Serotonin (5-HT) receptor subtypes mediate specific modes of 5-HT-induced signaling and regulation of neurosecretion in gonadotropin-releasing hormone neurons.

Serotonin (5-HT), the endogenous nonselective 5-HT receptor agonist, activates the inositol 1,4,5-triphosphate/calcium (InsP3/Ca2+) signaling pathway and exerts both stimulatory and inhibitory actions on cAMP production and GnRH release in immortalized GnRH neurons. The high degree of similarity between the signaling and secretory responses elicited by GnRH and 5-HT prompted us to target specific 5-HT receptor subtypes to deconvolute the complex actions of these agonists on signal transduction and GnRH release. Specific mRNA transcripts for 5-HT1A, 5-HT2C, 5-HT4, and 5-HT7 were identified in immortalized GnRH neurons (GT1-7). The rate of firing of spontaneous action potentials (APs) by hypothalamic GnRH neurons and cAMP production and pulsatile GnRH release in GT17 cells were profoundly inhibited during activation of the Gi-coupled 5-HT1A receptor. Treatment with a selective agonist to activate the Gq-coupled 5-HT2C receptor increased the rate of firing of spontaneous APs, stimulated InsP3 production and caused a delayed increase in GnRH release. Selective activation of the Gs-coupled 5-HT4 receptor also increased the rate of firing of APs, stimulated cAMP production, and caused a sustained and robust increase in GnRH release. The ability of 5-HT receptor subtypes expressed in GnRH neurons to activate single or multiple G proteins in a time- and dose-dependent manner differentially regulates the phospholipase C/InsP3/Ca2+, and adenylyl cyclase/cAMP signaling pathways, and thereby regulates the frequency and amplitude of pulsatile GnRH release. This process, in conjunction with the modulation of spontaneous electrical activity of the GnRH neuron, contributes to the control of the pulsatile mode of neuropeptide secretion that is characteristic of GnRH neuronal function in vivo and in vitro.

Neuroendocrine effect of toki-shakuyaku-san on ovulation in rats.

In this study, the experiments were designed to examine if Toki-Shakuyaku-San (TSS) has a neuroendocrine effect on ovulation. TSS (500 mg/kg Bwt in drinking water) was given to Sprague Dawley immature female rats on 25 days of age and during experimental sessions. Fifteen IU of the hMG (Gonadoryl 4; GNR 4) was injected intraperitoneally on the morning of 28 days of age, and ovulation occurred in 70% of rats on the following day. In TSS drinking immature female rats, administration of GNR 4 on 28 days of age results in ovulation twice: once on 29 days of age and again on 31 days of age. Administration of sodium pentobarbital (Nembutal) at 12:30 p.m. on 28 days of age did not block the first ovulation on 29 days of age, but administration at 12:30 p.m. on 30 days of age blocked the second ovulation on 31 days of age. However, delayed ovulation was observed on 32 days of age. This evidence indicates that GNR 4 acts on the ovary directly and induces first ovulation in TSS treated rats. However, TSS treatment modulates the chain of events in the neuroendocrine control of ovulation and results in the second ovulation.

[Qualitative and quantitative changes in the hypothalamus and the ependyma of the 3d cerebral ventricle in sheep after irradiation and hormonal stimulation]. / Kvalitatívne a kvantitatívne zmeny v hypotalame a v ependýme. III. mozgovej komory oviec po oziarení a hormonálnej stimulácii.

In the submitted work we concentrated our attention on the study of the changes in neurosecretion. PAS positive mucopolysaccharides in the hypothalamus and ependyma of the IIIrd cerebral ventricle and on studying the changes in the volume of the cell nuclei of the neurons of nucl. supraopticus (NSO), nucl. hypothalamicus centromedialis (NVM) and nucl. tuberomamillaris (NTM) of sheep after irradiation and after hormonal stimulation. We made our observations on 28 sheep of the Slovak Merino breed, of average live weight 35 to 40 kg, two- and three-years old in the period of physiological anoestrum. The first group of four ewes and the second group of four yearling lambs were controls. The third group of six ewes and the fourth group of six yearlings were exposed to irradiation all over with a dosage of 2.46 Gy (250 R) for a period of five days. To synchronize the oestrum of all the sheep we used agelline sponges which we fitted five days before irradiation. After the irradiation and removal of the sponges we gave the test sheep hormonal stimulation with 3 X 500 i. u. of serum gonadotropin. The fifth group of four ewes and the sixth group of four yearlings were only stimulated without irradiation. The fifth day after stimulation we slaughtered the sheep. We processed the brain samples by the usual histological methods. We carried out karyometric analysis with 3000X magnification and the measurement of 200 cells from one sample. We processed the values obtained mathematically according to Fischer and Inke (1956). We evaluated the quantity of neurosecretion material with a light microscope according to Nakahara (1962). We found that the multiplication of neurosecretion and the increased activity of the PAS reaction in the hypothalamic nuclei studied show that the irradiation and hormonal treatment stimulate the function of the hypothalamic structures. The results of the karyometric analysis in the yearlings also confirm this opinion. In the ewes the inhibitive influence of irradiation was probably manifest. The giving of hormones in combination with irradiation causes the multiplication of ependyma cells and the desquamation of surface layers.

