Circadian rhythms of melatonin release from individual superfused chicken pineal glands in vitro.

The pineal gland of birds contains one or more circadian oscillators that play a major role in overall temporal organization. We have developed a flow-through culture system for the isolated pineal by which we can measure the release of melatonin continuously from superfused glands over long periods of time. Chicken pineals release melatonin rhythmically, and these rhythms persist in vitro with a circadian oscillation. In light cycles the release of melatonin is strongly rhythmic; however, in constant conditions the amplitude of the rhythm is lower and appears to be damping. Light has at least two effects upon the isolated pineal: cyclic light input synchronizes the rhythm, and acute light exposure at night rapidly inhibits melatonin release. The cultured avian pineal clearly offers great potential as a model system for the study of vertebrate circadian oscillators and may open the way for an analysis of mechanism.     

Effects of season, temperature, and photoperiod on plasma melatonin rhythms in the goldfish, Carassius auratus

Abstract: Effects of season, environmental temperature, and photoperiod on plasma melatonin concentrations were studied in the goldfish, Carassius auratus. When goldfish were reared under natural conditions, melatonin levels at mid-dark exhibited seasonal changes, with higher levels obtained in June and September than in December and March. When fish were kept under light: dark (LD) cycle of 12: 12 at 5, 15, or 25°C during March-April, temperature-dependent increases in melatonin levels at mid-dark were observed. When animals were maintained under LD 16: 8 or LD 8: 16 in combination with temperature changes (5, 15, and 25°C) during January-February, the duration of nocturnal elevation in melatonin was controlled by the length of the scotophase while the amplitude was influenced by environmental temperature. These results indicate that plasma melatonin profiles in the goldfish exhibit seasonal changes that are regulated by both photoperiod and temperature.     

Examination of in vitro melatonin secretion from superfused trout (Salmo gairdneri) pineal organs maintained under diel illumination or continuous darkness

Melatonin secretion was measured from rainbow trout (Salmo gairdneri) pineal organs maintained individually under flow-through whole organ culture (superfusion) conditions. Radioimmunoassay of perfusate fractions collected during controlled photic conditions demonstrated that melatonin secretion in vitro remained basal during the photophase and underwent increases in titer during the scotophase. While amounts of melatonin (mel) secreted were characteristic of individual pineal organs, photophase values ranged between 0.25 and 0.75 ng mel/ml and scotophase values ranged from 6 to 10 ng mel/ml of perfusate. Diel melatonin secretion profiles reflected the illumination regimen, with light associated with low melatonin titer in the perfusate and darkness associated with high titer. Light pulses during a normal scotophase resulted in a depression in melatonin secretion regardless of whether it was administered early or late in the dark period. Pulses of darkness given early or late in a normal photophase resulted in increased melatonin secretion. Superfused trout pineal organs did not display endogenous rhythmicity in melatonin secretion when subjected to prolonged exposure to continuous darkness (DD), whether first exposed to entraining light/dark (LD) cycles prior to DD or exposed to DD at the initiation of superfusion. In both studies, elevated melatonin secretion gradually declined over time. But exposure to a 4:4LD cycle after DD resulted in decreased (with light) and increased (with darkness) melatonin secretion. These results demonstrate that the trout pineal organ can be maintained for extended periods of time in superfusion culture, that the trout pineal organ is very responsive to light or dark for regulating melatonin synthesis, and that an endogenous rhythm in melatonin synthesis when organs were maintained in DD was not detectable.     

