Changes in glucose, glycogen, thyroid activity and hypothalamic catecholamines in tench by starvation and refeeding.

The effects of short-term food deprivation (7 days) and refeeding (2 days) on different biochemical and neuroendocrine parameters were studied in tench. A 7-days fast resulted in a significant reduction of plasma glucose and glycogen hepatic content, supporting the key role of liver glycogen as energy depot for being consumed during fasting. The rapid recovery of normal values of blood glucose and glycogen stores by refeeding indicates a rapid replenishment of liver glycogen stores. The short-term starvation decreased circulating thyroid hormones (both T3 and T4) and T4 release from thyroid, supporting an interaction between nutritional state and thyroid function in tench. All these metabolic and hormonal changes were partial or totally reversed under refeeding conditions. An increase in hypothalamic content of norepinephrine and dopamine was found in fasted fish. This result might be a consequence of stress induced by starvation.

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.

Day/night variations of dopamine ocular content during Xenopus laevis ontogeny.

Concentration of dopamine (DA) and its metabolite, 3,4-dihydroxyphenylacetic acid is quantified by high-pressure liquid chromatography with a coulometric detection system in the eye of Xenopus laevis through ontogeny and in adults at two times during photocycle (midday and midnight). Ocular dopaminergic activity remains low during pre- and prometamorphosis and significantly rises in postmetamorphic froglets. This increase is more pronounced at midnight than at midday. The dualism of DA content versus DA release in Xenopus ocular tissue is studied in an eyecup culture system. On a 24-h cycle of DA release from adult Xenopus eyecups the highest DA release by eyecups is produced during daytime, and significantly decreases in darkness. From these results it can be concluded that in spite of the early development of the retinal dopaminergic system in the ontogeny of Xenopus, the final maturation must occur during the metamorphic climax. Endogenous DA release is significantly inhibited by light offset, which explains the higher ocular DA content found at midnight as compared to midday in postmetamorphic froglets and adults.

NPY receptors and opioidergic system are involved in NPY-induced feeding in goldfish.

The present study evaluated the effects of both intraperitoneal (i.p. ) and intracerebroventricular administration of selective Y(1) [(Leu(31), Pro(34))-NPY] and Y(2) [(Pro(13), Tyr(36))-NPY (13-36)] receptor agonists on food intake in satiated goldfish. Food intake (FI) was significantly increased by central administration of the Y(1) agonist (1 microg), but not by the Y(2) agonist, at 2 h postinjection. The feeding increase induced by (Leu(31), Pro(34))-NPY was in a similar magnitude to that obtained after ICV injection of the neuropeptide Y, and both feeding stimulations were reversed by the NPY (27-36), a general NPY antagonist. The i.p. administration of the agonists either did not significantly modify (Y(2) agonist) or decreased (Y(1) agonist) food intake in goldfish. These data indicate that it is the Y(1)-like (similar to Y(1) and/or Y(5)) receptor, and not Y(2), that is involved in the central modulation of the feeding behavior in goldfish. We also investigated the possible involvement of opioid peptides as mediators of the NPY stimulatory action on food intake in goldfish. The ICV administration of naloxone (10 microg), a general opioid antagonist, blocked the NPY-induced feeding in goldfish, suggesting that the opioidergic system is involved in feeding regulation by NPY.

Melatonin synthesis in the greenfrog retina in culture: I. Modulation by the light/dark cycle, forskolin and inhibitors of protein synthesis.

Melatonin is synthesized in the pineal gland and the retina of vertebrates. Retinal serotonin N-acetyltransferase (NAT) activity and melatonin show a daily rhythm with high levels during the dark phase of the photocycle. In some vertebrates, these retinal NAT and melatonin rhythms are maintained in vitro. The aim of present work is to develop an eyecup culture system for the greenfrog (Rana perezi), suitable to analyze the mechanisms of regulation of melatonin synthesis by simultaneous determination of NAT activity and melatonin release. The R. perezi eyecups released melatonin to the culture medium in a rhythmic manner at least over a 27-h period under photocycle conditions. NAT activity and melatonin rhythms were similar to that observed in vivo under natural environmental conditions. Rana perezi retina exhibits a pronounced photosensitivity in vitro. Forskolin increased up to 2-fold the NAT activity and 4-fold the melatonin production at any lighting conditions. The addition of the translation inhibitor, cycloheximide, to the medium reduced significantly both nocturnal NAT activity and melatonin release, suggesting that de novo protein synthesis is produced daily during darkness. Actinomycin D, a transcription inhibitor, needs a longer time of action, because pre-existing mRNA must be depleted before the inhibition of melatonin release can be observed. The eyecup culture system is highly sensitive to light and chemical factors, which makes it particularly suitable as a model for the neurochemical analysis of melatonin biosynthesis in the retina of Rana perezi.