Effect of thiourea on the hypothalamic nuclei of the mud eel Amphipnous cuchia (ham.).

Immersion of fish in thiourea solution (0.33 X 10(-6) ppm) caused atrophic changes in the NPO neurones only but no change in the cell bodies of the NLT was recorded.

Central nervous system action of bombesin to inhibit gastric acid secretion.

Bombesin or gastrin releasing peptide injected into the lateral, third, or fourth ventricle, or into the cisterna magna, inhibited gastric acid secretion induced by a wide variety of gastric acid stimulants in several animal models. Studies of bombesin microinfusion into specific hypothalamic nuclei of intact rats, or injection into the cisterna magna of midbrain transected rats, indicated that the peptide can trigger inhibition of gastric acid secretion from both forebrain and hindbrain structures. The neural pathways mediating bombesin action required the integrity of the cervical spinal cord; the vagus did not play an important role. Spantide, a substance P and bombesin receptor antagonist, was not useful in studying the physiological role of bombesin. This was due both to its inability to reverse the central action of bombesin on gastric secretion, and to its in vivo toxicity.

Neurosecretory and microstructural changes in the hypothalamus of rats following administration of lithium chloride and 3H-thymidin.

Adult male rats were intraperitoneally administered aqueous solution of lithium chloride (LiCl). Studies, including neurosecretory and microstructural changes within particular neurocytes in supraoptic (NSO) and paraventricular nuclei (NPV) were performed on hypothalamic sections. In the experimental rats the administered LiCl increased the level of GOMORI-positive neurosecretory material both in supraoptic and paraventricular nuclei. Great amounts of the neurosecretory material were markedly conspicuous in the above areas after 20 days of LiCl administration. Investigations carried out on cellular nuclei of particular neurocytes showed a significant enlargement of the nuclei, and statistical calculations revealed that, in comparison with the basic control, the difference was essentially significant (p less than 0.001). 3H-thymidin administration to the rats which had previously been on LiCl for 20 days demonstrated also that within supraoptic nuclei the incorporation of the isotope in cellular nuclei took a faster course than in control animals.

Physiological dependency between the hypothalamus and the thymus in Wistar rats. II. The neurosecretion level and microstructural changes following para-chlorophenylalanine administration. The differences in DL-5-hydroxy G-3H tryptophane incorporation in the hypothalamus of normal and thymectomized rats.

The experiments were carried out on male Wistar rats, both normal and thymectomized in which the thymus was excised in 12th h of life. 8 weeks later, the animals were administered parachlorophenylalanine [PCPA], i.e. a compound inhibiting tryptophane hydroxylase, in the amount of 200 mg/kg of body weight. The studied hypothalamus preparations, i.e. supraoptical [NSO] and paraventricular nuclei [NPV] revealed an increased level of Gomori-positive neurosecretory material in thymectomized rats when compared to control animals. It was observed that the former rodents manifested an elevated incorporation of the isotope, i.e. DL-5-Hydroxy [G-3H] Tryptophane. The calculations performed by the use of the Gosset's t-test ("Student's") showed that the difference was statistically significant (p less than 0.001).

[Reaction of the Gomori-positive hypothalamo-hypophyseal neurosecretory system of the rat to a single dose of thyroliberin]. / Reaktsiia Gomoripolozhitel'noi gipotalamo-gipofizarnoi neirosekretornoi sistemy krysy na odnokratnoe vvedenie tiroliberina.

A decrease in the functional neurosecretory cell activity of the rat hypothalamic supraoptical and paraventricular nuclei was seen 30 minutes after a single intramuscular thyroliberin injection (8 micrograms). The size of the neurosecretory cell nucleoli significantly diminished in both centers, while a large amount of intranuclear nucleoli-like bodies appeared in the paraventricular nucleus. The neurosecretory substance content in the posterior hypophysis significantly decreased, indicating an active neurohormone excretion. A possibility of negative feedback between the body thyroliberin level and the state of the Gomori-positive hypothalamic neurosecretory centers is discussed.

Gonadal steroids: humoral modulators of nerve-cell function.

