Adrenergic and peptidergic control of the regulation of cAMP efflux and melatonin secretion from perifused rat pineal gland.

Mammalian pineal gland receives peptidergic (e.g., vasoactive intestinal peptide [VIP]; peptide histidine isoleucine [PHI]; neuropeptide Y, NPY; substance P, calcitonin gene-related peptide [CGRP], arginine vasopressin [AVP] and oxytocin [OXT]) fibers in addition to sympathetic innervation. The dynamics of cAMP efflux and melatonin (MT) secretion were compared during the infusion of these peptides in our long-term perifusion system. VIP and PHI enhanced both pineal cAMP efflux and MT secretion in a dose-dependent manner (10 nM to 10 microM). However, the potency of PHI was slightly less. The peak of cAMP release always precedes that of MT production. The possible interactions between adrenergic and peptidergic compounds in the regulation of pineal cAMP efflux and MT secretion were also studied. VIP acts on specific peptidergic receptors, since its stimulatory effect could only be reduced by a VIP receptor antagonist. VIP has an additive effect at a lower (100 nM) concentration combined with norepinephrine (NE). NPY (100 nM) can completely block NE-induced MT secretion, but the decrease in cAMP efflux is less. However, NPY does not significantly influence VIP-stimulated cAMP efflux or MT secretion. These data suggest that NE, VIP, and NPY are differently involved in the cAMP and calcium signaling. The other neuropeptides are ineffective.

Development and control of the circadian pacemaker for melatonin release in the chicken pineal gland.

UNLABELLED: Melatonin (MT) release from explanted pineal glands of 3- to 20-week-old chicken was investigated in a 5-day perifusion system. Both the chicken and the explanted glands were exposed to various environmental lighting regimens. OBSERVATIONS: (1) The explanted chicken pineal is sensitive to direct light. Continuous illumination during the in vitro period abolishes the circadian rhythm of the MT secretion in 3 days. Continuous darkness has limited effect. (2) Reverse illumination completely reverses the MT cycle in 2 days. (3) Rhythmic illumination with short (6-h) periods only slightly modulates the MT release pattern: the basic, 24-h periodicity is preserved. (4) The circadian MT pacemaker develops normally and becomes synchronized to the day even if the chicken has never experienced alteration in the environmental illumination (those hatched and bred under continuous illumination). The explanted pineal from these chickens exhibits normal MT cycle and light sensitivity. Conclusion, Chicken pineal contains a complete, genetically coded circadian pacemaker with a fixed frequency. The pacemaker is synchronized to the day by the altered environmental illumination and by at least one other, unknown environmental factor. With altered illumination, in vitro, the 24-h periodicity of the pacemaker cannot be changed significantly, but its phase can be shifted. In contrast to conclusions obtained from in vivo observation in mammals, light seems to stimulate MT secretion from the avian pineal in vitro. For development and daily synchronization of the circadian MT pacemaker in the chicken pineal gland, periodic changes in the environmental illumination are not necessary.

Inhibition of cold stimulated thyrotropin release in rats by neurotropic drugs: specific or unspecific effect?

Male Wistar Olac rats were kept in stainless steel cages at 24 +/- 1 °C. Three days before the experiment they were transferred into another room and kept 3 days at 30 °C. On the day of experiment, groups of 14-16 animals each were injected i.p. with 10 mg/kg morphine hydrochloride (MO), 1.5 mg pimozid (PI), 10 mg/kg cyproheptadine (CY) or with the combinations of MO+PI and MO+CY in the doses indicated above. Exactly after 30 min each animal was transferred to individual plexiglass cage and then a half of each group (consisting of 7-8 animals) was transferred to the cold room (4 °C), while the other half was kept at 30 °C. Precisely after 60 min the animals were quickly decapitated, the trunk blood was collected and thyrotropin (TSH) was estimated in serum using specific rat TSH radioimmunoassay kit supplied by National Pituitary Agency, Bethesda, Md. It was found that the level of TSH significantly decreased (P < 0.01) in PI injected group even at 30 °C as compared with all other groups. The same was found at 4 °C. In addition, at 4 °C the groups injected with CY, PI+MO and CY+MO showed the TSH level significantly decreased (P < 0.01) as compared with "cold control" and even with the group injected MO only. Since the animals injected with PI and CY irrespectively of MO were deeply sleeping and showed decreased body temperature and blood pressure, it was suggested that the effect of these and possibly some other drugs using for the study of the central regulation of cold stimulated TSH release may be at least partly, if not completely, unspecific.

