Ultrastructural immunocytochemical localization of neurophysin and vasopressin in the median eminence and posterior pituitary of the guinea pig

Summary With the use of tissue prepared by freeze-substitution and the unlabelled antibody enzyme technique, neurophysin and vasopressin were localized at the ultrastructural level in the posterior pituitary and median eminence of the guinea pig. In the posterior pituitary neurophysin was found in the large neurosecretory granules (1300–1500 Å) of axons, Herring bodies, and nerve terminals. In some of these axons immunoreactive neurophysin was found outside of granules in the axoplasm. By light microscopy neurophysin was found in both the zona interna and zona externa of the median eminence; this was confirmed by electron microscopy. In the zona interna as in the posterior pituitary, neurophysin was localized both inside and outside the large neurosecretory granules. In the zona externa, immunoreactive deposit was primarily located in granules with a diameter of 900–1100 Å in nerve terminals abutting on the primary portal plexus. The distribution of vasopressin paralleled that of neurophysin except that the hormone was rarely extragranular. These results demonstrate for the first time that both neurophysin and vasopressin are present in granules of axons that are in contact with the hypophysial portal vasculature.The authors wish to thank Dr. Alan Robinson for the gifts of antiserum to bovine neurophysin I and for purified bovine neurophysin I; Dr. Ludwig Sternberger for the peroxidase-anti-peroxidase complex; and Dr. Robert Utiger for antiserum to lysine vasopressinSupported in part by U.S. Public Health Service grant RR-00167 to the Wisconsin Regional Primate Research Center from the National Institutes of Health. Primate Center publication No. 14-017.Recipient of NIH, NINDS Teacher-Investigator Award NS-1108.     

Ultrastructural characteristics of immunolabelled,corticotropin releasing factor (CRF)-synthesizing neurons in the rat brain

Summary The corticotropin releasing factor (CRF)-synthesizing perikarya and neural processes were detected at ultrastructural level in the hypothalamic paraventricular nucleus and in the median eminence of control and colchicine-pretreated rats. The unlabelled antibody peroxidase-antiperoxidase complex (PAP) immunohistochemical method was used in a pre-embedding manner, on thick, non-frozen sections. In CRF-perikarya, neurosecretory granules (80–120 nm in diameter), free ribosomes, and the rough endoplasmic reticulum were labelled. Unlabelled axon terminals formed asymmetric synapses on CRF-containing perikarya and dendrites. Immunolabelled axons terminated in the palisadic zone of the median eminence.     

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     

The lateral recess of the third ventricle in teleosts: An electron microscopic and golgi study

Summary The ependyma lining the lateral recess of the third ventricle of the teleost inferior lobe has been studied by light and electron microscopy, including Golgi impregnation methods. As many as five different cell types appear to line the ventricle, but some of these may be similar cells in different stages of activity. One cell type contains small dense-cored vesicles and appears to have processes extending into deeper portions of the lobe. Golgi preparations reveal subependymal cells with apical processes extending to the ventricle and basal extensions which may reach the pial surface. The present observations are discussed in relation to similar studies in other fishes, amphibians and mammals. Possible functions for the various cells observed are suggested.     

The zinc iodide-osmium tetroxide (ZIO) reaction on nerve endings in the median eminence of the rat under normal and experimental conditions

Summary The reaction of nerve endings in the median eminence of the rat to zinc iodide-osmium tetroxide (ZIO) staining was examined electron microscopically under normal and experimental conditions. The experimental condition of catecholamine exhaustion in the nerve endings was induced by the administration of H44/68 and reserpine. Vesicles in the terminals of catecholaminergic nerves reacted similarly to ZIO staining in both normal and experimental material. The majority of synaptic vesicles in various terminals gave a positive ZIO reaction. The neurosecretory elementary granules, however, failed to react with ZIO. On the other hand, some nerve terminals in the external layer of the median eminence showed a strong positive reaction in the cytoplasmic matrix, in mitochondria as well as in synaptic vesicles. These findings strongly suggest that the ZIO-positive substance in nerve terminals is not the transmitter itself, i.e. the monoamine, but rather represents a range of substances commonly found in various kinds of synaptic vesicles and is probably proteinaceous in nature. A brief discussion is also given on the difference in ZIO reactivity between neurosecretory elementary granules and small vesicles in the hypothalamo-hypophyseal tract.This work was supported in part by a research grant from the Ministry of Education, Japan     

Correlative scanning-transmission electron microscopic examination of the perinatal rat brain