Melatonin synthesis in the greenfrog retina in culture: II. Dopaminergic and adrenergic control.

Serotonin N-acetyltransferase (NAT) activity and melatonin show a daily rhythm with high levels at night. Although the rhythmic properties of NAT and melatonin are similar in pineal gland and retina, great differences in the light perception and transmission mechanisms exist. We have analyzed the effects of adrenergic and dopaminergic agents on greenfrog (Rana perezi) eyecup culture, in order to identify the receptors involved in the regulation of retinal melatonin synthesis. A D2-like receptor is directly involved in the regulation of NAT activity and melatonin release in R. perezi retina. Quinpirole mimics the effect of light, reducing the darkness-stimulated NAT activity and melatonin release, while sulpiride antagonized these actions. Neither D1-agonist (SKF 38393) nor D1-antagonist (SCH 23390) had effect on NAT activity. However, a significant inhibition of darkness-evoked melatonin release was produced by SKF 38393 after 6 hours of culture. The beta- and antagonist1-agonists showed a clear inhibition. However, a direct effect of beta, alpha1 and D1-agonists on photoreceptors is unproven, being more probable that the adrenergic actions imply a non-photoreceptor retinal cell. In conclusion, eyecup culture of Rana perezi revealed a dopaminergic control of melatonin synthesis and a possible modulation of dopaminergic tone by adrenergic receptors. Melatonin release is a more sensitive parameter than NAT activity to the action of neuroactive agents, suggesting that melatonin synthesis can be regulated by more than one enzymatic step in Rana perezi.

Neuropeptide Y has a stimulatory action on feeding behavior in goldfish (Carassius auratus).

The purpose of the present study was to elucidate the possible role of neuropeptide Y (NPY) in the feeding regulation in fish. We examined the effects of intracerebroventricular (i.c.v.) or intraperitoneal (i.p.) neuropeptide Y administration on food intake in satiated goldfish, at different time intervals postinjection (0-2, 2-8 and 0-8 h). Food intake was significantly increased by i.c.v. administered neuropeptide Y (1 microg) at 2 h postinjection, while no significant differences in food intake were observed after i.p. treatment. The neuropeptide Y receptor antagonist, neuropeptide Y-(27-36), totally counteracted the stimulatory action of neuropeptide Y on feeding. The possible involvement of neuropeptide Y in the eating behavior evoked by food deprivation has been investigated. Food deprivation by either 24 or 72 h significantly increased feeding, and the neuropeptide Y receptor antagonist attenuated such feeding stimulation. From our findings, we suggest, first, that neuropeptide Y is involved in feeding central regulation in goldfish, acting via specific neuropeptide Y receptors, and second, that hypothalamic neuropeptide Y would be released in response to food deprivation, contributing to generate the consequent eating behavior stimulation in Carassius auratus.

Alpha1-adrenergic and dopaminergic receptors are involved in the anoretic effect of corticotropin-releasing factor in goldfish.

This study investigates the noradrenergic and/or dopaminergic receptors subtypes involved in the anoretic action of CRF in goldfish. Agonists and antagonists of alpha1- and alpha2-adrenoceptors, and D1- and D2-dopaminergic receptors were intracerebroventricularly (i.c.v.) administered alone or in combination with CRF in the case of antagonists. Food intake and hypothalamic content of catecholamines and their metabolites were measured at 2 h postinjection. On one hand, alpha1-adrenergic receptor antagonist, but not alpha2, blocked the anoretic effect of CRF. Moreover, we found a blockade of CRF-induced anoretic action by pretreatment with specific D1- and D2-dopaminergic antagonists. On the other hand, the i.c.v. administration of CRF reduced hypothalamic norepinephrine (NE) and dopamine (DA) content, without modifications in their metabolism. Thus, our results suggest that the anoretic effect of CRF appears to be mediated by alpha1-adrenergic and dopaminergic receptors in fish. Second, the reduction in hypothalamic NE and DA synthesis could be due to a direct effect of CRF treatment and/or a secondary effect of food intake reduction.

