Neuropeptides in the median eminence

The presence and possible sources of more than 30 neuropeptides in the median eminence are summarized. The median eminence is the brain area which contains neuropeptides in the highest number and in the highest concentrations in the central nervous system. This area constitutes the final common pathway for signals from the brain to the pituitary. Many peptidergic fibers enter the median eminence and terminate around the pericapillary space and release their neuropeptides into hypophysial portal blood vessels. Other peptidergic fibers traverse the median eminence and terminate in the posterior pituitary. According to their origin, fibers in the median eminence can be classified as intra- or extrahypothalamic fibers. The neuropeptide-containing fibers in the median eminence are mainly intrahypothalamic, they reach the median eminence through either the lateral retrochiasmatic area or the tuberoinfundibular tract. Depending on the site of their action, neuropeptides may be either neurohormones acting on the anterior pituitary cells or neurotransmitters affecting the release of substances from other nerve terminals within the median eminence.     

Regeneration of the supraoptico-hypophyseal and paraventriculo-hypophyseal tracts in the hypophysectomized rat

Summary The effect of hypophysectomy on the nerve fiber pattern in the median eminence and infundibular stem of the rat has been investigated by a slightly modified Bodian technique. Postoperative changes in the distribution of neurosecretory material and connective tissue and changes in vascularity have also been studied.Extensive regeneration of the fibers of the supraoptico-hypophyseal and paraventriculo-hypophyseal tract could be demonstrated. It is most pronounced at the distal extremity of the infundibular stem but occurs also in the rostral part of the infundibular stem and in the median eminence. Regeneration starts in the second postoperative week and is completed about four weeks later. The nervous regeneration observed in the pituitary area after hypophysectomy is more extensive than is usually encountered after lesions elsewhere in the central nervous system.It could be demonstrated moreover that neurosecretory material accumulates at the same sites in which the terminals of the regenerated nerve fibers can be found. Hypophysectomy also causes an increase in capillary density and connective tissue content of the infundibular stem. Accumulations of neurosecretory material are always found in areas showing a high capillary density and a considerable amount of connective tissue.Factors which might be responsible for the extensive nervous regeneration in the pituitary area are discussed as are the factors determining the pattern of outgrowth of the regenerating nerve fibers. Morphological aspects of storage of posterior pituitary hormones are considered in the light of data in the literature and the results of the present work.Partly supported by a U.S. Public Health grant to Dr. E. Scharrer and a travel grant to the author from the Netherlands Organization for pure scientific Research (ZWO).     

Mitotic Figures in the Median Eminence of the Hypothalamus

The median eminence of the hypothalamus is part of the avenue by which neurosecreted hormones from the hypothalamic nuclei reach the pars nervosa (neural lobe) of the pituitary and eventually the bloodstream. Lithium treatment and osmotic stress increases the transport of neurosecretory hormones to the pituitary in the adult rat. Specialized astrocytes termed pituicytes in the pars nervosa of the pituitary participate in the secretory process and also develop considerable mitotic activity. The present work reveals similar mitotic figures in cells within the median eminence following 3 days of lithium treatment. The location and appearance of these mitoses add to the evidence that pituicytes are present in the median eminence. Moreover, mitoses occur within the ependymal (tanycyte) layer of the median eminence. Thus, the present results suggest that the tanycyte layer may contain pituicytes, indicating that the hypothalamus possesses specialized cells for modulating neurosecretion in response to osmotic challenges.     

