Axon guidance effect of classical morphogens Shh and BMP7 in the hypothalamo-pituitary system.

Hypothalamus plays a key role in homeostasis, and functions of the hypothalamus depend on the accurate trajectory of hypothalamic neuroendocrine axons. Thus, understanding the guidance of hypothalamic neuroendocrine axons is crucial for knowing how hypothalamus works. Previous studies suggest FGF10 deriving from the medial ventral midline of the hypothalamus plays an important role in axon guidance of the developing hypothalamus. Here we show that Shh and BMP7, which are from the anterior and posterior hypothalamic ventral midline respectively, together repel hypothalamic axons towards the medial ventral midline.

Activity-dependent remodeling of chondroitin sulfate proteoglycans extracellular matrix in the hypothalamo-neurohypophysial system.

The hypothalamo-neurohypophysial system (HNS) consisting of arginine vasopressin (AVP) and oxytocin (OXT) magnocellular neurons shows the structural plasticity including the rearrangement of synapses, dendrites, and neurovascular contacts during chronic physiological stimulation. In this study, we examined the remodeling of chondroitin sulfate proteoglycans (CSPGs), main extracellular matrix (ECM), in the HNS after salt loading known as a chronic stimulation to cause the structural plasticity. In the supraoptic nucleus (SON), confocal microscopic observation revealed that the immunoreactivity of 6B4 proteoglycans (PG) was observed mainly at AVP-positive magnocellular neurons but that of neurocan was seen chiefly at OXT-positive magnocellular neurons. The immunoreactivity of phosphacan and aggrecan was seen at both AVP- and OXT-positive magnocellular neurons. Electron microscopic observation further showed that the immunoreactivity of phosphacan and neurocan was observed at astrocytic processes to surround somata, dendrites, and terminals, but not synaptic junctions. In the neurohypophysis (NH), the immunoreactivity of phosphacan, 6B4 PGs, and neurocan was observed at AVP-positive magnocellular terminals, but the reactivity of Wisteria floribunda agglutinin lectin was seen at OXT-positive ones. The immunoreactivity of versican was found at microvessel and that of aggrecan was not detected in the NH. Quantitative morphometrical analysis showed that the chronic physiological stimulation by 7-day salt loading decreased the level of 6B4 PGs in the SON and the level of phosphacan, 6B4 PGs, and neurocan in the NH. These results suggest that the extracellular microenvironment of CSPGs is different between AVP and OXT magnocellular neurons and activity-dependent remodeling of CSPGs could be involved in the structural plasticity of the HNS.

Catecholaminergic input to the oxytocin neurosecretory system in the human hypothalamus.

Previous studies revealed that oxytocin release is increased by various forms of stress. Hypertonic saline injection, immobilization, and several other stressors elevated the blood level of oxytocin in rats. However, the mechanism of the stress-induced oxytocin release in human is not elucidated yet. Although numerous studies indicate that catecholamines play a pivotal role in modulating the release of oxytocin, there is a lack of data regarding the morphological substrate of this phenomenon. In order to reveal putative juxtapositions between tyrosine hydroxylase-immunoreactive (TH-IR) catecholaminergic and the oxytocinergic systems in the human hypothalamus, we utilized double-label immunohistochemistry in the present study. Numerous TH-IR axon varicosities abutted on oxytocin-IR neurons in the supraoptic and paraventricular nuclei, forming synapse-like associations. Close examination of these juxtapositions with high magnification failed to reveal any gaps between the contacting elements. In summary, the intimate associations between the TH-IR and oxytocin-IR elements may be functional synapses and may represent the morphological substrate of stress-influenced oxytocin release. The finding that several oxytocin-IR perikarya did not receive apparent TH innervation suggests that additional mechanisms may play significant roles in the oxytocin modulation by stressors.

[Endocytosymbiosis of microorganisms in mammal eukaryotic cells and factors of its regulation].

Morphofunctional equivalents of the process of long-term intracellular prokaryotes--eukaryotes interaction were studied by light and electron microscopy. The mechanisms for adaptation, elaborated in the course of evolution of bacteria-host interaction, were analysed on the ultrastructural level. A concept on the role of hypothalamic nonapeptides, as factors of regulation of intracellular persistence and symbiosis of prokaryotes, is discussed.

Interleukin-1beta immunoreactivity in identified neurons of the rat magnocellular neurosecretory system: evidence for activity-dependent release.