Effects of hypothalamic and thalamic lesions on prolactin secretion in goldfish (Carassius auratus)

The effects of hypothalamic and thalamic lesions on serum and pituitary prolactin levels in goldfish were studied. A radioimmunoassay was used to measure prolactin levels. Lesions in the nucleus lateral tuberis (NLT) pars anterioris, NLT pars posterioris, NLT pars inferioris, nucleus anterior tuberis, or in the nucleus preopticus did not affect serum or pituitary polactin levels compared to sham or normal control groups. Lesions in the NLT pars lateralis caused a significant increase in serum prolactin, but had no effect on pituitary prolactin levels. This suggests that the NLT pars lateralis is the origin of a factor that normally inhibits prolactin release from the pituitary. Large lesions that destroyed the medial thalamic and anterior hypothalamic region dorsal to the NLT caused a significant decrease in serum prolactin, but had no significant effect on pituitary prolactin levels. It is suggested that the lesions in this region may have interrupted neural afferents to the NLT pars lateralis that normally inhibit secretion of a prolactin inhibitory factor.     

Influence of catecholamines on gonadotropin secretion in goldfish, Carassius auratus

Serum gonadotropin (GtH) concentrations in female goldfish were measured before and at various times after intraperitoneal injection of drugs altering catecholamine synthesis and neural activities. Reserpine, a depleter of neurotransmitter stores, elevated serum GtH levels compared to controls, suggesting the general involvement of neurotransmitters in altering GtH release. 6-Hydroxydopamine, a catecholaminergic neurotoxin, increased serum GtH concentration, suggesting that catecholaminergic neurons inhibit GtH release. Blocking of L-DOPA and dopamine synthesis by alpha-methyl-para-tyrosine and carbidopa, respectively, but not norepinephrine by diethyldithiocarbamate, raised serum GtH values above those of controls. Injections of an alpha-agonist, clonidine, also increased serum GtH concentrations. These results suggest the existence of an inhibitory dopaminergic and a stimulatory alpha-adrenergic influence on GtH release in goldfish.     

Dopamine inhibition of gonadotropin and alpha-melanocyte-stimulating hormone release in vitro from the pituitary of the goldfish (Carassius auratus)

The purpose of the present investigation was to examine the receptor specificity of dopamine inhibition of gonadotropin (GtH) and alpha-melanocyte-stimulating hormone (alpha-MSH) release from the goldfish (Carassius auratus) pituitary in vitro. Pars distalis (PD) and neurointermediate lobe (NIL) fragments of the goldfish pituitary were superfused in vitro under various experimental paradigms; eluate from PD and NIL fragments was analyzed for (GtH) and (alpha-MSH), respectively. Spontaneous GtH release from PD fragments was relatively constant over 6 hr; continuous superfusion with dopamine reversibly inhibited spontaneous GtH release with an estimated ED50 of 10(-4.4) M. Domperidone, a specific D-2 receptor antagonist, reversed the inhibitory action of dopamine and increased spontaneous GtH release. Acute treatment of PD fragments with salmon GnRH (sGnRH) stimulated GtH release; dopamine inhibited GtH release from similarly treated fragments with an ED50 of 10(-7.5) M. The spontaneous release of alpha-MSH from NIL fragments was relatively constant over 6 hr; continuous superfusion with dopamine reversibly inhibited this release with an ED50 of 10(-7.2) M. Acute treatment of NIL fragments with thyrotropin-releasing hormone (TRH) caused acute dose-related increases in alpha-MSH release with an ED50 of 10(-8.2) M; dopamine reversibly inhibited alpha-MSH release from similarly treated fragments with an ED50 of 10(-7.7) M. Both stereoisomers of apomorphine, a dopamine agonist, inhibited GtH release from PD fragments treated with sGnRH; in contrast, alpha-MSH release from NIL fragments treated with TRH was stereospecifically inhibited by (-)-apomorphine, but not by (+)-apomorphine. Domperidone reversed (ED50 = 10(-6.6) M) dopamine (10(-6.3) M) inhibition of GtH release from PD fragments treated with sGnRH. In NIL fragments, the inhibitory action of dopamine (10(-6.3) M) was reversed by domperidone (ED50 = 10(-5.5) M), which restored the acute alpha-MSH release response to TRH. These results suggest the involvement of a low-affinity dopamine/neuroleptic receptor in dopamine inhibition of GtH and alpha-MSH release from the pituitary of the goldfish.     

