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     

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     

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     

T3 implantation mimics photoperiodically reduced encasement of nerve terminals by glial processes in the median eminence of Japanese quail

Photoperiodically generated triiodothyronin (T₃) in the mediobasal hypothalamus (MBH) has critical roles in the photoperiodic response of the gonads in Japanese quail. In a previous study, we demonstrated seasonal morphological changes in the neuro-glial interaction between gonadotrophin-releasing hormone (GnRH) nerve terminals and glial endfeet in the median eminence (ME). However, a direct relationship between photoperiodically generated T₃ and seasonal neuro-glial plasticity in the ME remained unclear. In the present study, we examined the effect of T₃ implantation into the MBH on the neuro-glial interaction in the ME. T₃ implantation caused testicular growth and reduced encasement of nerve terminals in the external zone of the ME. In contrast, no morphological changes were observed in birds given an excessive dose of T₃, which did not cause testicular growth. These results support the hypothesis that thyroid hormone regulates photoperiodic GnRH secretion via neuro-glial plasticity in the ME. T. Yoshimura was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) and a Grant-in-Aid for Encouragement of Young Scientists from the Ministry of Education, Science, Sports, and Culture, Japan.     

Monoamine fluorescence in the median eminence of the Japanese quail,Coturnix coturnix japonica,following medial basal hypothalamic deafferentation

Summary Monoamine fluorescence was examined in the ventral hypothalamus of the Japanese quail, Coturnix coturnix japonica after medial basal hypothalamic deafferentation. In sham-operated control birds, numerous yellow-green fluorescent fibers were observed in the median eminence and the nucleus tuberis. In the area of the paraventricular organ, a number of fluorescent fibers and cell bodies were observed. In birds with deafferented hypothalami, fluorescence disappeared both in the median eminence and the nucleus tuberis. In the area of the paraventricular organ, which was within the area of deafferentation, fluorescence of neuronal perikarya did not change, but fluorescent fibers decreased markedly in number. Disappearance of monoamine fluorescence in the median eminence and the nucleus tuberis is discussed in relation to the tanycyte absorptive function and gonadal development.Supported by Grants from the Ministry of Education to Professors T. Bando and H. Kobayashi, and a Grant from the Ford Foundation to Prof. H. Kobayashi.     

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     

Fine structure of ependymal cells in the median eminence of the frog and mouse revealed by freeze-etching

Summary Freeze-etched preparations of the ventricular surfaces of ependymal cells clearly reveal the presence of pinocytotic vesicles opening into the third ventricle and large vacuoles formed by broad cell projections. The density of the vesicular openings is approximately 20 per m². The ependymal cells in the median eminence of the frog are adjoined by tight junctions comprised of five to eight interconnected junctional strands, whereas near the median eminence in the mouse only one to two such strands form the tight junction of the ependymal cells. Gap junctions between the adjacent ependymal cells are detected near the median eminence in the mouse but not in the frog.This study was supported in part by a grant from the Japanese Ministry of Education (No. 067670)     

Distribution of LHRH in the rat and mouse brain with special reference to the tanycytes

Summary The distribution of luteinizing hormone-releasing hormone (LHRH) was studied in the rat and mouse brain by means of light and electron microscopic immunohistochemistry using the peroxidase-antiperoxidase method. An immunoreactive product to LHRH antiserum was found near the blood vessels of the vascular organ of the lamina terminalis. In the arcuate nucleus-median eminence region, an immunoreactive material occurred bilaterally in the hypothalamic tissue around the tuberoinfundibular sulci. Electron microscopy revealed that immunoreactive fibers observed light microscopically contain numerous granules 100–130 nm in diameter. No immunoreactive product was located in the tanycytes of the median eminence, the perikarya of hypothalamic neurons, and the parenchyma of several circumventricular organs (subfornical organ, subcommissural organ, pineal organ, area postrema).Supported by grants from the Ministry of Education of Japan and the Ford Foundation     