Studies on hypothalamo-pituitary corticoliberin system. II. Corticotropin-releasing factor (CRF) and neurophysin (NP) immunoreactive neurocytes in the hamster

The aim of the study was to investigate CRF- and neurophysin-immunoreactive neurocytes in hypothalamo-pituitary system of the hamster. CRF-immunoreactive nerve fibers were observed mainly in the outer layer of the median eminence and pituitary stalk and also in the neurohypophysis. On the contrary, neither intermediate lobe nor anterior pituitary contained CRF-immunoassayable substance. The pattern of distribution of neurophysin-immunoreactive fibres was different from CRF-immunoreactive fibres as far as a median eminence, pituitary stalk and neurohypophysis are concerned. Between the tannocytes of the III ventricle and nervous fibres forming the internal layer of the median eminence a CRF- and neurophysin-immunoreactive perikaryons of neurocytes were found. Results of the study suggest regulatory function of CRF-immunoreactive neurons of the hamster hypothalamo-pituitary system in controlling of ACTH secretion. Moreover, the distribution of CRF-immunoreactive substances in hamster hypothalamo-pituitary system shows some peculiarities if compared with other rodents.     

Evidence for hormonal participation in the natriuretic and kaliuretic responses to intraventricular hypertonic saline and norepinephrine.

To examine if hypothalamic or pituitary hormones are involved in the induction of the natriuresis which follows the injection of hypertonic saline or norepinephrine into the third ventricle, lesions were placed in the median eminence and the responses to intraventricular norepinephrine or hypertonic saline were evaluated. Sham lesions in which the electrode was lowered into the brain but stopped short of the hypothalamic region did not interfere with the natriuresis, kaliuresis, and antidiuresis induced by the third ventricular injection of either hypertonic sodium chloride or norepinephrine. Lesions in the median eminence which induced diabetes insipidus as evidenced by an increase in water consumption to approximately four times normal completely abolished the natriuresis and kaliuresis in response to intraventricular hypertonic saline or norepinephrine and diminished the antidiuresis. The observations suggest the possibility that a natriuretic hormone(s) is involved in the induction of central natriuresis.     

Effects of p-chloroamphetamine, a serotonin-depleting drug, on the median eminence and pituitary pars intermedia

p-Chloroamphetamine (PCA), an agent known to cause depletion of levels of brain serotonin in rodents, was administered to rats in three sequential injections (10mg/kg) to study effects on the hypothalamic median eminence and pituitary gland. One week following the initial sequence of injections of PCA, light and electron micrographs revealed degenerate fibers in the outer zone of the median eminence. Lower drug doses or single 10-mg/kg doses did not lead to morphologic changes. Neuronal processes located in the pituitary intermediate lobe appeared normal although there was a significant increase in the numbers of secretory granules contained within intermediate lobe cells drug-treated rats, as compared to controls. Fluorometric analysis of levels of catecholamine and indoleamine showed a decrease in serotonin in median eminence and pons-medulla, but no change in that of the pituitary. Levels of dopamine and norepinephrine remained unchanged after PCA treatment. The data suggest that fibers affected in the median eminence contain serotonin. Processes in the intermediate lobe may be resistant to the serotonin-lowering effects of PCA observed in brain tissue. In addition, PCA may directly affect granule release from pituitary cells, or may alternatively act on hypothalamic regions which affect the release of intermediate lobe cell hormones.     

Cholecystokinin varies in the posterior pituitary and external median eminence of the rat according to factors affecting vasopressin and oxytocin

Using radioimmunoassay (RIA), the content of gastrin-cholecystokinin family peptide immunoreactivity (G-CCK-IR), in the posterointermediate lobe (PIL) of the rat pituitary, has been determined in several experimental conditions. G-CCK-IR levels are significantly higher in males than in females. Salt loading induces a significant decrease of G-CCK-IR in animals of either sex. In males, G-CCK-IR levels are lower than controls 21 days after either castration or daily subcutaneous oestradiol injections. Using immunocytochemistry, G-CCK-IR disappears from the external median eminence 21 days after adrenalectomy. Our results show that, in addition to sex difference, factors affecting the vasopressin and/or oxytocin levels in the posterior pituitary and external median eminence also affect G-CCK-IR in the same regions. Cholecystokinin may therefore be of importance in functions related to these hormones.     

Isolated aplasia of the anterior pituitary as a cause of congenital panhypopituitarism. Case report.

