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The time course of gap junction formation and growth, following contraction synchronization of cardiac myocytes in culture, has been studied in a combined (electro) physiological and ultrastructural study. In cultures of collagenase-dissociated neonatal rat cardiocytes, pairs of spontaneously beating myocytes synchronized their contractions within one beat interval within 2-20 min after they apparently had grown into contact. 45 sec after the first synchronized beat an appreciable junctional region containing several small gap junctions was already present. In the following 30 min, neither the area of individual gap junctions nor their total area increased. 75 min after synchronization both the area of individual gap junctions and their total area had increased by a factor of 10-15 with respect to what was found in the first half hour. In the period between 75 and 300 min again no further increase in gap junctional area was found. In double voltage-clamp experiments, gap junctions between well-coupled cells behaved like ohmic conductors. In poorly coupled cells, in which the number of functional gap-junctional channels was greatly reduced, the remaining channels showed voltage-dependent gating. Their single-channel conductance was 40-50 pS. The electrophysiologically measured junctional conductance agreed well with the conductance calculated from the morphometrically determined gap-junctional area. It is concluded that a rapid initial gap junction formation occurs during the 2-20 min period prior to synchronization by assembly of functional channels from existing channel precursors already present in the cell membranes. It then takes at least another 30 min before the gap-junctional area increases possibly by de novo synthesis or by recruitment from intracellular stores or from nonjunctional membranes, a process completed in the next 45 min. [ABSTRACT FROM AUTHOR]

Recent studies have characterised a retrograde mechanism whereby the pineal hormone melatonin acts in the pars tuberalis (PT) of the pituitary gland to control thyroid hormone action in the hypothalamus, leading to changes in seasonal reproductive function. This involves the release of thyroid-stimulating hormone (TSH) from PT that activates type II deiodinase (DIO2) gene expression in hypothalamic ependymal cells, locally generating biologically active T3, and thus triggering a neuroendocrine cascade. In the present study, we investigated whether a similar regulatory mechanism operates in the European hamster. This species utilises both melatonin signalling and a circannual timer to time the seasonal reproductive cycle. We found that expression of βTSH RNA in the PT was markedly increased under long compared to short photoperiod, whereas TSH receptor expression was localised in the ependymal cells lining the third ventricle, and in the PT, where its expression varied with time and photoperiod. In the ependymal cells at the base of the third ventricle, DIO2 and type III deiodinase (DIO3) expression was reciprocally regulated, with DIO2 activated under long and repressed under short photoperiod, and the reverse case for DIO3. These data are consistent with recent observations in sheep, and suggest that the PT TSH third ventricle-ependymal cell relay plays a conserved role in initiating the photoperiodic response in both long- and short-day breeding mammals. [ABSTRACT FROM AUTHOR]

In mammals, the complex interaction of neural, hormonal, and behavioral outputs from the suprachiasmatic nucleus (SCN) drives circadian expression of events, either directly or through coordination of the timing of peripheral oscillators. Melatonin, one of the endocrine output signals of the clock, provides the organism with circadian information and can be considered as an endogenous synchronizer, able to stabilize and reinforce circadian rhythms and to maintain their mutual phase‐relationship at the different levels of the circadian network. Moreover, exogenous melatonin, through an action on the circadian clock, affects all levels of the circadian network. The molecular mechanisms underlying this chronobiotic effect have also been investigated in rats. REV‐ERB α seems to be the initial molecular target. [ABSTRACT FROM AUTHOR]

Using quantitative autoradiography, we have studied the influence of maternal plasma melatonin on the expression and density of melatonin receptors in the brain and pituitary of rat offspring. At birth, the same structures displayed melatonin receptors whether the rats were born to and reared by intact or pinealectomized dams. The receptor density was, however, about 20% lower in the group born to pinealectomized dams. At postnatal day 9, when the pups of both groups synthetize rhythmically their own melatonin, this difference was suppressed. These results indicate that melatonin does not appear to be a requirement for the expression of its receptors, but seems to play a stimulatory role in their synthesis. [ABSTRACT FROM AUTHOR]