Regulation of pineal melatonin secretion: comparison between mammals and birds.

The melatonin secretory pattern of the perifused rat and chicken pineal was compared in response to different lighting conditions and norepinephrine administration. The following main differences were observed. 1. Explanted (perifused) rat pineal showed, after a rapid initial surge, a steady continuous basal secretion of melatonin. This was independent from the day-night (light-dark) periods. Chicken pineal, however, showed a characteristic daily rhythm of melatonin production with peak in the dark and nadir in the light phase. 2. This daily rhythm could not be extinguished by keeping the pineal donor birds in constant light- or constant dark environment for at least 2 weeks immediately before sacrifice. 3. Short light impulse (5 min), applied in the middle of the dark phase, was ineffective in birds. Keeping the perifusion chambers in continuous light or darkness, however, depressed the amplitude of the circadian rhythm even in the next cycle. 4. Rat pineal responds to norepinephrine stimulation with a dose-related increase of melatonin release, independently from the phase of the day, while in the chicken, norepinephrine slightly inhibits both the diurnal and the nocturnal level of melatonin secretion. 5. It can be inferred that melatonin secretion of the mammalian pineal gland is primarily regulated by a peripheral (sympathetic) innervation. This is modulated, under in vivo circumstances, by different environmental factors, mainly by light conditions transmitted by neural mechanisms. In contrast, the primary secretory process of the avian pineal is based on an intrinsic circadian rhythm. This might be genetically coded or maintained by yet unknown neurohormonal mechanisms and/or external factors (e.g. magnetic fields). This fairly stable circadian rhythm is only modulated by environmental lighting conditions.

Influence of serotonergic neurons and of pineal gland on the development of the neonatal androgen sterilization syndrome in the rat.

The effect of the removal of pineal gland, performed in adult age or during perinatal life, was investigated in the neonatal androgen sterility (NA-CEA) syndrome, in combination with drugs acting on serotonergic neurons. Perinatal pinealectomy (Px) was more potent in preventing the development of NA-CEA state than Px performed in the adult age. These data indicate that the masculinized hypothalamus becomes less sensitive to pineal influences during the lifespan. Effect of Px was potentiated by drugs increasing the central serotonergic tone. The results lead to the assumption that pineal hormones are influential on the maturation of serotonergic neurons, which might interfere with the sterilizing property of neonatal androgen treatment during the "critical" period of sexual differentiation.

Ontogeny of galanin-immunoreactive neurons in the central nervous system of the chicken.

1. The ontogenic development and differentiation of the galanin-immunoreactive (GA-IR) neuron systems in the avian brain was studied by the aid of immunocytochemistry. 2. The first traces of GA-immunoreactivity can be detected already on day 2 of embryonic life within the neural tube, and the system appears to be fully developed around day 16. 3. GA-IR neuron system develops parallel in the hypothalamus and in extrahypothalamic sites. 4. The first well-defined groups of GA-IR perikarya are visible within the diencephalon and myelencephalon as early as day 6. 5. In the embryonic hypothalamus, the periventricular and the tuberal area are especially rich in GA-IR neurons, while in extrahypothalamic regions, the mesencephalon and lower brain stem is prominent in this respect. 6. Since similarities have been observed in developmental patterns of the GA-IR system and the releasing-hormonal systems, it might be assumed that the galaninergic system might have functional relationship with these latter neuronal structures during development.

Long-term effect of perinatal neurochemical lesions of brain monoaminergic neurons on body growth, growth hormone secretion and thyroid activity.