The gonadal steroids, estradiol, testosterone and progesterone, alter neuroendocrine function and behavior in adult mammals and do so by acting on the central nervous system and pituitary gland. Besides intracellular receptor sites, various cellular and chemical actions of gonadal steroids are being studied within the brain and pituitary. The review summarizes the present state of such research and points as well to new advances in our understanding of the cellular basis of developmental events such as brain sexual differentiation and puberty, which are influenced by gonadal steroids.

Responses of paraventricular and supraoptic units to angiotensin II, sar1-ile8-angiotensin II and hypertonic NaCl administered into the cerebral ventricle.

Extracellular recordings of action potentials were made from neurones antidromically identified as neurosecretory cells in the paraventricular and supraoptic nuclei of urethane-anesthetized female rats. Eighty-six neurones were examined for their responsiveness to 10 ng of angiotensin II (AII) injected into the third cerebral ventricle and 78 (91%) of them increased their firing rate following the AII injection. None of the neurosecretory cells tested showed a response to the intraventricular (IVT) injection of isotonic NaCl. Thalamic neurones and non-neurosecretory hypothalamic neurones did not respond to the AII given IVT. Firing activity of 13 neurosecretory neurones was recorded during reflex milk ejection induced by suckling pups in the lactating rats. Seven of them were classified as oxytocinergic cells because they showed a burst of activity before reflex milk ejections and the remaining 6 neurones which gave no burst of firing before milk ejections were classified as nonoxytocinergic neurones. The IVT application of AII resulted in activation of all the oxytocinergic neurones and 5 of the 6 non-oxytocinergic neurones. The effect of AII on the firing of the neurosecretory cell was inhibited by the simultaneous application of Sar1-Ile8-AII (1 microgram), a competitive AII antagonist. The IVT injection of the antagonist alone inhibited the spontaneous firing of the neurosecretory cells, but it did not affect the firing of thalamic or non-neurosecretory hypothalamic neurones. Hypertonic NaCl (0.85 M NaCl, 1 mu1 IVT) also activated 13 of 20 neurosecretory cells tested. Combined application of AII and hypertonic NaCl elicited a marked potentiation of the response of neurosecretory cells to each of the stimuli. These findings indicate that AII activates neurosecretory cells stimulating specific AII receptors in the brain and that AII has a synergistic action with hypertonic NaCl. Inhibition of spontaneous activity of neurosecretory cells by a competitive AII antagonist suggests that endogenous AII may participate in the maintenance of basal activity of neurosecretory cells.

[Histochemical changes in the neurosecretory hypothalamic nuclei as result of an intoxication with mercury compounds (author's transl)]. / Histochemische Veränderungen in den neurosekretorischen Hypothalamuskernen als Folge einer Intoxikation mit Quecksilberverbindungen.

The effect of experimental poisoning by organic compounds, frequently used in agriculture as fungicides, on the histochemical and histoenzymic pattern of the hypothalamic neurosecretory system has been studied. The experimental rats were fed by means of a gastric tube with the following compounds: Phenylmercuryacetate, 0.1 g daily, for 10 days; Aethylmercury-p-toluenesulphanilide, 0.2 g daily, for 10 days, and Methoxyethylmercurychloride (Ceresan), 0.1 g daily, for 6 days. The histochemical and histoenzymic investigations have shown that ingestion of Phenylmercuryacetate brought about an increase in the release of antidiuretic hormone (ADH) with a concomitant enhancement of production of neurosecretory substances. The peroral administration of Aethylmercury-p-toluenesulphanilide and of Methoxyethylmercury chloride instead, resulted in the accumulation of the neurosecretion within the hypothalamic-hypophyseal system with a parallel inhibition of ADH release. The experimental poisoning of Ceresan had also a stimulatory effect on the activity of many enzymes in the neurosecretory nuclei of the rat hypothalamus. Morphological changes resulting from the experimental intoxication were only rarely observed in the neurosecretory cells of the investigated hypothalamic nuclei.

Release of immunoreactive alpha-MSH by synaptosome-enriched fractions of homogenates of hypothalami.

Immunoreactive alpha-melanocyte-stimulating hormone (alpha-MSH) was found to be concentrated in a synaptosome-enriched fraction prepared by differential centrifugation of rat hypothalamic homogenates. The release of the hormone from this preparation was investigated. After incubation, the synaptosomes were isolated by ultrafiltration and alpha-MSH in the ultrafiltrate was determined by radioimmunoassay. Particle-bound alpha-MSH, recovered by extraction with acid ethanol, and alpha-MSH released from the synaptosome preparation, were immunologically similar to synthetic alpha-MSH and had an accompanying melanotropic activity. Less than 10% of the particle-bound alpha-MSH was released during incubation in 0.32 M sucrose. However, in the presence of 2 mM Ca2+, alpha-MSH release increased with increasing concentrations (30-150 mM) of K+. The stimulatory effect of 60 mM K+ was complete within 2 min and was potentiated by increasing Ca2+ concentrations over the range of 0 to 2 mM. K+-induced release of alpha-MSH was independent of temperature from 1 to 30 degrees C, and neither glucose (10 mM) nor dopamine (10(-10)-10(-2) M) had any effect on the release of the peptide. It is concluded that a synaptosome-enriched fraction from the hypothalamus contains a releasable pool of immunoreactive alpha-MSH that is mobilized by depolarizing concentrations of K+ in a Ca2+-dependent manner.