Inhibitory effect of serotonin on feeding behavior in goldfish: involvement of CRF.

The possible action of 5-HT on feeding behavior in goldfish has been studied. Food intake was significantly reduced by intracerebroventricular (ICV) injection of serotonin (5-HT, 10 microg) at 2 h postinjection. After peripheral (intraperitoneal) administration of 5-HT (1 and 10 microg/g bw), no significant modifications in food intake were detected. Thus, it can be concluded that there is a central anoretic action of 5-HT in teleost fish. Taking in mind the inhibitory effect of corticotropin releasing factor (CRF) on feeding in teleosts and the interactions between 5-HT and CRF described in mammals, we investigated the possible involvement of CRF as mediator of the 5-HT anoretic action in goldfish. The ICV pretreatment with alpha-Helical CRF[9-41](20 microg) partially blocked the inhibitory effect of 5-HT on food consumption in goldfish. These results show that CRF mediates, at least in part, the 5-HT-induced feeding inhibition in goldfish. On the other hand, the alterations in hypothalamic indoleamines content evoked by ICV treatments would suggest that the activation of CRF neurons by 5-HT appears to inhibit hypothalamic serotoninergic transmission, supporting the intermediate role of this neuropeptide in the central anoretic effect of 5-HT in goldfish.

Changes in thyroid hormone concentrations and total contents through ontogeny in three anuran species: evidence for daily cycles.

Three anuran species (Rana perezi, Xenopus laevis, and Bufo calamita) of different phylogenetic origins and ecological habitats have been studied during ontogeny with respect to day/night changes in whole-body concentrations and total content of extrathyroidal thyroxine (T4) and triiodothyronine (T3). There were no significant day/night changes in thyroid hormones (TH) during embryonic stages. Daily cycles in TH with higher nocturnal values appeared during premetamorphosis in R. perezi and X. laevis. Cyclicity disappears for T3, while it is reversed for T4, in prometamorphic R. perezi and X. laevis. In contrast, there were significantly higher T3 (0.74 +/- 0.13 ng/g) and T4 (2.08 +/- 0.54 ng/g) levels at night in prometamorphic B. calamita. Significant daily changes in T3 and T4 with higher nocturnal values (T3, 788.29 +/- 118.38 pg/g; T4, 1.95 +/- 0.4 ng/g) were again seen in X. laevis at the end of climax, while in B. calamita low TH values appeared at early scotophase and there were no significant changes in R. perezi at this time. Similar daily profiles were observed for TH whole-body concentrations and total contents.

Effect of alpha-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 alpha-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.

Effect of constant and fluctuating temperature on daily melatonin production by eyecups from Rana perezi.

We analysed the effect of daily temperature cycles in relation to constant temperature on day/night melatonin synthesis in frog eyecups in culture. Eyecups were cultured for 24 h under 12L:12D photoperiod and two thermal regimes, constant temperature (25, 15 and 5 degrees C) and thermoperiod (WL/CD, thermophase coinciding with photophase and cryophase coinciding with scotophase; and CL/WD, cryophase coinciding with photophase and thermophase coinciding with scotophase). A negative correlation between ocular serotonin N-acetyltransferase activity and culture temperature for both diurnal and nocturnal activities has been observed. This effect of increased ocular activity at low temperature is more pronounced than the well-known stimulatory effect of darkness, and it does not depend on the photoperiod phase. The lack of interactions between the phase of photoperiod and culture temperature indicates that the effects of both factors are independent. Night-time temperature is the key factor in determining the amplitude of the melatonin rhythm in the Rana perezi retina. However, daytime temperature can not counteract the inhibitory effect of light on ocular melatonin synthesis.

Daily changes in thyroid activity in the frog Rana perezi: variation with season.