In the present experiments, extended studies were performed on long-term effect of perinatal neurotoxic damage of brain monoaminergic neurons exerted on Grown Hormone (GH) and on Thyrotrophic Hormone (TSH) secretion. Neurotoxins were applied for selective destruction of the different components of the monoaminergic neuron system. Deficient GH secretion and reduced TSH-mobilizing capacity were observed in consequence of perinatal injury of dopaminergic neurons, meanwhile in perinatal serotonergic lesion bearing rats, reduced GH secretion was associated with increased reactivity of the TSH-mobilizing mechanism. The results show that perinatal damage of monoaminergic neurons induce long-lasting alteration of the neural mechanisms regulating GH and TSH secretion.

Differences in hormone release patterns from the anterior pituitary and the pineal gland.

Patterns of hormone release from peptide- and monoamine-hormone secreting cells were compared. LH, GH and PRL secretion from rat anterior pituitary cells and melatonin secretion from the rat pineal gland fragments were studied in a dynamic, in vitro system. The following fundamental differences were found: 1. Adenohypophysial cells respond to a specific stimulus (releasing hormones) with hormone secretion within seconds. There is a 90-minute lag, however, between the beginning of specific stimulation (norepinephrine, 1 microM for 30 min) and the onset of melatonin (MT) release from the pineal cells. 2. Hormone secretion from the pituitary cells returns to the basal value five to 10 minutes after the stimulus has been stopped. Once initiated, MT release from the pineal lasts for five to seven hours even if the NE stimulus was stopped one hour before the beginning of the MT response. 3. A transitory increase in potassium concentration (by 50-100 mM) induces a sharp, distinct rise of hormone secretion from pituitary cells, similar in shape and size experienced as response to releasing hormone stimulation. Pineal cells do not respond to elevation of potassium concentration at all. 4. High concentration of tropic hormones can be extracted from pituitary cells at the end of the superfusion experiment. Their hormone content is equivalent to the amount released from non-stimulated cells in a 50 to 80 hours period (LH and GH cells). No significant quantity of MT can be extracted from non-stimulated, disintegrated pineal cells. This diversity in the control of hormone secretion from anterior pituitary and the pineal gland can be considered as a model for peptide and monoamine-hormone producing glands.

Galanin-like immunoreactivity in the chicken brain.

The anatomical distribution of neurons containing galanin has been studied in the central nervous system of the chicken by means of immunocytochemistry using antisera against rat galanin. Major populations of immunostained perikarya were detected in several brain areas. The majority of galanin-immunoreactive cell bodies was present in the hypothalamus and in the caudal brainstem. Extensive groups of labeled perikarya were found in the paraventricular, periventricular, dorsomedial and tuberal hypothalamic nuclei, and in the nucleus of the solitary tract in the medulla oblongata. In the telencephalon, immunoreactive perikarya were observed in the preoptic area, in the lateral septal nucleus and in the hippocampus. The mesencephalon contained only a few galanin-positive perikarya located in the interpeduncular nucleus. Immunoreactive nerve fibers of varying density were detected in all subdivisions of the brain. Dense accumulations of galanin-positive fibers were seen in the preoptic area, periventricular region of the diencephalon, the ventral hypothalamus, the median eminence, the central gray of the brainstem, and the dorsomedial caudal medulla. The distributional pattern of galanin-immunoreactive neurons suggests a possible involvement of a galanin-like peptide in several neuroregulatory mechanisms.

Long-Term Dynamic in vitro System for Investigating Rat Pineal Melatonin Secretion.