Identification of MSH release-inhibiting elements in the neurointermediate lobe of the rat.

Neurointermediate lobes of rats comprise elements which, when excited in vitro, bring about an inhibition of the release of melanocyte stimulating hormone (MSH). Superfusion of neurointermediate lobes of intact donor rats with medium containing 45 mM K+ induced a stimulation of the release of oxytocin, arginine-vasopressin and dopamine (DA) and inhibited the release of MSH. Fluorescence histochemical observations and the results of release studies indicate that electrothermic lesions in the mediobasal hypothalamus induced a more rapid degeneration of dopaminergic than of peptidergic terminals in the neurointermediate lobe. Dopaminergic nerve terminals and the stimulated release of DA had vanished completely on the second day after these lesions, which coincided with the disappearance of K+-induced inhibition of MSH release. Frontal hypothalamic deafferentations resulted in disappearance of peptidergic nerve terminals as evidenced by the development of diabetes insipidus and the strong decline of depolarization-induced release of oxytocin and vasopressin from neurointermediate lobes in vitro. In contrast, the dopaminergic plexus was left intact, as was the K+-induced stimulation of DA release and inhibition of MSH release. We conclude that the K+-induced inhibition of MSH release is mediated by DA rather than by neuropeptides from terminals in the neurointermediate lobe. The results are in agreement with the proposed MSH release-inhibiting role of the dopaminergic tuberoinfundibular neurones.

Neural control of reproduction.

The feedback loops involved in the neural control of reproductive cyclicity in primates are reviewed. A combination of morphological, endocrinological, and surgical experimental approaches was used to identify the different mechanisms involved. The main signal for pituitary secretion of gonadotropins is the estrogen secreted by the ovary. Estrogen probably affects the hypothalamic secretion of gonadotropin-releasing hormone (GnRH), which in turn stimulates the pituitary production of gonadotropins. These may in turn, through a short feedback loop, regulate the secretion of GnRH. Evidence for a direct effect of estrogen on the pituitary is presented. Future possible areas of research, including extrahypothalamic influences on reproductive mechanisms, are briefly outlined.

Changes in the distribution of non-neuronal elements in rat median eminence and in anterior pituitary hormone secretion after activation of tuberoinfundibular dopamine neurones by brain stimulation or nicotine.

In order to investigate the posssibility of acute functional changes in non-neuronal elements (mainly tanycytes) of the median eminence, the proportion of portal capillary surface covered by such elements was measured by quantitative electron microscopy in ovariectomized, oestrogen-progesterone-pretreated rats. In some of these animals, the functional state of the tuberoinfundibular dopamine (DA) neurones was assessed by histochemical microfluorimetry. Serum concentrations of luteinizing hormone (LH), growth hormone (GH) and prolactin were determined by radioimmunoassay. Two different types of treatment, i.e. systemic administration of nicotine (1 mg/kg, s.c.) or electrical stimulation in the medial amygdaloid nucleus, markedly reduced the percentage of capillary surface covered by non-neuronal profiles within 20 and 15 min, respectively. At the same time, the tuberoinfundibular DA system responded by an increase in cellular fluorescence intensity, reflecting neuronal activation. Medial preoptic stimulation had basically the same effect but with more variability in the change in capillary coverage by tanycytes. The action of nicotine was prevented by pretreatment with the DA receptor blocking agent, pimozide (5 mg/kg), which indicates (1) that a dopaminergic mechanism was involved in the nicotine effect and (2) that the tanycyte response was elicited by DA released from nerve terminals acting at some receptor site. Nicotine also lowered serum levels of GH and prolactin. Pimozide antagonized only the effect on prolactin. While the reaction of DA neurones and capillary coverage by tanycytes were correlated with each other in individual rats, no statistically significant correlation was observed between tanycyte response and hormone levels, so that no conclusions can as yet be drawn as to the neuroendocrine significance of the tanycyte reaction. These results indicate that rapid changes in the proportion or portal capillary surface covered by non-neuronal profiles can be elicited by stimulation of extrahypothalamic brain areas or by activation of cholinergic mechanisms. The tanycyte response appears to be mediated at least in part by the tuberoinfundibular DA neurones.