We document a male infant with congenital panhypopituitarism as detected at birth, in whom the adenohypophysis was totally absent by magnetic resonance imaging and all the anterior pituitary hormones were undetectable. His neurohypophysis was, by contrast, identified ectopically at the median eminence and antidiuretic hormone was appropriately secreted.     

Role of the central nervous system neuropeptides in body fluid homeostasis

Several peptides are produced by central nervous system neurons, many of these are involved in the control of body fluid homeostasis. The presence of neuropeptides in the median eminence and circumventricular organs, in the neurosecretory hypothalamic cell groups and in the baroreceptor centres are briefly summarized.     

The hypophyseal pars tuberalis is enriched with distinct phosphotyrosine-containing proteins not detected in other areas of the brain and pituitary

The regulation of cell activity, growth and metabolism by a number of growth factor receptors and proto-oncogene products involves tyrosine kinase activity resulting in autophosphorylation of the receptors and production of phosphorylated tyrosine-containing protein substrates. The identification and precise localization of phosphotyrosine (PY)-containing proteins are first steps in elucidating the functional role of tyrosine kinases in the modulation of the central nervous system and related areas. In the present report, we describe PY-containing proteins in the median eminence and adjacent pars tuberalis of the rat adenohypophysis by immunocytochemistry using light and electron microscopy, and by Western blotting analysis. PY-immunoreactivity was found to be most intense throughout the cytoplasm of a population of epithelial pars tuberalis cells. Polyacrylamide gel electrophoresis and Western blotting of tissue extracts from various brain and pituitary regions demonstrated a general pattern of 4 major bands of PY-proteins, with an additional dense band representing a 44 kDa protein that was highly phosphorylated on tyrosines and that was exclusively found in the pars tuberalis. Additional investigation for the presence of insulin receptors, a tyrosine kinase previously correlated with the distribution of PY-proteins, demonstrated a receptor localization in axons and nerve terminals in the external and internal zone of the median eminence. However, the large amount of different PY-proteins present in the secretory cell population of the pars tuberalis could not be attributed to the insulin receptor. Our findings demonstrate that there is a large amount of cell-specific tyrosine kinase activity in the median eminence and contacting the pars tuberalis; these may play a significant role for transduction of biological signals or metabolic regulation in the neuroendocrine region.This paper is dedicated to Professor Dr. Leonhardt on the occasion of his 75th birthday     

Effect of protein deficiency on some hypothalamic and pituitary hormones in growing male lambs. An immunohistochemical study

Growing male lambs were fed with diets containing 14.0, 10.8 and 7.6% protein for 3 months to determine their effects on the content of hypothalamic LHRH and SRIH and pituitary LH and GH, using immunohistochemical methods. Lowering the concentration of dietary proteins caused marked changes in the immunoreactivity of these hormones. The immunoreactive (IR) content of LHRH stored in the median eminence was enhanced, and the proportion of LH cells and their IR content increased. Opposite effects were observed in the SRIH/GH system; IR SRIH content stored in the median eminence markedly diminished, and, although hyperplasia of GH cells was observed, their IR content was diminished. This study indicates that prolonged restrictions of protein in the diet of growing male sheep affects the immunoreactive content of the investigated hormones. It seems that they suppress LHRH/LH release and augment GH release, possibly via suppression of hypothalamic somatostatin.     

Pituitary adenylate cyclase activating polypeptide (PACAP): discovery and current status of research.