Abstract Several details regarding the control of melatonin (MT) secretion from the pinealocytes are still to be clarified. To obtain more data on the mechanism and kinetics of MT secretion and on the interactions between bioactive materials affecting MT production, a perifusion system has been developed in our laboratory. In this dynamic in vitro system the surviving pinealocytes maintain their full responsiveness for at least 5 days. In order to determine the MT contents of large numbers of samples collected in the perifusion system, a sensitive MT radioimmunoassay was set up utilizing our specific MT antibody. In our perifusion system the basal MT release does not change significantly during the 5-day experiments. Norepinephrine (NE) was used at 1 muM concentration for 30 min as a marker of the responsiveness of pinealocytes, given at the beginning and at the end of the same experiments. No significant difference was found in the MT responses to NE stimulation over 5 days. The kinetics of MT response and the dose-response relationship were investigated after NE exposure at various concentrations (100 nM to 10 mM). NE at 100 nM was found to be ineffective. Between 1 muM and 1 mM concentrations NE increased the MT release in a dose-dependent manner. No significant difference was found between the responses above 1 mM concentration. NE seems to be a specific stimulator of pineal MT production. The MT production reached the maximum value after a relatively long lag (2 to 3 h) when NE application had been stopped, and returned to basal values after 5 to 6 h. This prolonged time-course of MT secretion, in contrast with the fast responses of pituitary cells to releasing hormones, suggests that NE stimulated the synthesis of MT rather than the release of stored hormone. The modulatory effects of light-dark cycle on basal and stimulated MT release of perifused pineals was also investigated: Neither basal nor NE-stimulated (100 nM to 10muM) MT release was influenced significantly by light-dark conditions showing that the light-dark cycle does not have a direct modulatory effect on MT secretion under in vitro circumstances. Based on our observations, this perifusion system should be a useful tool for investigating: 1) hormone interactions on the regulation of pineal MT release, and 2) accurate kinetics of MT response.

Localization of different releasing hormone immunoreactive (LH-IR) neurons and their projections in central nervous system of birds. A mini-review.

The aim of the present review is to summarize the question of the co-existence of two or more releasing hormones within a neuron or nucleus of the avian central nervous system (CNS). Furthermore, we attempt to differentiate between the character and functional significance of hypothalamic and of extrahypothalamic releasing hormone containing neurons. In order to approach these important questions, we have to summarize the localization and distribution of the different releasing hormone immunoreactive (RH-IR) structures in the avian brain, compared to the much more thoroughly investigated mammalian releasing hormone system. This mini-review comprises data obtained by immunohistochemical approach, exclusively. Other data, based on radioimmunoassay or other morphological methods, will be omitted here.

Impaired thyroid function provoked by neonatal treatment with drugs affecting the maturation of monoaminergic and opioidergic neurons.

The aim of the present work was to study the basal secretion rate and the reactivity of the TSH-thyroid axis in adult rats neonatally exposed to drugs influencing monoaminergic and opioidergic neurons. The early postnatal administration of drugs antagonistic with the dopaminergic or serotoninergic neurons resulted in a persistent higher rate of basal secretion of TSH, while the administration of drugs synergistic with the monoaminergic neuron systems was weakly influential in this respect. The exposure to opioids in the perinatal period resulted in a permanent reduction of serum TSH levels which was even more pronounced when the exposure to morphine was advanced to the fetal period of life. These data raise the possibility that the permanent TSH depressing effect of perinatal administration of opioids is due to their effect exerted on the maturation of the monoaminergic neurons. From the other hand, our results lead to assume that there is a perinatal critical period in the maturation of monoaminergic neurons regulating TSH secretion in the adult age. In accordance with this assumption, the data obtained in rats bearing perinatal neurotoxic destruction of catecholaminergic neurons contribute to the concept that the disturbed maturation of monoaminergic neurons in the supposed critical period of development might lead to permanent deficiency also in the reactivity of the TSH-thyroid axis.

Studies and reevaluations of some aspects on thyroid function after superior cervical sympathetic gangliectomy in rats.