Differential permeability of pineal capillaries to lanthanum ion in the rat (Rattus norvegicus), gerbil (Meriones unguiculatus) and golden hamster (Mesocricetus auratus)

Summary Capillaries in the pineal gland of the rat (Rattus norvegicus), gerbil (Meriones unguiculatus) and golden hamster (Mesocricetus auratus) were investigated by means of electron-microscopical tracer studies using lanthanum. The tracer was administered together with the fixative solution by perfusion via the left cardiac ventricle. In the rat, endothelial junctions of fenestrated capillaries are permeable to lanthanum. In the gerbil the tracer does not leave the capillaries, which are of the non-fenestrated type throughout the organ. In the golden hamster the two portions of the pineal system have different types of capillaries. While in the superficial pineal fenestrated capillaries permeable to lanthanum predominate, the deep pineal possesses capillaries that are, without exception, devoid of fenestrations and impermeable to the tracer. It is suggested that differences in the structure of the capillaries are related to differences in the extent of a specialized contact of the pineal gland to the third ventricle.The authors dedicate this paper to Professor H. Rollhäuser, Münster, on the occasion of his 65th birthday     

Immunocytochemical demonstration of rod-opsin,S-antigen,and neuron-specific proteins in the human pineal gland

Summary The aim of this study was to examine whether rod-opsin and S-antigen immunoreactions were present in the pineal organ of adult man and how these immunoreactions were correlated with neuronal markers, e.g., synaptophysin, and neurofilaments L, H and M. Three perfusion-fixed epithalamic regions including the pineal organ and five pineal glands obtained at routine autopsy were used. The specimens were taken from female or male patients, 25 to 85 years of age. All immunoreactions were performed using highly specific, well-characterized antibodies. Rod-opsin and S-antigen-immunoreactive pinealocytes occurred in all pineal organs investigated; however, the immunoreaction was restricted to small subpopulations of pinealocytes (rod-opsin immunoreaction: approximately 3%–5%; S-antigen immunoreaction: approximately 5%–10% of the total population). In contrast, immunoreactions for synaptophysin and neurofilaments M and H were present in numerous pinealocytes. Immunoreactivity for neurofilament L was not found. These data suggest that the cellular composition of the human pineal organ is heterogeneous. Moreover, the presence of rod-opsin and S-antigen immunoreactions in the human pineal organ indicates that it may be affected by autoimmune retinal diseases that are provoked by antibodies against these proteins, as is the case in rodents and non-human primates.     

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     

Effects of adrenalectomy and excess hydrocortisone on the incorporation of 35S-labelled cysteine in the hypothalamic-neurohypophyseal neurosecretory system of the rat

Summary The incorporation of ³⁵S-labelled cysteine in the hypothalamic-neurohypophyseal system was studied in normal and adrenalectomized rats and in rats treated with excess hydrocortisone. Labelled cysteine was intraperitoneally administered and grain counts were made of autoradiographs produced from sections of the supraoptic and paraventricular nucleus, median eminence and neurohypophysis of animals killed 45 min., 4 hours and 24 hours after administration of the labelled substance. On the whole, lower incorporation levels of the label were noted in the adrenalectomized rats, compared with the controls. In the rats treated with excess hydrocortisone, the grain counts at 45 min and 4 hours after injection were higher and those at 24 hours were lower than those of the controls.The findings are discussed, among other things, in terms of rate of uptake vs. time and related to previous reports on the cysteine uptake and neurosecretory activity of the hypothalamic-neurosecretory sytem.This study was financially supported by the Sigrid Jusélius Foundation, Helsinki, Finland and the National Research Council for Medical Sciences, Finland.     