Intraneuronal transport in the hypothalamoneurohypophysial system of the goldfish (Carassius auratus)

The oxytocin-like hormone of the goldfish was labeled by intraventricular injection of [³H]tyrosine. Ion-exchange chromatography allowed the isolation of this peptide with a high degree of isotopic purity from groups of six pituitaries. Time course studies showed that radioactive hormone began to arrive in the gland 2–3 hr after intraventricular injection of the isotope, suggesting an intraaxonal transport rate of the order of 1 mm/hr.     

Circadian rhythms of pineal function in rats

In pineal glands melatonin is synthesized daily. Melatonin synthesis in rats kept in most light-dark cycles occurs during the subjective night. This rhythm, which persists in constant dark, is a circadian rhythm which may be a consequence of another circadian rhythm in the pineal gland, of N-acetyltransferase activity (NAT). The NAT rhythm has been studied extensively in rats as a possible component of the system timing circadian rhythms. The NAT rhythm is driven by neural signals transmitted to the pineal gland by the sympathetic nervous system. Environmental lighting exerts precise control over the timing of the NAT rhythm. In rats, there is enough data to describe a daily time course of events in the pineal gland and to describe a pineal "life history." Hypothetical schemes for generation of the NAT rhythm and for its control by light are presented.     

Circadian rhythms in serum melatonin from infancy to adolescence

The circadian rhythm of circulating melatonin was determined in 38 children, aged 1-18 yr. Serum melatonin concentrations at 2400 and 0300 h in children aged 1-5 yr were significantly (P less than 0.001) higher than those in older children [6-10 yr, puberty stage 1 (P1)]. Nocturnal melatonin levels also were significantly (P less than 0.01) higher in P1 subjects than in P2 subjects aged 9-12 yr. A further small decline was found from P2 to P4/P5 (P less than 0.05). Thus, the decline in the nocturnal melatonin surge is not exclusively related to pubertal development, but begins in infancy.     

Prostaglandin synthesis in goldfish heart, Carassius auratus

Potential precursors for prostaglandin (PG) synthesis were measured in goldfish heart and skeletal muscle by gas chromatography. Heart tissue contained docosahexaenoic, arachidonic, eicosapentaenoic, and eicosatrienoic acids in concentrations of 3223 +/- 128, 1216 +/- 7.8, 260 +/- 72.8, and 250 +/- 14 ng/mg wet wt, respectively. 14C-Labeled substrates were examined for their ability to be converted to prostaglandins. Eicosatrienoic and docosahexaenoic acid were not synthesized into prostaglandins, with 66 and 72% of the substrate remaining as free fatty acids, respectively. In contrast, both arachidonic and eicosapentaenoic acids were converted predominantly to PGFs and PGIs. The conversion was time dependent and complete by 30 min. The conversion patterns with eicosapentaenoic acid and arachidonic acid were essentially the same. The data suggest that goldfish cyclooxygenase can utilize two of the four potential substrates for prostaglandin synthesis. As fatty acid levels in fish vary with environmental temperature, substrate availability rather than cyclooxygenase preference may dictate the types of prostaglandins which are produced.     

Gonadotropin-releasing hormone induction of apoptosis in the testes of goldfish (Carassius auratus)

