Immuno-cytochemical demonstration,in the external region of the amphibian median eminence,of separate vasotocinergic and mesotocinergic nerve fibres

Summary By means of single and double immuno-enzyme cytochemical staining techniques, it was shown that the external region of the amphibian median eminence contains separate vasotocinergic and mesotocinergic nerve fibres. Moreover, it was demonstrated that the vasotocinergic fibres also contain neurophysin. In animals in which the hypothalamic magnocellular neurosecretory preoptic nuclei had been completely removed, the immuno-reactive vasotocinergic and mesotocinergic fibres of the median eminence had disappeared. From this result, it is concluded that, at least the great majority of the vasotocinergic and mesotocinergic fibres of the external region of the amphibian median eminence are processes of neurosecretory perikarya located in the hypothalamic magnocellular preoptic nuclei. On the other hand, our results do not exclude the possibility that a minority of these neurosecretory fibres originate from small immuno-reactive perikarya which were found in the tuber cinereum. The observation that both kinds of processes accumulate around blood capillaries of the hypophysial portal system strongly suggests that they play a role in the control of the activity of the pars distalis of the hypophysis.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Identification,in the external region of the rat median eminence,of separate neurophysin-vasopressin and neurophysin-oxytocin containing nerve fibres

Summary Immuno-enzyme cytochemical investigations, using single and double staining techniques, showed that the external region of the rat median eminence contains separate neurophysin-vasopressin fibres and neurophysinoxytocin fibres. These neurophysin-hormone containing nerve fibres are influenced by bilateral adrenalectomy and by colchicine treatment. The external region of the median eminence of the homozygous Brattleboro rat contains neurophysin-oxytocin fibres. It does not contain immuno-reactive neurophysin-vasopressin fibres. Bilateral adrenalectomy also influences the neurophysin-vasopressin containing neurons of the suprachiasmatic nuclei. In the neurons of the parvicellular part of the rat hypothalamic paraventricular nuclei, staining for vasopressin and for oxytocin is completely absent.     

Identification of separate vasopressin-neurophysin II and oxytocin-neurophysin I containing nerve fibres in the external region of the bovine median eminence

Summary Immuno-enzyme histochemical investigations showed the presence, in the external region of the bovine median eminence, of accumulations of vasopressin-neurophysin II-and oxytocin-neurophysin I-complexes. These two hormone-neurophysin complexes are located in separate fine varicose nerve fibres. The results strongly plead against an important role of tanycytes in the transport of vasopressin, oxytocin and neurophysins from the cerebrospinal fluid to the hypophysial portal blood-vessels.This work was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek.     

The origin of somatostatin fibers in the median eminence and neural lobe of Rana temporaria

Summary A light microscopic immunocytochemical study of the brain of frogs with hypothalamic lesions was performed in order to obtain evidence concerning the origin of somatostatin fibers in the median eminence and neural lobe of the hypophysis. The results indicate that the somatostatin fibers of the neural lobe originate from somatostatin perikarya located in the prechiasmatic part of the hypothalamus and possibly also in the telencephalon. The somatostatin neurons of the pars ventralis tuberis cinerei do not send axons to the neural lobe. The frog median eminence contains axon terminals of somatostatin neurons located in the pars ventralis of the tuber cinereum. Many other somatostatin fibers of the frog median eminence originate from somatostatin neurons located outside the tuber cinereum. Most of these neurons probably lie in the preoptic hypothalamic region.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

The functional and structural border of the neurohemal region of the median eminence

