locations of goldfish maintained under different lighting conditions. The current results show circadian rhythms in Aanat-2 and Hiomt-2 transcripts in liver and hindgut, suggesting a local melatonin synthesis in goldfish. Moreover, the analysis of daily expression of gAanat-2 under different lighting conditions, including continuous light (24L) and darkness (24D) revealed light-dependent rhythms in the pineal and retina, as expected, but also in liver and hindgut. The persistence in hindgut of these gAanat-2 rhythms under both constant conditions, 24L and 24D, suggests expression of this transcript is governed by a circadian clock and entrained by nonphotic cues. Finally, the current results support the existence of melatonin synthesis in gut and liver of the goldfish. (Author correspondence: mjdelgad@bio.ucm.es)

Light-dark cycle and feeding time differentially entrains the gut molecular clock of the goldfish (Carassius auratus).

The aim of the present study was to investigate how photocycle and feeding-time cues regulate the daily expression of Per1a, Per2a, Per3, and Cry3 in the goldfish hindgut. For this purpose, we studied the daily rhythmicity of these genes in fish maintained under different lighting conditions and under different feeding regimes (scheduled or not). We also studied whether the timing of just one meal is able to reset the hindgut molecular clock. In a first experiment, randomly fed fish were divided into four groups and kept under different light conditions for 30 d: 12 h light and 12 h dark (12L:12D), an inverted photoperiod (12D:12L), constant darkness (24D), and constant light (24L). In a second study, fish maintained under 24L were divided into four groups fed at different time points for 35 d: (1) fish scheduled-fed once a day (at 10:00 h); (2) fish fed with a 12-h shifted schedule (at 22:00 h), (3) fish fed at 10:00 h throughout the experiment, except the last day when fed at 22:00 h; and (4) a randomly fed group of fish. Fish were sacrificed every 6 h throughout a 24-h cycle. In both experiments, gPer1a, gPer2a, gPer3, and gCry3 transcripts were quantified using Real Time-qPCR in the hindgut. Results show the clock genes gPer1a, gPer2a, and gCry3 are synchronized by both zeitgebers, the photocycle and feeding regime, in goldfish hindgut. Moreover, such clock genes anticipate light-on and food delivery, when these cues appear in a cyclic manner. In the absence of both zeitgebers, gCry3 and gPer2a rhythmicity disappeared. In contrast, the gPer1 rhythm was maintained under 24L and random feeding conditions, but not always, suggesting that food when randomly supplied is able to reset the clock depending on other factors, such as the energetic and metabolic conditions of the fish. The expression of gPer2a was not activated during the light phase of the cycle, suggesting the hindgut of goldfish is a non-direct photosensitive organ. In contrast to the other three genes, gPer3 expression in the goldfish hindgut seemed to be dependent on the timing of the last food delivery, even in the presence of a photocycle. This gene was the only one that maintained daily rhythms under both constant lighting conditions (24D and 24L), although with lower amplitude than when a photocycle was present. This indicates that, although the acrophase (peak time) of the gPer3 expression rhythm seems to be driven by feeding time, there is an interaction of both zeitgebers, food and light, to regulate its expression. In conclusion, present data indicate: (1) the hindgut of goldfish can be synchronized in vivo by both the photocycle and feeding time; (2) food is a potent signal that entrains this peripheral oscillator; and (3) both environmental cues seems to target different elements of the molecular clock. (Author correspondence: eisornaa@bio.ucm.es)

Serotonin-induced contraction in isolated intestine from a teleost fish (Carassius auratus): characterization and interactions with melatonin

Background  Serotonin (5‐HT) plays a critical role in several gastrointestinal functions in vertebrates. In teleosts lacking enterochromaffin cells, intestinal 5‐HT originates from serotonergic enteric neurons. In the present study, the foregut of a stomachless teleost, the goldfish (Carassius auratus), was used to evaluate the in vitro effect of 5‐HT on fish intestinal motility. We also studied the role of melatonin (MEL), an indoleamine sharing the biosynthetic pathway with 5‐HT, as regulator of serotonergic activity.

In Situ Localization and Rhythmic Expression of Ghrelin and ghs-r1 Ghrelin Receptor in the Brain and Gastrointestinal Tract of Goldfish (Carassius auratus)

Ghrelin is a gut-brain peptide hormone, which binds to the growth hormone secretagogue receptor (GHS-R) to regulate a wide variety of biological processes in fish. Despite these prominent physiological roles, no studies have reported the anatomical distribution of preproghrelin transcripts using in situ hybridization in a non-mammalian vertebrate, and its mapping within the different encephalic areas remains unknown. Similarly, no information is available on the possible 24-h variations in the expression of preproghrelin and its receptor in any vertebrate species. The first aim of this study was to investigate the anatomical distribution of ghrelin and GHS-R1a ghrelin receptor subtype in brain and gastrointestinal tract of goldfish (Carassius auratus) using immunohistochemistry and in situ hybridization. Our second aim was to characterize possible daily variations of preproghrelin and ghs-r1 mRNA expression in central and peripheral tissues using real-time reverse transcription-quantitative PCR. Results show ghrelin expression and immunoreactivity in the gastrointestinal tract, with the most abundant signal observed in the mucosal epithelium. These are in agreement with previous findings on mucosal cells as the primary synthesizing site of ghrelin in goldfish. Ghrelin receptor was observed mainly in the hypothalamus with low expression in telencephalon, pineal and cerebellum, and in the same gastrointestinal areas as ghrelin. Daily rhythms in mRNA expression were found for preproghrelin and ghs-r1 in hypothalamus and pituitary with the acrophase occurring at nighttime. Preproghrelin, but not ghs-r1a, displayed a similar daily expression rhythm in the gastrointestinal tract with an amplitude 3-fold higher than the rest of tissues. Together, these results described for the first time in fish the mapping of preproghrelin and ghrelin receptor ghs-r1a in brain and gastrointestinal tract of goldfish, and provide the first evidence for a daily regulation of both genes expression in such locations, suggesting a possible connection between the ghrelinergic and circadian systems in teleosts.