The results of 11 experiments in a total of 571 rats (initial body weight of 150-250 g) are reported and some findings differing from those by others are discussed. It was repeatedly found that the animals after bilateral or even unilateral superior cervical sympathetic gangliectomy (GX) did not gain body weight during the first week after surgery. Though they started to grow later, for several weeks their body weight remained significantly less than that of sham operated controls (SH). Though such phenomenon has not yet been described, it may well explain the increase of thyroid weight (as expressed per body weight) after gangliectomy alone or combined with antithyroid drug treatment or hypophysectomy as described by others. It was suggested that such changes may depend on general metabolic changes resulting in a striking inhibition of body weight gain rather than on some specific effect of GX on the thyroid. This view was supported by evaluating the data on absolute and relative thyroid weight from 4 experiments in a total of 265 animals. The level of thyroxine (T4) and thyrotropic hormone (TSG) was repeatedly found to be significantly decreased after GX for until about 72 h and 24 h after surgery, respectively, which was in agreement with the data reported by others. However, the onset of such decrease was repeatedly found to appear at 6 or 8 h after surgery (in one experiment even at 3 h after surgery) which is also contrasting to the onset of T4 decrease at 14 h after surgery as found by others who suggested a correlation of such thyroid depression with a depletion of noradrenaline from the thyroid and may be even from median eminence. In these experiments, however, a decrease of T4 level was found several hours before the depletion of noradrenaline from the thyroid which appeared at 12 h after surgery and remained at similar level until 40 days, while no remarkable changes of that were found in SH animals (with the excretion of slight increase after 24 h). Between about 4 and 40 days after surgery no significant changes in T4 and TSH levels after GX were found as compared with SH animals is in agreement with others.4+n one experiment the increase of T4 at 2 h after TRH injection, resulting apparently from the effect of endogenous TSH, was significantly inhibited in GX animals at 8 days after surgery, while in other experiments (at 8 and 40 days after surgery) no difference in T4 level increase was found in GX animals as compared with SH ones. In general, it may be suggested that superior cervical sympathetic gangliectomy may result in some temporary and perhaps transient changes in pituitary-thyroid function in rats.

Ontogenetic development of thyrotropin-releasing hormone (TRH)-immunoreactive structures in the brain of the mallard embryo.

Developmental changes of thyrotropin-releasing hormone (TRH)-immunoreactive structures in the brain of mallard embryos were studied by means of immunocytochemistry (PAP technique). The primary antibody was generated against synthetic TRH. Immunoreactive neurons were first detected in the hypothalamus of 14-day-old embryos. By day 20, increasing numbers of immunoreactive perikarya were observed in the paraventricular nucleus, anterior preoptic region and supraoptic region. Immunoreactive fiber projections were seen in the median eminence as early as embryonic day 20; they occurred also in some extrahypothalamic regions (lateral septum, accumbens nucleus). The number and staining intensity of the cell bodies increased up to hatching, and continued to increase during the first week after hatching.

Circannual serum creatine kinase patterns after ganglionectomy and pinealectomy of the Wistar rat.

Previous investigations have shown that an inverse correlation exists between serum thyroxine levels (T4) and serum creatine kinase activity (CK) both in hypothyroid and hyperthyroid states. In the present study, serum T4 levels and total serum CK activity were analysed after thyroidectomy (TX), ganglionectomy (extirpation of the superior cervical sympathetic ganglion) (GX), or pinealectomy (PX) four times a year (March, June, September, and December). Blood samples were taken four times daily (at midlight, middark, and at 1 hour after the onset of the light and dark periods). Animals were kept under an artificial light schedule that simulated natural light-dark periods. The annual main values of serum T4 levels were significantly decreased after the operations, whereas serum CK activities were increased. Our circannual investigations demonstrated an inverse correlation between serum T4 levels and total serum CK activity (under hypothyroid circumstances). In addition, the circannual pattern of the serum CK activity demonstrated maxima under short-day (winter) conditions and minima under long-day (summer) conditions. GX and PX provoke an unimportant shift (a little longer than 2 months) in maxima and minima of the annual rhythm. Circannual curves exhibited differences in the degree of the increase of serum CK activity of GX and PX groups. These results indirectly confirm the assumption that experimental sympathetic denervation or removal of the pineal gland will induce a hypothyroid state over the long term.

New data on the immunocytochemical localization of thyrotropin-releasing hormone in the rat central nervous system.