Summary In stressed rats the tanycytes of the ventrolateral wall of the third ventricle exhibit by light microscopic immunohistochemistry a positive staining for neurohormones which is distinctly limited to the distal perivascular end of the tanycyte process. Since by electron microscopic immuncytochemistry the tanycyte cytoplasm does not show any reaction product, the light microscopic reaction most likely results from a labeling of the intercellular space in the direct vicinity of the subendothelial cleft. Whether this subendothelial space is permeable to neurohormones was tested by injection of HRP¹. In the region of the arcuate nucleus 30 min after intravenous application, the marker is affixed to the membranes of the perivascular tanycyte processes in the subendothelial cleft of capillaries possessing non-fenestrated endothelia. Occasionally, HRP penetrates for a short distance between the tanycytes. Then the labeling of the intercellular cleft ends abruptly. Here, several parallel ridges of tight junctions between the perivascular distal tanycyte processes are found by the freezeetching technique. Since HRP cannot reach the subendothelial clefts of this region by passing through capillary walls due to the presence of a blood-brain barrier, it is suggested that the marker penetrates from the median eminence this far via the subendothelial extracellular space. It is prevented from spreading further by the tight junctions of the perivascular tanycyte endings. The same way may be taken by the neurohormones. Hence, a border area exists adjacent to the dorsolateral aspect of the neurohemal region of the median eminence where the tanycytes isolate the neuropil from the cerebrospinal fluid not only by their apical tight junctions, but also by basal tight junctions from the subendothelial cleft. This communicates with the perivascular space of the portal vessels.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr. 569/2) and Stiftung VolkswagenwerkDedicated to Professor Dr. R. Ortmann on the occasion of his 65th birthday.The skilful technical assistance of Miss K. Bielenberg, Mrs. A. Hinz and Mrs. H. Prien is thankfully acknowledged     

Immunohistochemical localization of thyrotropin-releasing factor in the rat median eminence

Summary Antibodies raised in rabbits against a synthetic preparation of thyrotropin-releasing factor (TRF) were used in association with the immunoperoxidase histochemical technique to localize TRF in the rat median eminence. Using coronal sections cut through the mid-arcuate region, specific immunoreactive material was observed in the medial and lateral regions of the superficial layer of the external median eminence.This work was financed by a grant from the Medical Research Council of New Zealand     

Electron microscopic immunocytochemical demonstration of separate vasotocinergic and mesotocinergic nerve fibres in the median eminence of the frog hypophysis

Summary With the use of the unlabelled antibody peroxidase-antiperoxidase complex (PAP) technique at the electron microscopic level, it was shown that both the internal and the external regions of the median eminence of the frog contain separate vasotocinergic and mesotocinergic nerve fibres. This observation confirms the results of previous immunocytochemical studies at the light microscopic level. The mean size of the neurohypophysial hormone-containing granules in the external region of the median eminence is significantly smaller than that of the neurohypophysial hormone-containing granules in the internal region of the median eminence. No significant difference could be found between the mean granule size of the vasotocinergic and mesotocinergic fibres of either the internal or the external region of the frog median eminence.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

The tanycyte of the rat median eminence

Summary The present ultrastructural study proves the existence of nerve terminals closely apposed to the plasmalemmata of tanycytes in the rat median eminence. Several of these axo-tanycytic endings display remarkable accumulations of agranular endoplasmic reticulum in the form of pleomorphic vesicles which are closely apposed on either side of the plasma membrane of each cell compartment. Some of these vesicular profiles give the impression of structural continuity across both membrane systems. This phenomenon is discussed in the context of being a potential substratum for communication between both cell compartments.Supported by NIH Grant NS13717 and NSF Grant BNS 78-11820     

Electron microscopic immunocytochemical demonstration of separate neurophysin-vasopressinergic and neurophysin-oxytocinergic nerve fibres in the neural lobe of the rat hypophysis

Summary By means of the unlabeled antibody peroxidase-antiperoxidase (PAP) technique at the electron microscopic level, it was demonstrated that the hormones of the neural lobe of the rat hypophysis are located in separate neurophysin-vasopressinergic and neurophysin-oxytocinergic nerve fibres. These observations confirm the results of our previous immunocytochemical studies at the light microscopic level.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Sizes of granular vesicles in the rat median eminence,with special reference to the zona granulosa

Summary A quantitative evaluation has been made of the sizes of neuronal granular vesicles (GV) in different (sub-)zones of the rat median eminence. On the basis of the frequency distribution of the mean sizes of the GV in the individual nerve profiles, at least 4 tentative categories of nerve profiles may be distinguished. However, available data do not permit characterization of nerve fibre categories in terms of neurohormone and/or neurotransmitter storage. From dorsal to ventral the palisade zone shows a decrease in mean size of GV; furthermore, the relative number of nerve profiles characterized by small GV (mean size smaller than 112 nm) increases. In contrast, the GV in the zona granulosa, that part of the external zone of the median eminence ventral to the palisade zone, are of relatively large size. Qualitative observations indicate that the zona granulosa may be regarded as an area for both storage and release of biologically active substances. We are grateful to Mrs. R.M.Y. Hartsteen for technical assistance, to Miss P.C. Delfos and Mr. W. van den Oudenalder for photographic assistance and to Prof. J. Moll for helpful criticism.     