Effect of estrogen and its antagonist on the expression of arginine vasotocin (AVT) and its oxytocic-like receptor VT3 in the shell gland of Japanese quail, Coturnix coturnix japonica

Avian neurohypophysial hormone arginine vasotocin (AVT) is known to regulate shell gland contractility during oviposition. While studying the role of estrogen in the expression and regulation of AVT and its oxytocic-like receptor VT3, using in situ hybridization and immunohistochemistry, it was observed that the expression of AVT and its receptor was not detected in the shell gland of sexually immature Japanese quail. However, administration of estrogen to these birds not only stimulates the growth and activity (as assessed by increased mucosal fold length, total protein content and alkaline phosphatase level) of the shell gland but also upregulates the expression of AVT and VT3. Further, administration of estrogen antagonist tamoxifen to sexually mature bird shows opposite results. On the other hand, localization of ir-AVT, observed in the ovary of sexually mature bird, was not detected in the estrogen treated sexually immature quail. It is concluded that estrogen not only affects the growth and differentiation of avian oviduct, but also regulates the expression of shell gland AVT and its receptor VT3. Present findings suggest that the locally synthesized AVT acts in a paracrine way to upregulate VT3 receptor and thus facilitates the endocrine function of neurohypophysial AVT during oviposition.     

Retrograde and orthograde axon transport by brief-exposure to nickel chloride: Methodology and parameters for success in the brain-retrocerebral complex of the cockroach Diploptera punctata

Exposure of the intact brain-retrocerebral complex of the cockroach Diploptera punctata to 500 mM NiCl₂ in 0.1% bovine serum albumen for 10 min resulted in the filling of neurones connecting the components of the brain-retrocerebral complex. Following severance of one corpus cardiacum from the brain prior to exposure to NiCl₂, a larger number of neurones filled in the contralateral side of the pars intercerebralis. Reducing exposure time to NiCl₂ to 30s did not result in backfilling of intact preparations. However, if the nervi corporis allati I (NCA 1) were sectioned, then exposed to NiCl₂ for 30s and incubated in culture medium at 4°C for 24 h, neurones of the severed NCA I backfilled to somata originating in the pars intercerebralis and pars lateralis. In addition, the axons distal to the cut also filled with the tracer in the orthograde direction, revealing a complex network of axon terminals investing the corpora allata. Sectioned nervi corporis allati I exposed to the tracer solution, then incubated in culture medium for 0.5 h, rapidly backfilled at a rate of 30–100 mm/day. Rapid backfilling did not occur in the absence of BSA. One per cent fluorescein isothiocyanide-labelled BSA backfilled as rapidly as the $\text{NiCl}{}_2\text{BSA}$ solution. 2,4-Dinitrophenol and cytochalasin B inhibited brief-exposure backfilling whereas colchicine had no effect in short term incubations but partially inhibited backfilling in long term incubations. Backfilling is thus a metabolically mediated event and the rate of transport of the tracer is similar to that of retrograde fast axonal transport of proteins.     

Topography of short portal vessels in the rat pituitary gland: a scanning electron-microscopic and morphometric study of corrosion cast replicas

We applied scanning electron microscopy combined with imaging and morphometric techniques to analyze the dorsal topography and morphology of short portal vessels linking the capillary beds of the pituitary neural and anterior lobes in adult male albino rats. The pituitary microvasculature was replicated by intracarotid injection of Batson's No. 17 compound producing plastic casts that were advantageous for comprehensive morphometric analyses using an imaging device. The analysis revealed the existence of two types of portal vessels having quantitatively different morphological properties. The bilateral venular plexus of 3–4 vessels located at the base of the infundibular stalk (each venule measuring 300 m in length and 32 m in diameter) appears to be the major part of the short portal system in the dorsum of the rat pituitary gland. Narrower capillary-like shunt vessels (6.8 m in diameter), of about the same length as the venules, were situated throughout other subregions of the intermediate lobe cleft. The short portal vessels of both types made direct anastomoses with the capillary networks in the neural and anterior lobes. The neural lobe capillaries were twice as numerous (1324 per mm²), and only half as wide (6.2 m), as the sinusoidal capillaries in the anterior lobe (density of 637 per mm²; diameter of 13.7 m). The topographical position of the portal venular system suggests that the caudolateral subregions of the pituitary neural and anterior lobes have a functional relationship dependent on rapid interlobe transfer of neurohumoral factors such as hormones via the portal blood. This process appears to be supplemented throughout the rest of the cleft between the two lobes by a small number of capillary shunts that supply the epithelial cell lobules of the intermediate lobe in situ. The findings collectively indicate that this portal system provides a constant stream of neurohumoral information that is shared moment-by-moment between the pituitary neural and anterior lobes.     