For last 2 years since PACAP was first discovered, many important findings on PACAP have been reported. cDNAs encoding the precursor proteins of PACAP in sheep, human and rat were cloned, and the precursor proteins characterized. PACAP was found in a high concentration in the central nervous system, adrenal medulla and testis. Immunohistochemical study indicated that PACAP containing neural fibers are present throughout the brain, including both internal and external zones of the median eminence. In the hypothalamus many PACAP positive cell bodies were demonstrated in the supraoptic nucleus and the paraventricular nucleus in various species. Four types of high affinity PACAP receptor were demonstrated. PACAP receptors in the central nervous system, pituitary, adrenal medulla and germ cells of the testis are highly specific for PACAP, and not shared with VIP. The PACAP receptor was solubilized and cross-linking of 125I-PACAP27 with the binding protein suggest that the molecular weight of the receptor is around 57,000. Various biological actions of PACAP were reported, but the physiological cellular events linked with PACAP-induced activation of adenylate cyclase remain to be investigated.     

Organization of tyrosine hydroxylase-immunoreactive neurons in the di-and mesencephalon of the American bullfrog (Rana catesbeiana) during metamorphosis

Summary We examined the immunocytochemical distribution of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis, in the di-and mesencephalon of developing bullfrog tadpoles. Special attention was given to catecholaminergic innervation of the median eminence and pituitary. In premetamorphic tadpoles, tyrosine hydroxylase-immunoreactive neurons were visualized in the suprachiasmatic and infundibular hypothalamus, the ventral thalamus, and midbrain tegmentum by Taylor-Kollros stage V. The number of labeled neurons in all these areas increased as metamorphosis progressed. By mid-prometamorphosis, labeled neurons appeared in the preoptic recess organ as well as in the posterior thalamic nucleus. The majority of cells in the preoptic recess organ, as well as occasional neurons in the suprachiasmatic nucleus, exhibited labeled processes which projected through the ependymal lining of the preoptic recess to contact cerebrospinal fluid. The modified CSF-contacting neurons of the nucleus of the periventricular organ were devoid of specific staining. By late prometamorphosis, labeled fibers from the suprachiasmatic nucleus were observed projecting caudally to enter the hypothalamo-hypophysial-tract en route to innervating the median eminence and pituitary. Labeled fibers arising from the dorsal infundibular nucleus projected ventrolaterally to contribute to catecholaminergic innervation of the median eminence and pituitary. Immunoperoxidase staining of tyrosine hydroxylase-immunoreactive fibers and terminal arborizations in the median eminence were restricted to non-ependymal layers, while labeled fibers in the pituitary were observed in the pars intermedia and pars nervosa. Staining of tyrosine hydroxylase-immunoreactive fibers in the median eminence and pituitary was sparse or absent in premetamorphic tadpoles, but became increasingly more intense as metamorphosis progressed.     

Efferent projections from the retrochiasmatic area to the median eminence and to the pars nervosa of the hypophysis with special reference to the A15 dopaminergic cell group in the sheep

Anterograde tracers, viz. Phaseolus vulgaris leucoagglutinin and fluorescein dextran, were used in conjunction with tyrosine hydroxylase immunohisto-chemistry to study the projections of the A15 dopaminergic cell group towards the median eminence and pituitary in sheep. After injection of the tracers in the retrochiasmatic area, which contains the cell group A15, fibres containing anterograde tracer were observed in the internal zone of the median eminence and in the pars nervosa of the pituitary. Numerous tyrosine hydroxylase immunoreactive fibers were present in the external zone of the median eminence and in the pars intermedia and the pars nervosa of the pituitary, with characteristic patterns of organisation in each area. Most tyrosine hydroxylase-immunoreactive fibres containing fluorescein dextran were located in the pars nervosa, whereas only a few were observed in the internal zone of the median eminence. It was concluded that at least part of the dopaminergic innervation of the pars nervosa originated from the A15 group. These results provide morphological evidence for (1) the role of dopaminergic neurons of the A15 cell group in the seasonal control of prolactin secretion via the release of dopamine in the pars nervosa, and (2) putative physiological interactions between dopamine and the secretion of neurohypophysial hormones in sheep.     