Relationship of the central ACTH-immunoreactive opiocortin system to the median eminence and the pituitary gland of the rat

Summary By immunocytochemical methods, the present study describes ACTH-immunoreactive fibers in the pituitary stalk and neural lobe. This opiocortin-hypothalamo-neural lobe projection arises in a bed nucleus of perikarya in the basal hypothalamus, follows supraoptico-hypophyseal fibers in the zona interna of the median eminence, and distributes throughout the neural lobe. No ACTH-immunoreactive fibers project to the zona externa; some are present in the subependymal layer and at the lateral margins of the median eminence. Further studies must identify the role of these fibers in posterior lobe function. It remains also to be determined whether this system terminates upon primary pituitary portal capillaries and delivers opiocortin neuropeptides to the adenohypophysis.Supported by NIH Grants HD-07962, NS-15345 and AM-22029The skillful technical assistance of Donna Wilson, Nancy Dembs and Jay Hocton is thankfully acknowledged     

Ultrastructural characterization of exocytotic release sites in different layers of the median eminence of the rat

Summary The release of neuronal secretory products by exocytosis in different layers of the median eminence of the rat was investigated ultrastructurally after perfusion with Ringer solution containing tannic acid. Exocytotic images were observed in all layers studied. Neurohaemal release sites were found in the pars externa of the palisade layer, where they occurred not only against the basal lamina of the pericapillary space, but also opposite, adjacent to neuronal and glial elements. In the lateral portion of the pars externa of the palisade layer most release sites were separated from the pericapillary space or the pial surface by ependymal or glial processes. In the pars interna of the palisade layer, and in the reticular, fibre and subependymal layers, release was observed in different types of axonal processes without morphological synaptic specializations. We suggest that products released in the pars externa of the palisade layer are destined to reach the capillaries of the primary portal plexus. Although the non-vascular release sites may serve a similar hormonal function, they may alternatively represent the morphological correlate of axoaxonal contacts or of paracrine, non-synaptic release sites.     

Histochemically demonstrable monoamines in the pituitary gland and median eminence of the female rat during the postnatal development

Summary The postnatal development of formaldehyde induced fluorescence (FIF) was studied in the pituitary glands of female rats. The effects of 3,4-dihydroxyphenylalanine (L-dopa), D,L-5-hydroxytryptophan (DL-5-HTP) and dopamine (DA) treatments on the FIF were followed during the postnatal period.The appearance of specifically fluorescing monoamines into the cells of the pars intermedia occurred postnatally and the level of the adult fluorescence was reached at 4–5 weeks' age. The intensity of the fluorescence was independent on the density of the fluorescing nerves. Among the fluorescing nerves droplet fibres were regularly observed from the age of 3 weeks, which confirms the theory that these fibres are caused by toxic factors when the blood-brain barrier is not functioning.There was no change postnatally in the number of fluorescing cells in the pars distalis.The fluorescing innervation of the median eminence, developed most rapidly at the age of 1–3 weeks and the level of the adult fluorescence was reached at the age of 4–5 weeks.The first specifically fluorescing cells after L-dopa treatment were observed at 6 days age. A remarkable increase in the number of fluorescing cells was seen between 12 and 18 days. After DL-5-HTP treatment fluorescent cells were seen but at later stages. These observations suggest that the cells in the pituitary gland, which store amine-precursors and monoamines developmentally differ from the APUD-cells. The rapid increase of the fluorescing cells between 12 and 18 days and the simultaneous development of the fluorescing innervation of the median eminence suggest the following correlations: the development of dopaminergic innervation of the median eminence — the secretion of releasing hormones — the activity of PAS-positive cells (FSH, LH and TSH secretion) — the uptake of L-dopa and DL-5-HTP into the PAS-positive cells.Dopamine was not uptaken into the cells of pars distalis. The walls of the blood vessels began to show fluorescence suggesting a barrier mechanism, which prevents the DA-uptake into the PAS-positive cells.This work was supported by the Grant for Young Research Workers, University of Helsinki.     