An antiserum raised against the synthetic tripeptide pyroglutamyl-histidyl-proline (free acid) was used to localize thyrotropin-releasing hormone (TRH) in the rat central nervous system (CNS) by immunocytochemistry. The distribution of TRH-immunoreactive structures was similar to that reported earlier; i.e., most of the TRH-containing perikarya were located in the parvicellular part of the hypothalamic paraventricular nucleus, the suprachiasmatic portion of the preoptic nucleus, the dorsomedial nucleus, the lateral basal hypothalamus, and the raphe nuclei. Several new locations for TRH-immunoreactive neurons were also observed, including the glomerular layer of the olfactory bulb, the anterior olfactory nuclei, the diagonal band of Broca, the septal nuclei, the sexually dimorphic nucleus of the preoptic area, the reticular thalamic nucleus, the lateral reticular nucleus of the medulla oblongata, and the central gray matter of the mesencephalon. Immunoreactive fibers were seen in the median eminence, the organum vasculosum of the lamina terminalis, the lateral septal nucleus, the medial habenula, the dorsal and ventral parabrachial nuclei, the nucleus of the solitary tract, around the motor nuclei of the cranial nerves, the dorsal vagal complex, and in the reticular formation of the brainstem. In the spinal cord, no immunoreactive perikarya were observed. Immunoreactive processes were present in the lateral funiculus of the white matter and in laminae V-X in the gray matter. Dense terminal-like structures were seen around spinal motor neurons. The distribution of TRH-immunoreactive structures in the CNS suggests that TRH functions both as a neuroendocrine regulator in the hypothalamus and as a neurotransmitter or neuromodulator throughout the CNS.

Thyrotropin-releasing hormone (TRH)-immunoreactive structures in the brain of the domestic mallard.

The distribution of immunoreactive thyrotropin-releasing hormone (TRH) in the central nervous system of the domestic mallard was studied by means of the peroxidase-antiperoxidase technique. After colchicine pretreatment, the highest number of TRH-immunoreactive perikarya was found in the parvocellular subdivision of the paraventricular nucleus and in the preoptic region; a smaller number of immunostained perikarya was observed in the lateral hypothalamic area and in the posterior medical hypothalamic nucleus. TRH-immunoreactive nerve fibers were detected throughout the hypothalamus, forming a dense network in the periventricular area, paraventricular nucleus, preoptic-suprachiasmatic region, and baso-lateral hypothalamic area. TRH-containing nerve fibers and terminals occurred in the organon vasculosum of the lamina terminalis and in the external zone of the median eminence in juxtaposition with hypophyseal protal vessels. Scattered fibers were also seen in the internal zone of the median eminence and in the rostral portion of the neural lobe. Numerous TRH-immunoreactive fibers were detected in extrahypothalamic brain regions: the highest number of immunoreactive nerve fibers was found in the lateral septum, nucleus accumbens, olfactory tubercle, and parolfactory lobe. Moderate numbers of fibers were located in the basal forebrain, dorsomedial thalamic nuclei, hippocampus, interpeduncular nucleus, and the central gray of the mesencephalon. The present findings suggest that TRH may be involved in hypophysiotropic regulatory mechanisms and, in addition, may also act as neuromodulator or neurotransmitter in other regions of the avian brain.

Changes in the daily rhythm of serum testosterone levels following superior cervical sympathetic ganglionectomy in the cold-exposed rat: the role of the pineal.

The effect superior cervical sympathetic ganglionectomy (Gx) exerted on the daily rhythm of serum testosterone levels was investigated in cold-exposed rats. Rhythmic changes in pineal and pituitary weights were also measured. 1. Exposure to cold (10 degrees C for 72 h) resulted in a significant decrease of serum testosterone level and in an increase of the pineal weight. 2. In neutral ambient temperature (24 degrees C) Gx, 30 d after operation, led to a moderate, statistically insignificant increase of serum testosterone levels and to decreased pineal weights (statistically significant). 3. The reactions provoked by cold exposure were counteracted by Gx. Testosterone levels, as well as the pineal weight, showed no remarkable change in the Gx, cold-exposed animals. 4. These results confirm our assumption that experimental manipulations of the pineal gland can provoke significant changes in the neuroendocrine system only under special loading circumstances, e.g., cold exposure. Sympathetic denervation of the pineal gland counteracts the cold-induced decrease of testosterone levels by counteracting the pineal antigonadotropic activity. 5. The empirical regression curves of the investigated parameters indicate that Gx or cold exposure provide a shift in the upper and lower limits of the daily rhythm. Partly inverted rhythms were also observed. 6. The presented results are discussed in relation to the parallel changes previously described in serum thyroxin, cholesterol, thyrotropin (TSH), and pituitary TSH levels. Thyroidal-gonadal interactions, as well as cold exposure as a stress-generating factor, have been considered in the possible explanation of the data herein reported.