Tanycyte and vascular patterns in the basal hypothalamus of Coturnix quail with reference to their possible neuroendocrine significance

Summary A well developed system of ependymal glial cells with long basilar processes stretching to the surface of the brain (tanycytes, Horstmann, 1954) has been described in the basal hypothalamus of Coturnix quail. The tanycytes both in the median eminence and the ventro-lateral hypothalamus form a link between the third ventricle and the hypophysial circulation. The processes of the ventro-lateral tanycytes terminate in the region of the infundibular sulcus in apposition to a loose network of vessels which are continuous with the primary plexus of the hypophysial portal system.Within the median eminence, the subependymal capillary network connects the vasculature of the contra-lateral sides of hypothalamus. There are no direct connections with the hypophysial portal vessels.With the aid of the light and electron microscope the ventricular ependyma was divided into a dorsal typical region and two glandular regions (ventro-lateral and ventral). Each region contains different forms of tanycyte. One of the two forms of tanycyte (designated type 3) associated with the ventro-lateral glandular ependyma has no contact with the third ventricle.Ultrastructural studies on the glandular ependyma failed to show any obvious differences between castrated, oestrogen or testosterone implanted, and sexually mature or immature quail.The possibility that the tanycyte-vascular system may have a neuroendocrine role is discussed.I am indebted to Professor A. Oksche, Dept. of Anatomy, University of Giessen for providing research facilities and to The Royal Society for additional financial support.     

Twenty-four-hour changes in pinealocytes,capillary endothelial cells and pericapillary and intercellular spaces in the pineal gland of the mouse

Summary Semiquantitative electron-microscopic observations on the pineal gland of dd-mice were carried out to determine whether 24-h rhythms exist in pinealocytes, pericapillary and intercellular spaces and capillary endothelial cells. Nuclear and cytoplasmic areas of pinealocytes and the area of condensed chromatin in pinealocytes showed inversely related circadian rhythms; the former two increased, whereas the latter decreased, during the light period. The extent of pericapillary and wide intercellular spaces exhibited 24-h changes, with an increase and decrease occurring during the light period and the dark period, respectively. The cross-sectional area of endothelial cells decreased and the number of fenestrae increased during the light period; this was reversed during the dark period. The results suggest that the increase in the nuclear and cytoplasmic areas of pinealocytes, the area of pericapillary and wide intercellular spaces and the number of fenestrae, and the decrease in the area of condensed chromatin and endothelial cells during the light period may be related to an increase in synthetic activity of pinealocytes in the mouse.     

Neuronal components of the circadian clock in the scorpion,Androctonus australis: Central origin of the efferent neurosecretory elements projecting to the median eyes

Summary The central origin of efferent neurosecretory fibers (ENSF) supplying the median eyes of the scorpion, Androctonus australis, with circadian information was investigated at the light- and electron-microscopic levels by the use of several tracer techniques. The results obtained convincingly show that the central neurons stained via the optic nerve are the source of the ENSF in the retina. Almost all of these neurons give rise to two different axon collaterals, one of which projects into the ipsilateral, the other into the contralateral median-eye nerve. Three protocerebral contact zones of the ENSF system are described, and their functional role within the circadian clock system is discussed.This investigation represents a portion of the Ph. D. thesis of one of the authors (S.H.) and was supported by grants to G.F. from the Deutsche Forschungsgemeinschaft (SFB 45).