Correlative ontogenetic development of catecholamine- and LHRH-containing nerve endings in the median eminence of the rat

Summary The ontogenetic development of catecholamine (CA)-and LHRH-containing nerve endings in the median eminence of the rat was investigated by combining fluorescence histochemistry and immunohistochemistry in the same tissue section. LHRH-terminals appeared earlier than CA-terminals and were already detectable in the lateral part of the external layer of the central ME on the first day after birth. CA-nerve endings were first seen in a corresponding region of the ME on the seventh postnatal day. At this stage both types of terminals showed the earliest manifestation of a correlative pattern of their distribution. Subsequently the development of both types of nerve endings proceeded rapidly, and at 14 days their distribution pattern corresponded to that in adult animals. The authors conclude that at this stage the CA-neurons play a constant and significant role in the release of LHRH into the portal capillaries. The correlation between both types of nerve endings and the ontogenetic development of the capillary plexuses of the hypophysial portal system is discussed.This work was supported in part by a grant (No. 248093, 321426) from the Ministry of Education, Science and Culture, Japan     

Cellular organization of the lateral and postinfundibular regions of the median eminence in the rat

Summary The structural organization of the rostral, lateral and postinfundibular regions of the median eminence (ME) of 5-day cyclic diestrous rats was studied with light and electron microscopic methods. The ependymal cells lining (i) the floor of the infundibular recess (IR) at rostral levels, (ii) the lateral extensions of the IR, and (iii) the floor of the premammillary recess appear to represent the same type of tanycyte ependyma (1 tanycytes). In the entire width of the rostral and postinfundibular palisade regions, as well as in the lateral palisade region of the preinfundibular ME, the processes of the 1 tanycytes form a continuous cuff. This cuff separates the nerve endings from the blood vessels and the pars tuberalis. At this level, synaptoid contacts between neurosecretory axons and the ependymal cuff can be observed. The ultrastructural characteristics of the 1 tanycytes are described and their ependymal endings tentatively classified into three types. In the lateral regions of the ME, the Golgi study revealed the presence of two fiber systems: (i) one possessing a latero-medial trajectory and distributed in the subependymal region; (ii) the other formed by a loose longitudinal tract originating from neurons of the arcuate nucleus. Some functional implications of the cellular organization of the rat ME are discussed.Supported by Grants from PLAMIRH (92.171.2.77) and from the Dirección de Investigaciones, Universidad Austral (S-77-28)The authors wish to thank Miss Rosario Andrade, Mrs. Elizabeth Santibáñez and Mr. Armando Bilbao for their assistance     

Effect of salt loading and salt deprivation on the vasopressin and oxytocin content of the median eminence and the neural lobe in adrenalectomized rats

Summary In adrenalectomized rats the influence of salt loading or salt deprivation on the vasopressin and oxytocin content of the median eminence (ME) and the neural lobe (NL) was studied by means of various methods: (1) morphometric and microphotometric analysis of aldehyde fuchsin-stained sections of ME and NL; (2) immunohistochemical demonstration of neurophysin, oxytocin, and vasopressin in the ME and in the NL; (3) radioimmunological measurement of oxytocin and vasopressin in the ME and in the NL. Adrenalectomy in salt-substituted rats raised the vasopressin content of the outer layer of the ME (OLME) but had no influence on the amount of vasopressin in the inner layer of the ME and in the NL. Osmotic stimulation of adrenalectomized rats by hypertonic saline markedly diminished vasopressin and oxytocin in the inner layer of the ME and in the NL but did not, or only slightly reduced vasopressin in the OLME. Withdrawal of salt supplementation in adrenalectomized rats resulted in a decrease of plasma sodium and plasma volume. It did not change the vasopressin or oxytocin content of the inner layer of the ME and of the NL, but it was correlated with a decrease of vasopressin in the OLME. The present findings may suggest that vasopressin in the OLME is involved in salt and/or volume regulation by influencing the hypophysial-adrenal axis.The study was supported by the Deutsche Forschungsgemeinschaft (Bo 392/6-5¹ and SFB 90, Cardiovasculäres System, A5²). The morphometric measurements with the TAS plus were carried out at the Max-Planck-Institut für Psychiatrie, Munich, FRG. We are particularly indebted to Prof. G. W. Kreutzberg and Prof. P. Schubert for their help     