Summary The ultrastructural organization of the perinatal hypothalamus and the dynamics of neuronal and ependymal growth and plasticity were examined in this investigation. The brains of fetal rats 16, 17 and 18 days in utero and those of postnatal rats 1–16 days post partum were fixed with aldehyde fixatives and prepared for combined SEM/TEM analysis. By day 17 in utero the ventricular (ependymal) surfaces of the fetal thalamic wall, cerebral vesicle and rhomboid fossa were relatively well differentiated with cilia and microvilli. Type II histiocytes were the first supraependymal cell to appear upon the ventricular lumen and were evident by day 17 in utero. In contrast, the apical surfaces of tanycytes of the infundibular recess as well as those of most other circumventricular organs were poorly differentiated and unremarkable. Tanycytes of the infundibular recess exhibited a simple hexagonal mosaic pattern of apposed plasmalemmata and even by day 1 post partum few cilia or microvilli were evident.By day 5–6 post partum Type I supraependymal neurons and axonal processes began to make their appearance with some emerging from the underlying parenchyma of the median eminence. By day 16 post partum the ventricular surface of the infundibular recess was comparable with that of the adult.The Type I supraependymal neurons are remarkably similar in their ultrastructural organization with parvicellular neurosecretory neurons elsewhere in the endocrine hypothalamus. Their emergence at day 5–6 post partum suggests a possible correlation with the critical period of sexual differentiation and a potential receptor role for this cell line. On the contrary this phenomenon may simply be a developmental anomaly. Nonetheless, the mergence of such elements upon the lumen of the third cerebral ventricle underscores a remarkable degree of neuronal plasticity in the perinatal hypothalamus.Supported by USPHS Program Project Grant NS 11642-04 and USPHS-BRSG Grant RR-05403.The authors wish to thank N. Kutryeff for her excellent technical assistance     

A system of intraependymal cisternae along the margins of the median eminence in the rat: Structure,three-dimensional arrangement and ontogeny

Summary Structure, three-dimensional arrangement and ontogeny of large intracellular cisternae located in the median eminence region of the rat hypothalamus were studied using toluidin-blue stained semithin sections and electron microscopy. The cisternae occur along the projections of ependymal cells lining the ventral portion of the third ventricle (infundibular recess). Small cisternae can be seen close to the ventricle, whereas larger ones, divided into smaller compartments by thin septa, cluster near the surface of the hypothalamus. The cisternae are encompassed by a thin layer of cytoplasm to which axon terminals containing synaptic and dense core vesicles are closely related. Cisternae are arranged around the median eminence in a characteristic pattern. They occupy the midline in the retrochiasmatic area, flank both margins of the median eminence and extend caudally behind the origin of the pituitary stalk. The cisternae appear first between the 15th and 17th postnatal days. At about the 30th day their size and distribution resemble the situation observed in adult animals. The ependymal cisternae are suggested to be closely related to the luteinizing-hormone releasing-hormone (LH-RH)-containing fibers.     

Light and electron microscopic observation on the appearance of immunoreactive LHRH in perinatal rat hypothalamus

Summary The appearance and localization of LHRH were studied in the developing hypothalamus of perinatal rats using the unlabelled antibody method. By light microscopy, immunoreactive LHRH was first detected as brown dots on day 18.5 of gestation in the OVLT and on day 19.5 in the median eminence, respectively. When the median eminence was examined by the preembedding immunohistochemistry technique for electron microscopy, the occurrence of immunoreactive LHRH fibers could be demonstrated on day 18.5. These fibers were thin and very occasionally encountered near the surface of the lateral regions of the median eminence. The axoplasm contained a few immunopositive secretory granules and also extragranular immunoreactive products. With development, a gradual increase was noted both in number and size of nerve fibers with a concomitant accumulation of secretory granules within the axoplasm.A possible physiological significance of LHRH is discussed in relation to the onset of hypothalamo-hypophysial system in fetal life.     

Ultrastructural characteristics of vasopressin-containing neurons in the paraventricular nucleus of the hypothalamus

Summary Vasopressin-containing neurons, identified by immunocytochemistry, are located predominantly in the posterior magnocellular division of the paraventricular nucleus of the rat hypothalamus. By electron microscopy, the immunoreaction product is seen within the cell bodies and neuronal processes. In the perikarya and dendritic processes, the immunoreactive material is associated primarily with neurosecretory granules. Axonal processes, identified by their content of microtubules and accumulation of neurosecretory granules, show the immunoreaction product in association with both of these organelles. Afferent axo-dendritic, axo-somatic and putative axo-axonic synapses with immunostained vasopressinergic neurons can be identified. The presynaptic profiles do not contain immunoreactive material. This study contributes to the ultrastructural characterization of vasopressinergic neurons in the paraventricular nucleus and of their afferent synaptic input.Supported by NIH Grants HD-12956 and 2SO7RR05403     