Using quantitative autoradiography, we have studied the relationship between melatonin receptor density in the pars tuberalis (PT) and photoperiodic changes in sexual activity in a photoperiodic (Syrian hamster), and a non photoperiodic (rat) rodent. Syrian hamsters exposed to short photoperiod (SP) for 13 weeks or kept in long photoperiod (LP) with daily melatonin injections for 8 weeks, had both gonadal regression and a significant decrease in 2-I-melatonin binding site density in the PT when compared to controls. In contrast, when the animals were sexually active, photorefractory Syrian hamsters exposed to SP for 27 weeks, or rats kept for 13 weeks in SP, the PT melatonin receptor density was similar to that of control animals kept in LP. These results show clearly that a correlation exists between gonadal status and density of melatonin receptors in the PT and suggest that the PT could be the site where melatonin mediates its effects on seasonal function. [ABSTRACT FROM AUTHOR]

5-Methoxytryptamine (5-MT), 5-methoxytryptophol (5-ML) and melatonin (Mel) were measured in the plasma after 2, 5, and 8 weeks administration of 25 Μg 5-MT to golden hamsters kept under long photoperiod. 5-MT showed a one compartment kinetic profile in the plasma with half lives of 14.8 min after 2 weeks, 15 min after 5 weeks and 19.1 min after 8 weeks. A rapid metabolism of 5-MT was shown, Mel and 5-ML being detected in the plasma following 5-MT administration. However it was also shown that the gonadal atrophy observed after 5-MT administration cannot be due to its metabolism into these 2 compounds. Indeed when exogenously administered at a dose generating the same plasma concentration as that observed after 5-MT, the gonadal regression observed after the association of 5-ML and Mel is much less than that observed after 5-MT. 5-MT is thus a compound of great physiological interest. [ABSTRACT FROM AUTHOR]

Male golden hamsters were exposed to short photoperiod at either 20‡C or 5‡C. After 4 weeks a complete gonadal inhibition was observed in animals kept at 5‡C while in animals kept at 20‡C such an inhibition was much less. No significant difference in the pattern of pineal and plasma melatonin concentrations was observed between hamsters kept at 20‡C and 5‡C. If in the golden hamster pineal melatonin secretion is implicated in the transduction of the photoperiodic information, it is probably not implicated in the transduction of thermal information. [ABSTRACT FROM AUTHOR]

In the European hamster (Cricetus cricetus, L.) pinealectomy as well as a constant high level of either circulating melatonin or 5-methoxytryptamine induce marked changes in the immunocytochemically demonstrable central vasopressinergic innervation. When compared to control animals, a drastic decrease of AVP-immunoreaction was observed in the diagonal band of Broca, the lateral septum, the medial amygdala and the ventral hypothalamus. The results obtained suggest that part of the central vasopressinergic innervation is involved in pineal dependent seasonal functioning of the animal. [ABSTRACT FROM AUTHOR]

A prominent innervation of the pineal gland of the European hamster with nerve fibres containing neuropeptide Y (NPY) and tyrosine hydroxylase (TH) was demonstrated by means of immunohistochemistry. Nearly all the TH- and NPY-immunoreactive nerve fibres in the superficial pineal gland disappeared after bilateral superior cervical ganglionectomy, showing that the majority of NPY- and TH-immunoreactive nerve fibres belonged to the sympathetic nervous system. Since, in the European hamster, preliminary studies of the NPY-fibre density in the pineal gland had indicated seasonal changes, the density of NPY-immunoreactive nerve fibre profiles was ascertained in the superficial pineal gland in a series of animals between the first part of November and late April. The highest density of NPY-immunoreactive nerve fibre profiles was observed during midwinter. On the other hand, during the same period of the year, the number of sympathetic TH-immunoreactive sympathetic nerve fibre profiles did not exhibit seasonal variation, nor did substitution of testosterone, during the sexually inactive period, affect the density of NPY-containing nerve fibres in the gland. Our results show the presence of a testosterone-independent annual variation in the content of NPY in the sympathetic nerve fibres innervating the pineal gland of the European hamster. This variation can be correlated with the changes in the daily pattern of melatonin production observed by others in the same species at this period of the year. [ABSTRACT FROM AUTHOR]