Quantitative radioautographic light and electron microscopic analysis of the localization of monoamines in the median eminence of the rat

Summary Serotonin containing structures in the median eminence of the rat have been studied by quantitative light and electron microscopic radioautography following intraventricular infusion of tritiated 5-hydroxytryptophan. One hour after injection of the tracer the highest density of silver grains was recorded in the ependymal and external zones, especially in the lateral palisade zone. The proportion of labelled neurosecretory terminals was also larger in the lateral palisade zone (29%) as compared with the medial palisade zone (13%), although the mean number of developed silver grains per one terminal was higher in the latter. On the average, 16% of neurosecretory terminals sequestered radiolabelled 5-hydroxytryptophan in the external zone of the rat median eminence. It is suggested that serotonin, like catecholamines, is discharged from neurosecretory terminals localized in the external zone and via the portal circulation affects the function of the anterior pituitary. The sites of origin of serotoninergic structures of the median eminence as well as the possible role of monoamine (catecholamine and indolamine) neurohormones in a dual peptidergic and monoaminergic control of anterior pituitary functions are discussed.This work was presented at the joint session of the Society of Anatomists, Histologists and Embryologists, the Society of Physiologists, and the Society of Neuroendocrinologists dedicated to the memory of Professor Wolfgang Bargmann, held in Leningrad, January 17, 1979     

Intragranular colocalization of arginine vasopressin and methionine-enkephalin-octapeptide in CRF-axons in the rat median eminence

Summary Ultrastructural appearances of axonal terminals containing corticoliberin (CRF) were examined in the rat median eminence prepared by a freeze-drying procedure. Immunolabeling was performed by using 5-, 8-, or 15-nm gold-antibody complexes for CRF, arginine vasopressin (VP) and methionine-enkephalin-octapeptide (Enk-8), singly or in combination. In intact animals, the CRF-containing secretory granules were only slightly labeled with goldanti-VP or -Enk-8. In adrenalectomized rats, granules within single axons appeared to be labeled with all the immunogold complexes. This intragranular colocalization of the three antigens was confirmed by using three neighboring sections of the same axon terminals which were stained separately with each one of the antibodies and visualized with the avidin-biotin-peroxidase complex method. The granules labeled for CRF had decreased 9 days after adrenalectomy but had increased again by day 21, while those labeled for VP steadily increased after adrenalectomy. However, this did not correspond with the appearances of cell bodies in the paraventricular nucleus; the cell bodies labeled for both CRF and VP steadily increased in number and in stainability. By contrast, Enk-8 immunoreactivity in the axonal terminals and cell bodies was not affected by adrenalectomy. These findings suggest that although the three peptides could be released simultaneously from the axonal terminals, VP may play some special role in the expression of CRF activity.     

Effects on gonadal function by lesioning tanycytes in the median eminence of the rat and Japanese quail

Summary The ependymal linings of the median eminence were destroyed by electric cautery or intraventricular injection of picric acid in the rat and Japanese quail. In these animals the ventricular lumen near the median eminence disappeared due to adhesion of lesioned walls on both sides of the third ventricle. Electric lesions of the ependymal layer containing tanycytes did not induce appreciable disturbance in the estrous cycles. Rats in which tanycytes were lesioned by picric acid displayed 4-day estrous cycles after prolonged diestrus (10–22 days). After destruction of tanycytes in the quail, a photostimulated gonadal growth was observed. It is concluded that the tanycyte transport of the ventricular fluid to capillaries of the portal vessels appears unnecessary for maintenance of adenohypophysial gonadotrophic activities.Supported by a grant from the Ministry of Education of Japan and by a grant from the Ford Foundation. The authors wish to express their gratitude to Dr. B.K. Follett for providing chicken LH (IRC2) and to the late Mr. T. Asai for assay of serum LH