Plasma triiodothyronine (T3) and thyroxine (T4) levels, as well as thyroid free (f) and bound (b) thyroid hormones (TH) content, were determined by radioimmunoassay (RIA) in adult Rana perezi frogs during a 24 h cycle in winter, spring, summer, and autumn. Significant daily changes in plasma T3 levels were present in all the seasons except for winter, being the lowest values observed during the scotophase. In contrast, plasma T4 only showed significant changes in spring, following a similar pattern to the one described for T3. Thyroid fT3 content did present day/night significant changes only in spring showing high contents at early scotophase. Mean fT4 content was higher at the beginning of light phase than during the rest of daily photocycle in spring and autumn, but significant differences appeared only in autumn. Regarding the thyroid bound content of TH, bT3, and bT4 presented significant daily changes in spring and autumn. However, different profiles were observed in these two seasons. High bound contents were found at early photo- and scotophase with lower values at late dark phase in spring, whereas higher contents were detected at this time in autumn. The present results indicate the existence of seasonally changing daily fluctuations in thyroid activity in Rana perezi and it seems that an interaction between photoperiod and temperature plays a role in the regulation of these daily changes.

Mu-opioid receptor is involved in beta-endorphin-induced feeding in goldfish.

The present study evaluated the central effects of selective opioid receptor subtype agonists and antagonists on food intake in satiated goldfish. Significant increases in feeding behavior occurred in goldfish injected with beta-endorphin, the kappa agonist, U-50488, the delta agonist, [D-Pen2,D-Pen5]enkephalin (DPEN), and the mu agonist, [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO). On the other hand, the different receptor antagonists used: nor-binaltorphamine (nor-BNI) for kappa, 7-benzidilidenenaltrexone (BNTX) for delta 1, naltriben for delta 2, beta-funaltrexamine (beta-FNA) for mu, and naloxonazine for mu 1, by themselves, did not modify ingestion or slightly reduced it. The feeding stimulation by beta-endorphin was antagonized by beta-FNA and naloxonazine, but not by nor-BNI, BNTX, or naltriben. These data indicate that the mu-opioid receptor is involved in the modulation of the feeding behavior in goldfish.

CRF effect on thyroid function is not mediated by feeding behavior in goldfish.

In the present study we examined the effects of acute corticotropin-releasing factor (CRF) administration and refeeding treatment on glucose levels and thyroid hormones (plasma levels and thyroid contents) in 48-h food-deprived goldfish. Central CRF administration (2 micrograms) decreased food intake and the thyroid T3 free fraction, without significantly modifying either thyroid hormones bound fractions (T3 and T4) or plasma glucose levels. Subsequently, we tested whether CRF affects thyroid activity by itself, or whether this effect is mediated by CRF-induced feeding reduction. CRF treatment in fasted fish reduced thyroid-free T3 and increased thyroid-free T4, which could be mediated by a decreased intrathyroidal 5'-monodeiodinase activity. These data suggest a CRF effect upon thyroid activity independent of feeding reduction. On the other hand, refeeding after 48-h fasting caused a significant increase in thyroid free T4 content and plasma thyroid hormone levels. Thus, a relationship between nutritional status and thyroid function, which could overlap with CRF effects, cannot be discarded. Plasma glucose levels were only significantly modified by refeeding, which seems to be the signal triggering the increase in glucose titers. Our results support the existence of both CRF-thyroid activity and nutritional status-thyroid function interactions in goldfish.

Alterations in food intake and thyroid tissue content by corticotropin-releasing factor in Tinca tinca.

The present experiments test the effects of intracerebroventricular injections of ovine corticotropin-releasing factor (1 microgram) on food intake, plasma glucose levels and thyroid function at 8 h postinjection in tench. Food intake and thyroid triiodothyronine (T3) content were significantly decreased after CRF treatment. Thyroid thyroxine (T4) content and plasma glucose levels were not modified by this neuropeptide. The present results suggest that CRF plays a role on food intake regulation and thyroid gland activity in tench.

The galanin-induced feeding stimulation is mediated via alpha 2-adrenergic receptors in goldfish.