The development of the retinohypothalamic tract (RHT) labeled with cholera toxin and of the vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) systems was studied in the hypothalamus of neonatal mink by using immunohistochemistry. Retinal fibers were observed in the suprachiasmatic nucleus (SCN) from birth and were adult-like by day 14. VIP and PHI immunoreactivity was also present from birth. Melatonin binding was studied by autoradiography using [125I]melatonin as a ligand. A specific binding was detected in near-term fetal and neonate brains in the olfactory epithelium, various thalamic nuclei, the pineal gland, and the pars tuberalis of the pituitary gland, but never in the SCN. These results are discussed in the context of the potential role of daylight cycles and/or melatonin in entraining circadian rhythms in neonate mink. [ABSTRACT FROM AUTHOR]

By use of antibodies raised against leu-enkephalin and met-enkephalin immunoreactive, opioidergic bi- and multipolar cells were demonstrated in the pineal gland of the European hamster. Ultrastructural analysis of these opioidergic cells revealed them to be pinealocytes. Processes emerged from the cell bodies and terminated in club-shaped swellings containing many small clear and some larger granular vesicles. Some of the terminals made synapse-like contacts with non-immunoreactive pinealocytes. The presence of the opioidergic pinealocytes strongly indicates that the pineal gland of the European hamster, in addition to its pinealopetal nervous regulation, is regulated by intrapineal peptidergic pinealocytes via a synaptic mechanism. A possible paracrine role of the opioidergic cells must also be considered. [ABSTRACT FROM AUTHOR]

High affinity melatonin-binding sites have been described, by means of autoradiography with 2-I-melatonin as the ligand, in more than 60 brain areas of about 20 mammalian species, with dramatic variations in the nature and number of labelled structures among the different species studied. As melatonin is involved in the synchronization of biological rhythms, we have tried to correlate the brain areas containing melatonin-binding sites with some rhythmic functions typical of give species. Therefore, we have studied the location of melatonin-binding sites in the complete brain of five long-day breeders with hibernation cycles, viz. one insectivore and four rodents. With the exception of the suprachiasmatic nuclei and the pars tuberalis of the pituitary, both of which contain binding sites in all five species, few reactive structures are common, even among species from the same family, e.g. the edible dormouse and the garden dormouse. [ABSTRACT FROM AUTHOR]

When two spontaneously beating neonatal rat heart cells in tissue culture were allowed to grow together they synchronized their originally independent beats to a common rhythm, as measured with an opto-electronic technique. Both single isolated cells and cell pairs exhibited a highly irregular beating pattern. Beating irregularity was strongly and positively correlated with mean interbeat interval. Synchronization of beating occurred in 50% of the pairs studied within one beating interval. In the remaining cell pairs, the first synchronized beat was followed by a 4-65 s period of partial synchronization. The time difference between contraction moments of two cells in a pair respective to each other (latency) changed upon synchronization from a random value to a fixed value. In a few cases the latency decreased during 20 to 30 s after the first synchronized beat before a steady-state value was reached. The mean interbeat interval (IBI) of the synchronized cell pairs was governed by the mean IBI of the originally faster beating cel. In 83% of the cases the mean IBI of the cell pairs was between that of the originally isolated beating cells. We conclude from the experiments described that physical coupling (i.e. gap junction formation) is virtually complete before beating synchronization occurs. [ABSTRACT FROM AUTHOR]

Temporal patterns of hibernation were studied by continuous monitoring of body temperature by radiotelemetry over 6 months in European hamsters, Cricetus cricetus, at constant temperature and photoperiod. Entrances into hibernation occurred mostly at the end of the night (0000-0800 hours), while arousals were randomly distributed between day and night. This is at variance with a control of bout duration by a clock with a period of 24 h. Consequently, the timing of entrances implies a phase-resetting of the circadian clock on each arousal. Persistence of circadian rhythmicity with a period different from 24 h during deep hibernation was investigated examining whether the durations of torpor bouts were integer multiples of a constant period. A non-parametric version of the classical contingency test of periodicity was developed for this purpose. Periods ranging from 21 to 29 h were tested. Nine animals out of ten showed at least one significant period in this range ( P<0.01), either below 24 h (21.8±0.5 h, n=4) or above (27.3±0.5 h, n=7). However, we have found a theoretical model of bout durations for which the contingency test of periodicity sometimes gives false significant results. This indicates that the power of the test is weak. With this reservation our results suggest that a circadian oscillator controls the duration of a bout of hibernation, which would occur after an integer, but variable and possibly temperature-dependent number of cycles. [ABSTRACT FROM AUTHOR]