Apoptosis, or programmed cell death, can occur via death receptor or mitochondrial pathways. Normal spermatogenesis in mammals involves apoptosis mediated, in part, by the death receptor fas and its ligand. The regulation of programmed cell death in the gonads has been shown to be dependent on a number of locally produced factors, including GnRH. Whereas the role of GnRH in the control of apoptosis and follicular atresia has been documented in the mammalian ovary, GnRH regulation of testicular apoptosis remains obscure. A previous study in our laboratory demonstrated the involvement of GnRH on the induction of DNA fragmentation in mature, perispawning testis. In this study, we tested the hypothesis that GnRH plays a differential regulatory role during male gamete maturation by studying the effect of GnRH on the induction of apoptosis during goldfish spermatogenesis. Treatment with GnRH resulted in DNA fragmentation only during late stages of spermatogenesis as assessed by oligonucleotide detection and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assays. The GnRH-induced apoptosis in the goldfish testis was found to be mediated by increased levels of fas and fas ligand-like proteins as well as elevated activity of caspase-3 (an executioner caspase) and -8 (a death receptor-activated caspase). The results suggest the involvement of the death receptor pathway in GnRH-induced apoptosis, providing support for the hypothesis that GnRH plays an important role in the control of spermatogenesis in the goldfish testis.     

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, alpha-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 microg/g), or the cortisol synthesis inhibitor, metyrapone (100 and 200 microg/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 alpha-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 microg/g) and metyrapone (200 microg/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.     

Effects of third ventricle injection of prostaglandins on gonadotropin secretion in goldfish,Carassius auratus

The effects of injection of prostaglandin (PG) E₁, PGE₂, and PGF_2α into the third ventricle on serum gonadotropin (GTH) concentrations in the goldfish were tested. Blood samples were taken at 30 min postinjection for radioimmunoassay of serum GTH. PG dosages of 0.5 and 1.0 μg were ineffective. However, PGE₂ and PGF_2α at the 2.0-μg dosage significantly suppressed serum GTH. PGE₁ at a 2.0-μg dosage had no effect. There were no effects on serum GTH when 2.0 μg of PGE₁, PGE₂, or PGF_2α were injected intraperitoneally. The results indicate that PGE₂ and PGF_2α suppress gonadotropin secretion by some action, presumably on the hypothalamus. However, action of PGE₂ and PGF_2α by diffusion from the site of injection to some other brain site or the pituitary cannot be eliminated in the present study.     

Melatonin secretion from goldfish pineal gland in organ culture

Pineal glands were removed from goldfish reared under 12L-12D at 25 degrees for 2 weeks. These were incubated for 6 days under (1) normal 12L-12D (lights on 0600-1800 hr), (2) reversed 12L-12D (lights on 1800-0600 hr), (3) continuous dark, or (4) continuous light condition at 25 degrees. The incubation medium was changed at 12-hr intervals (0600-1800 and 1800-0600 hr) and secreted melatonin (MLT) was measured by RIA. Under 12L-12D or reversed 12L-12D, MLT secretion was active in the dark phase and was suppressed in the light phase of a given photoperiod. Under a continuous dark condition, a large amount of MLT was secreted into the medium, although the amount gradually decreased. The MLT secretion was more active in the period corresponding to the dark phase of the acclimatory photoperiod than in the period corresponding to the light phase. This pattern in secretion remained for 4 days. Under a continuous light condition, MLT secretion was suppressed, but the secretion was rapidly increased after changing the photoperiod from the light to the dark condition. These findings clearly indicate that MLT secretion in the organ-cultured pineal gland is photosensitive. It is active under dark and inactive under light conditions. The existence of a circadian rhythm in MLT secretion is also suggested.     

Evidence for a gonadotropin-releasing hormone binding protein in goldfish (Carassius auratus) serum

The binding of salmon gonadotropin-releasing hormone (sGnRH) and its superactive analog, [D-Arg6, Pro9-NEt]-sGnRH, to a macromolecular component in goldfish serum was studied, using 125I-[D-Arg6, Pro9-NEt]-sGnRH and 125I-sGnRH as labeled ligands. Bound was separated from free labeled ligand by gel filtration with Sephadex G-50. The binding of labeled ligand to goldfish serum was dose-dependent. The results indicate a single class of binding site having low affinity and high capacity. The existence of a GnRH binding protein in serum may, in part, contribute to the long-lasting pharmacological action of GnRHs in goldfish.