Ultrastructural study of the uptake of peroxidase by the rat median eminence

An active role of the ependymal cells (tanycytes) of the median eminence in the transport of hypothalamic hormones has been recently suggested. In order to investigate the fate of material present in the cerebrospinal fluid, a protein tracer, horse-radish peroxidase (HRP) was injected into the left lateral ventricle of rats. Two minutes after the injection, HRP had largely diffused between tanycytes and hypendymal cells. As soon as 5 min after the injection, HRP had completely penetrated all the layers of the median eminence. A few labelled vesicles and lysosomes were occasionally seen in ependymal and glial cells. At longer time intervals (20 min, 1 and 4 hrs), a reaction was observed in the lumen of fenestrated capillaries of the pituitary portal plexus. In many nerve endings of the external zone, vesicles and lysosomes were seen to contain HRP. An interesting observation was the localization of HRP between nerve endings and cells in both the pars nervosa and the pars intermedia of the pituitary gland. No reaction was recorded in the anterior pituitary and the kidney. Seventeen hours after the injection, the extracellular space was free of reaction but a few positive intracellular structure were still found. These results clearly indicate that some material from the third ventricle can rapidly diffuse between cells and axons of the median eminence to reach the fenestrated capillaries of the pituitary portal plexus and the posterior pituitary without involving an active transport by tanycytes.     

Distribution of vitamin D binding protein expressing neurons in the rat hypothalamus

We observed immunostaining for vitamin D binding protein (DBP) in rat hypothalamus. Part of the supraoptic and of the paraventricular neurons showed DBP immunoreactivity, in part colocalized with Arg-vasopressin. DBP was also observed in widespread axonal projections throughout the lateral hypothalamus, the median eminence and the posterior pituitary lobe. A portion of ependymal cells, the choroids plexus epithelium and some of the endocrine cells in the anterior pituitary lobe contained DBP immunoreactivity. In situ hybridization of semithin sections with a synthetic oligonucleotide probe to DBP mRNA resulted in staining of magnocellular hypothalamic neurons, but not of ependymal cells or anterior lobe cells. Our observations indicate an intrinsic expression of DBP in the rat hypothalamus. DBP may be synthesized and transported along with the classical neurohypophyseal hormones. The multiple locations of DBP-expressing neurons indicate multiple functional properties: DBP may be released from in the posterior lobe, it may act as a hypophyseotropic factor and as a central neuroactive substance.     

Neurotensin-induced activation of hypothalamic dopaminergic neurons is accompanied by a decrease in pituitary secretion of prolactin and alpha-melanocyte-stimulating hormone.

The effects of neurotensin on the activity of hypothalamic tuberoinfundibular and periventricular-hypophysial dopaminergic (DA) neurons, and on the secretion of pituitary hormones that are tonically regulated by these neurons (i.e. prolactin and alpha-melanocyte-stimulating hormone [alpha MSH], respectively) were examined in estrogen-primed ovariectomized rats. The activity of tuberoinfundibular and periventricular-hypophysial DA neurons was estimated by measuring concentrations of the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the terminals of these neurons in the median eminence and intermediate lobe of the posterior pituitary, respectively. Intracerebroventricular administration of neurotensin caused a dose- and time-related increase in DOPAC concentrations in both the median eminence and intermediate lobe, and a concurrent decrease in plasma levels of prolactin and alpha MSH. These results suggest that neurotensin-induced inhibition of secretion of prolactin and alpha MSH from the pituitary may be due to the stimulatory action of this neuropeptide on the release of dopamine from tuberoinfundibular and periventricular-hypophysial neurons.     