In the mammalian pineal gland, serotonin (5-HT) is located both in the pinealocytes and in the noradrenergic nerve terminals. Pineal 5-HT can be metabolized by three different routes, one of these being its deamination, catalized by monoamine oxidase (MAO). MAO is known to exist as two isozymes, MAO-A and MAO-B. Using two different cytochemical methods at the ultrastructural level, we have localized the presence of MAO in the pineal gland of the rat. The use of selective inhibitors of A-type (clorgyline) and B-type (deprenyl) has shown that MAO-A is localized in the noradrenergic nerve terminals, while pinealocytes contain MAO-B. Taking into account that 5-HT is only deaminated by MAO-A, the specific association of each MAO isozyme with a defined cell type implicates that two cellular compartments are needed in the pineal gland for the biosynthesis of 5-methoxytryptophol and 5-methoxyindole acetic acid, while for the synthesis of melatonin and 5-methoxytryptamine just one cellular compartment, the pinealocyte, is appropriate. [ABSTRACT FROM AUTHOR]

This study shows for the first time that perfusion of rat or hamster brain with a cyclohexylamine-paraformaldehyde mixture makes possible the observation by autoradiography of melatonin binding sites in structurally well-preserved fixed tissues. This result is a first step in the identification of melatonin-receptor-containing cell types by cytoautoradiography. [ABSTRACT FROM AUTHOR]

The pinealocytes of the pig contain conspicuous dense bodies, the nature and role of which are not yet fully elucidated. The aim of this study was to demonstrate whether or not these structures are involved in the secretion process. The tannic acid-Ringer incubation (TARI)-method, which allows a clear-cut ultrastructural study of secretory discharge by exocytosis, has been used. The results indicate that pig pinealocytes release the content of the dense bodies with an amorphous inner structure into the extracellular space via exocytosis and that this secretion is quantitatively important. The secreted material is proteinaceous in nature; this indicates that polypeptides are released by the pineal. [ABSTRACT FROM AUTHOR]

It is demonstrated that systolic and diastolic depolarization rate are correlated with the percentage of myofilaments in the cells of the rabbit sinoatrial node. It appears that, in the rabbit sinoatrial node, little-excitable transitional cells exist in the zone of propagation at the septal side of the typical nodal cells. [ABSTRACT FROM AUTHOR]

Abstract Melatonin, secreted only during the night by the pineal gland, transduces the photoperiodic message to the organism. One important target for the hormone is the pars tuberalis (PT) of the adenohypophysis which displays a very high number of melatonin binding sites in mammals and is implicated in the seasonal regulation of prolactin secretion. To gain insight into the mechanism by which the melatonin signal is decoded in the PT, we studied the effect of photoperiod on the PT cells expressing the MT1 melatonin receptor in a highly photoperiodic species, the European hamster. Recently, we showed that, in the rat, the MT1 receptor mRNA is expressed in PT-specific cells characterized by their expression of β-thyroid stimulating hormone (β-TSH) along with the α-glycoprotein subunit (α-GSU). As the cellular composition of the PT shows variability among species, we first identified the cell type expressing the MT1 receptor in the European hamster by combining immunocytochemistry and nonradioactive in situ hybridization for the MT1 receptor mRNA. Our results show that, in the European hamster, as in the rat, the MT1 receptor is only expressed by the PT-specific-cells, β-TSH and α-GSU positive. In a second step, we analysed the effects of photoperiod on the MT1 mRNA, and on β-TSH and α-GSU both at the mRNA and protein levels. Our data show that, compared to long photoperiod, short photoperiod induces a dramatic decrease of MT1, β-TSH and α-GSU expression. Protein levels of β-TSH and α-GSU were also dramatically reduced in short photoperiod. Together, our data suggest that melatonin exerts its seasonal effects in the PT by signalling to PT specific-cells through the MT1 receptor subtype. [ABSTRACT FROM AUTHOR]