In the present study we examined the effects of intracerebroventricular (ICV) or intraperitoneal (IP) galanin administration on food intake in satiated goldfish, at different time intervals after injections, 0-2, 2-8 and 0-8 h. We found that food intake was increased by ICV administered galanin (1 and 3.33 micrograms) at 2 and 8 h postinjection, while no modifications on feeding were observed after intraperitoneal injection in any of the studied time intervals. The galanin receptor antagonist, galantide, blocked the galanin-induced feeding increase. Pretreatment with yohimbine (alpha 2-adrenergic receptor antagonist), but not with prazosin (alpha 1-adrenergic receptor antagonist) antagonized the stimulatory effect of galanin on ingestive behavior. These results suggest that galanin is involved in the central regulation of feeding in goldfish, being the food intake stimulatory effect mediated by the alpha 2-adrenergic system.

Differential characteristics and regulation of arylamine and arylalkylamine N-acetyltransferases in the frog retina (Rana perezi).

Arylamine N-acetyltransferase activity (A-NAT: E.C.2.3.1.5) from Rana perezi retina was studied using p-phenetidine as specific substrate. Enzyme characteristics and regulation were compared with respect to the arylalkylamine N-acetyltransferase (AA-NAT: E.C.2.3.1.87) from the same tissue. A-NAT activity is distributed in both neural retina and choroid-pigmented epithelium complex, showing a 10-fold higher specific activity in neural retina. In contrast, AA-NAT activity is restricted to neural retina. Subcellular localization in neural retina indicated that both enzymatic activities are in the supernatant fraction (39,000 g, 20 min). p-Phenetidine acetylation was linear as a function of the neural retina amount in the assay (1/16 to 1 retina), and it is insensitive to phosphate buffer pH in the range 6.5-8.4. A-NAT kinetic showed a hyperbolic shape for both cosubstrates. Kinetic constants were KM = 11.2 microM, Vmax = 0.49 nmol/h/mg prot. for p-phenetidine (50 microM acetyl-CoA), and KM = 113.4 microM, Vmax = 3.1 nmol/h/mg prot. for acetyl-CoA (5 mM p-phenetidine). The additivity test for both enzymatic activities in retina homogenates demonstrated that both acceptor amines do not compete for the catalytic sites. Serotonin addition in the assay modifies differentially the kinetic characteristics of both enzymes. Serotonin acted as a strong mixed inhibitor, mainly competitive in nature (competitive Ki = 18.1 microM; non-competitive Ki = 1.9 mM) for AA-NAT. However, it acted as a weak inhibitor with respect to A-NAT, mainly non-competitive, (competitive Ki = 5.7 mM; non-competitive Ki = 8.7 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Seasonal changes in thyroid activity in male and female frog, Rana perezi.

Plasma T3 and T4 levels, and thyroid free (f) and bound (b) thyroid hormones contents, were determined by radioimmunoassay in adult male and female Rana perezi over 1 year. Plasma T3 levels show a significant seasonal cycle in both male and female frogs, increasing from January to peak in July (71-74 pg/ml) to decrease in August-October. Plasma T4 concentrations also varied seasonally, and sex differences were apparent. Basal T4 levels from the end of summer to the end of winter and higher values (682-651 pg/ml) in spring and early summer occur in female frogs; males have lowest levels in October (59.78 +/- 13.92 pg/ml) and higher levels during the rest of the year, except for a significant decrease in April. The thyroid content of fT3 and fT4 displayed similar seasonal patterns, with peak values in April and July-August. Sex differences are again present. The thyroid contents of bT3 are high between March and July in both sexes, with values in females being greater than those in males. The bT4 content follows a similar seasonal pattern in males and females and shows a minimum in March and a maximum in summer. Thyroid releasing capacity seems to change depending on the season and sex.

Central administration of beta-endorphin increases food intake in goldfish: pretreatment with the opioid antagonist naloxone.

The effect of intraperitoneal or intracerebroventricular beta-endorphin administration on food intake has been studied in satiated goldfish. Food intake was evaluated at different time intervals after injections, 0-2, 2-8 and 0-8 h. The 0.1 and 1 micrograms doses of beta-endorphin intracerebroventricularly administered induced an increase in food intake during the first 2 h postinjection, while no modifications on feeding were observed in the next 6 h. These same doses of beta-endorphin used increased cumulative food intake at 8 h postinjection. In contrast, intraperitoneal injection of 1 micrograms of beta-endorphin did not modify food intake in any of the studied time intervals. Naloxone, an opioid antagonist, attenuated the beta-endorphin-induced feeding increase. These results suggest that opioids may play a role in modulation of feeding central regulation, acting via opioid receptors in goldfish.