Postnatal development of the suprachiasmatic hypothalamic nucleus of the rat

Summary Light and electron microscopy of newborn, four day, one, two, three and five week old rats revealed principally a progressive increase in the diversity and number of synaptic contacts in the suprachiasmatic nucleus (SCN). The major increase in synaptic diversity occurred between four days and one week of age. Correlation between this finding and the adult synaptic morphology of SCN (Güldner, 1976) on the one hand, and the ontogeny of circadian rhythms on the other were made. This suggested that the retinal afferents arriving on day four form asymmetrical contacts with dendrites. While increase in synaptic number was progressive, it was most marked between three and five weeks of age. By five weeks, most features of the adult SCN were present. No significant morphological effects were evident as a result of neonatal retinal lesions.Supported in part by grants NS-12265, NS-12267, HD04583 and HD-08658 from the National Institutes of Health, USPHS. The electron microscopic facilities of the California Regional Primate Center, supported by NIH grant RR-00169, were utilized. The technical assistance of Mrs. Viviana Wong is gratefully acknowledged. A preliminary report of a portion of this data was given at the Society for Neuroscience, November, 1974 in St. Louis, Missouri.     

Uptake of peroxidase from the third ventricle by ependymal cells of the median eminence

Summary Peroxidase injected into the subarachnoid space in mice is absorbed by ependymal cells of the median eminence. The ependymal cells of the median eminence of the rat and Japanese quail absorb peroxidase injected into the third ventricle. The processes of these ependymal cells terminate at the capillaries of the primary plexus or those surrounding the ventromedial nucleus of the hypothalamus. In all three species, peroxidase is absorbed by the ependymal cells of the paraventricular organ and by those in close proximity to it. Some ependymal cells send processes to the capillaries in the lateral nucleus of the hypothalamus. These phenomena are discussed in relation to adenohypophysial function.A part of this investigation was effected while the senior author held a Visiting Professorship at the University of Giessen [Department of Anatomy (Professor A. Oksche, Director)].     

Ultrastructural immunocytochemical localization of luteinizing hormone-releasing hormone (LH-RH) in the median eminence of the guinea pig

Summary LH-RH was localized at the ultrastructural level in axons and nerve terminals of the median eminence of the male guinea pig. LH-RH positive neuronal profiles were most concentrated in the medial-dorsal aspect of the infundibular stalk and in the post-infundibular median eminence at the level immediately following separation of the stalk from the base of the brain. LH-RH containing axon profiles were most abundant in the palisade zone; nerve terminals in contact with the hypophysial portal vasculature were relatively rare. The hormone was present within granules that measured 900–1,200 Å in axons of the palisade zone and 400–800 Å in nerve terminals abutting on the portal plexus. The differently sized granules represent heterogeneous populations.Supported in part by U.S. Public Health Service grant HD-09636 from the National Institutes of Health and RR-00167 to the Wisconsin Regional Primate Research Center from the National Institutes of Health. Primate Center Publication No. 15-031The authors wish to thank Dr. Sandy Sorrentino, Jr. for the gift of antiserum to LH-RH and Dr. Ludwig Sternberger for the peroxidase.antiperoxidase complex     

Myelinated Herring bodies in the posterior part of the median eminence of the mouse

Summary The posterior part of the median eminence of the albino mouse (CF # 1-JCL) contains a cluster of myelinated axons beneath the tanycyte layer. Among them, small Herring bodies surrounded by myelin sheaths are revealed by electron microscopy. These structures contain electron-dense bodies, lamellar bodies, autophagic bodies, autophagic vacuoles, and neurofilaments. A few neurosecretory granules and mitochondria are also present. Some myelinated axons contain mostly accumulated neurosecretory granules.     

Ependymal specializations

Summary The primary plexus of the toad hypothalamic-adenohypophysial portal system has two types of loops. The short loops are localized in the external region of the median eminence and surrounded by nerve endings and glial cells. The long loops approach the ependymal lining of the median eminence. The ascending and descending branches of these loops are surrounded by nerve and ependymal endings and glial cells. The actual subependymal portion of the long loops is virtually in contact with ependymal processes only, which form a cuff interposed between this portion of the long loops and the fibres of the hypothalamic-neurohypophysial tract. Many of the vascular endings of the ependymal processes have electron dense granules whose diameter ranges between 700 and 1400 Å. The ultrastructure of the ependymal cells suggests that these granules are transport material and not secretory material.This anatomical arrangement linking the ependyma of the median eminence and the long loops of the primary plexus of the hypothalamic-adenohypophysial portal system makes the possibility of an interrelationship between the cerebrospinal fluid and the portal blood very considerable.Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina. The author takes great pleasure in thanking Prof. H. Heller for his constant interest and criticism.     