Superior cervical ganglionectomy effects on median eminence and anterior and mediobasal hypothalamic taurine content in male rats: effects of hyperprolactinemia

The neuroendocrine sequelae of acute or chronic superior cervical ganglionectomy in control or pituitary-grafted rats were studied by analyzing both plasma prolactin, growth hormone (GH) and ACTH levels, and taurine (TAU) content in the hypophysiotropic area of the hypothalamus or the median eminence. As expected, after either acute or chronic ganglionectomy, norepinephrine (NE) content decreased in the brain areas studied, although the values remained higher in hyperprolactinemic rats. TAU content was differentially modified by acute vs. chronic surgeries, thus indicating the possible existence of hypothalamic interactions between TAU and NE to regulate pituitary hormone secretion. Indeed, associated differential changes in plasma prolactin, GH and ACTH levels may be due to the observed TAU changes. As expected, pituitary grafting increased plasma prolactin, GH and ACTH levels, so that the presence of a pituitary graft differentially interferes with the effects of either surgery not only on TAU content but also on the plasma levels of the hormone studied. Globally, ongoing studies confirm the differential effects of acute and chronic superior cervical ganglionectomy on plasma prolactin, GH and ACTH levels, and provide new evidence about its effects on TAU content in the hypophysiotropic area of the hypothalamus and the median eminence that may partially explain the changes observed in the pituitary hormones studied.     

Developmental correlation between hypothalamic somatostatin and hypophysial growth hormone

Summary The objective of the present study was to determine, by means of immunocytochemistry, the age in fetal development at which GH is first detectable in the pituitary gland and somatostatin in the median eminence, and to correlate temporally the development of these two hormones throughout the remainder of pregnancy. Mice were studied at 15–19 days of gestation with the peroxidase-antiperoxidase (PAP) technique of Sternberger. Somatotropes in the pars distalis were initially detected at 16 days of gestation and by 17 days they were a prominent component of the parenchymal cell population of the hypophysis. These cells were ovoid and distributed uniformly throughout the pars distalis; many were located adjacent to sinusoidal capillaries. Their number and staining intensity increased by 19 days. Somatostatin was not consistently observed in the median eminence until 19 days of gestation. Reaction product indicative of the presence of somatostatin in presumptive nerve endings was located on the ventral surface of the median eminence and in the external lamina of the infundibulum in proximity to the superficial portal capillaries. Results of the present investigation support the concept that the potential for neuroendocrine control of GH secretion exists in the mouse by the end of fetal development. Several hypotheses concerning the temporal relationship between the appearance of somatostatin in the hypothalamus and of GH in the anterior pituitary gland are discussed.Supported by a Biomedical Research Support Grant (NIH RR 5417). Appreciation is extended to the National Pituitary Agency, NIAMDD for the following radioiodination-grade hormones: hGH, rPRL, rTSH, rFSH and hCG     

Aminergic regulation of neuroendocrinological functions: theoretical background and some clinical examples

Remarkable progress has been made during recent years in the central regulation of the hypothalamic releasing and inhibiting factors and the respective anterior pituitary hormones. There are two nearly universal inhibitory organizations: short tuberoinfundibular dopamine (TIDA) neurons and somatostatinergic system originating from the periventricular hypothalamus and terminating to the median eminence. It is now known that e.g. dopamine, noradrenaline and acetylcholine enhance while 5-hydroxytryptamine and GABA inhibit somatostatin secretion. These transmitters are also involved in the regulation of all releasing factors and pituitary hormones. Clinical applications have been developed based on the regulation of prolactin and growth hormone. Inhibitory TIDA neurons are undoubtedly the major determinants of prolactin secretion. Hyperprolactinaemia is one of the most common endocrinological side-effects of the drugs antagonizing dopaminergic transmission. Expectedly, dopaminergic drugs (bromocryptine, lergotrile, piribedil, dopamine and levodopa) are quite effective in reducing high prolactin levels regardless of the reason. The secretion of growth hormone is predominantly under dual dopaminergic control: hypothalamic stimulation and pituitary inhibition. The former masters the function of the normal gland, while the peripheral inhibitory component takes over in acromegalic gland. Hence dopaminergic drugs are able to reduce elevated growth hormone levels in 30-50% of the acromegalic patients. In normal man, dopamine agonists increase growth hormone levels. An analogous situation can be seen in Cushing's disease regarding ACTH secretion.