Regulation of AHI1 expression in adult rat brain: Implication in hypothalamic feeding control

Recent studies revealed that Abelson helper integration site 1 (AHI1) plays a role in brain development. However, little is known about the role of AHI1 in adult brain. To directly assess the role of AHI1 in the adult brain, we cloned full-length cDNA of rat AHI1 and observed prominent expression of AHI1 in the hypothalamus, which contributes mainly to the control of energy homeostasis. Furthermore, we demonstrated that food deprivation caused induction of AHI1 in the hypothalamus and subsequent re-feeding down-regulated AHI1 expression, suggesting the involvement of AHI1 in feeding control. Moreover, the expression of AHI1 was increased in serum-depleted Neuro2A cells and restored by subsequent insulin treatment. Furthermore, treatment in food-deprived rat with intraperitoneal glucose also reduced the increased AHI1 expression. These results demonstrate that AHI1 expression can be regulated through diet and suggest the novel role of AHI1 in feeding behavior.     

Immunoelectronmicroscopic localization of vasopressin in the rat suprachiasmatic nucleus

Summary The classical areas for arginine-vasopressin (AVP) synthesis are the magnocellular supraoptic (SON) and paraventricular nuclei. More recently AVP was also demonstrated in neurons of the parvocellular suprachiasmatic nucleus (SCN) of the rat. This result was substantiated in the present study by means of immunoelectron microscopy, by subjecting sections to antivasopressin plasma. Conventional electron microscopy revealed dense-core vesicles in the SCN cell bodies and fibres (mean diameter 94.7±0.9 nm and 84.0±1.1 nm respectively). These vesicles were infrequent within the cell bodies and could not be accumulated by ethanol administration. Immunoelectron microscopy showed a positive reaction in the cell bodies and fibres within vesicles of 93.7±1.1 nm and 98.5±1.2 nm respectively. By comparison, the cell bodies and fibres of the SON showed immunoreactive granules of 143.0±1.8 and 147.3±1.8 nm respectively. The presence in the SCN of AVP in vesicles of different size than those in the SON suggests that synthesis of this substance is indeed occurring within the SCN cells.Supported by The Foundation for Medical Research FUNGOThe authors wish to thank Dr. L.A. Sternberger (Edgewood Arsenal, Md., U.S.A.) for the peroxidase-anti-peroxidase complex, Dr. J.G. Streefkerk (Free University, Amsterdam) and the members of our project group Neuroendocrinology for their suggestions, Mr. P.S. Wolters and Miss A. van der Veiden for their skilled assistance     

Post-coital depletion of dense-core vesicles from the external layer of the rabbit median eminence

Summary The effects of mating on the depletion of osmiophilic material from dense-core vesicles (DCV) within the external layer of the rabbit median eminence (ME) were investigated. Two different populations of DCV were demonstrated within the ME external layer of the non-mated control animals. A plot of the diameters of these vesicles showed two peaks, one at 90–100 nm, and the other at 120–130 nm. These two populations of DCV were found in separate axons and axon terminals in contact with, or near the perivascular spaces of, portal capillaries. Within these axons and terminals an occasional membrane profile or vesicle ghost was also observed.The same two populations of DCV were evident in the ME external layer of animals that were sacrificed at 10 minutes post-coitus. In these experimental animals, the number and size of the smaller population of DCV were the same as those of the none-mated controls. However, there was an obvious decrease in the number of the large (120–130nm) DCV, together with a simultaneous marked increase in the number of vesicle ghosts. These ghosts had a mean diameter of 137±14 nm. On the basis of their size, it is suggested that the vesicle ghosts represent large DCV that have been depleted of their content following mating. Ultrastructural evidence for the depletion of osmiophilic material from the large DCV of mated animals is provided.According to previous writers the smaller DCV within the ME are aminergic, whereas the larger DCV may contain releasing factors (RF). Our findings provide further morphological evidence to support this hypothesis. Furthermore, RF and biogenic amines appear to be contained within separate nerve terminals in the rabbit ME.This investigation was supported by grants to Drs. D. G. Montemurro and R. P. Singh from the Medical Research Council of Canada. The authors gratefully acknowledge the technical assistance of Miss A. Overweel and Mrs. S. Griffin.