Interleukin-1beta has been demonstrated in neurons of the rat hypothalamus, including cells of the magnocellular neurosecretory system and tuberoinfundibular system (Lechan et al., [1990] Brain Res. 514:135-140). Despite its potential importance to regulation of neuroendocrine function, however, neither the specific cell types that express interleukin-1beta or the conditions that may result in its release have yet been described. Therefore, we utilized a combination of immunocytochemical and immunoelectron microscopic localization, in conjunction with Western blot analysis, on normonatremic, hypernatremic, and lactating rats to assess the site of synthesis and potential secretion characteristics of interleukin-1beta in the rat magnocellular neurosecretory system. Interleukin-1beta immunoreactivity was localized within both oxytocin and vasopressin neurons in the paraventricular, supraoptic, accessory and periventricular hypothalamic nuclei. Additionally, interleukin-1beta immunoreactive fibers were localized in the zona interna and zona externa of the median eminence and in the neurohypophysis. Immunoelectron microscopic analysis revealed that interleukin-1beta immunoreactivity is associated with small spherical structures, distinct from neurosecretory granules, in neurosecretory axons within the neurohypophysis. Furthermore, stimulation of heightened neurosecretory activity via chronic osmotic challenge and lactation resulted in a marked diminution in levels of interleukin-1beta immunoreactivity in the neurohypophysis with a subsequent return to normal levels after cessation of the stimuli. Western blot analysis confirmed the existence of interleukin-1beta protein in the neurohypophysis and provided further evidence for reduction in levels of IL-1beta immunoreactivity after stimulation of secretory activity. These results suggest an endogenous neuronal source of interleukin-1beta exists within the rat magnocellular neurosecretory system under normal physiological conditions. The potential for activity-dependent release of IL-1beta and implications for the involvement of interleukin-1beta in regulation of neurosecretory activity are discussed.

Size and degeneration increase in herring bodies during aging in hamsters.

The hypothalamo-neurohypophysial tract of young, adult and aged male hamsters was studied at lateral and ventral regions of hypothalamus by means of electron microscopy. Neurosecretory swelling axons (Herring bodies) were usually found as classically described containing abundant neurosecretory granules, mitochondria, few microtubules and profiles of smooth endoplasmic reticulum in all groups of age. However, in aged hamsters, starting at 18-month-old subjects, we observed that the size of some neurosecretory axons was highly increased. Autophagic and degenerative features were seen in the larger ones. These data could suggest abnormal axonal storage or axonal transport blocked during aging. The implications in the role of hypothalamus-neurohypophysial system during aging are discussed.

Regeneration of neurosecretory axons into various types of intrahypothalamic grafts is promoted by the absence of blood brain barrier: fine structural analysis.

Isogenous grafts of neural lobe and optic nerve and autologous grafts of sciatic nerve were placed into contact with the intrahypothalamically transected hypothalamo-neurohypophysial tract, and their fine structural characteristics examined at various time periods thereafter. The vascular bed of neural lobe grafts is composed primarily of fenestrated capillaries, that are permeable to blood-borne HRP throughout the entire experimental period. The microvasculature of sciatic nerve grafts consists of continuous, as well as fenestrated capillaries, which are similarly permeable to HRP. Fenestrated capillaries and HRP leakage in optic nerve grafts are observed at 10 days, but only in grafts located ventrally in the hypothalamus at 30 days. Neurosecretory axon regeneration is seen only in grafts or adjacent hypothalamus where the blood-brain barrier is breached. Regenerating axons are closely associated with the specific glial cells of the respective graft. Based on these observations, we conclude that blood-borne factors are necessary to initiate and sustain regeneration of transected neurosecretory axons, and that such regeneration occurs only in the presence of glial cells.

The magnocellular neurosecretory system of the hamster hypothalamus: an ultrastructural and morphometric study during lifetime.

A quantitative study regarding the age-related changes occurring in the nucleus and the somatic organelles of neurosecretory magnocellular neurons of the hypothalamo neurohypophyseal system (HNS) was carried out in the hamster at six age-points during animal life. The magnocellular cells of both parts of the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) of male Syrian hamsters between 3 and 30 months of age were examined ultrastructurally. Cells of all age groups present the same morphological ultrastructure. Standard manual morphometric techniques are used to calculate the following parameters related directly or indirectly with cellular activity: nuclear area, nucleolar area, nuclear invagination index and volumetric fractions of some intracellular structures (Golgi apparatus, mitochondria, rough endoplasmic reticulum and lipofuscin). With respect to the cell nucleus, the parameters are not modified during aging. No significant differences in the volume density of subcellular components, except lipofuscin, were detected at the age groups studies. However, there is a positive linear trend among all parameters and age except for the rough endoplasmic reticulum. Our results suggest maintenance of the synthetic activity of the magnocellular neurons in the hamster during aging but in no case an increase in their metabolic activity.

Fetal development and regulation of pituitary cell types.

The ontogenesis of the pituitary gland is considered from anatomical and functional points of view. Embryogenesis of the hypothalamo-pituitary unit involving development of the hypothalamo-hypophyseal portal system is complete during early life as shown in several mammalian species. The ultrastructural characteristics of the different cell types during development are described according to observations made by using immunochemical techniques. The patterns of differentiation of the cell types are reviewed according to studies of pituitary glands from human anencephalic fetuses and encephalectomized rat fetuses as well as in vitro studies of cultured pituitary primordia in synthetic media. The maturation of the neuroendocrine mechanisms controlling the secretion of fetal hormones is also analyzed. During fetal life, the factors implicated in the regulation of pituitary hormone secretion are generally the same as in adults, but the intensity of the response of pituitary cells to their action is variable according to the species, thus reflecting an immaturity in the functioning of certain cell types.

An immunohistochemical and fine-structural analysis of peptidergic hypothalamic neurosecretory axon regeneration into the leptomeninges of the rat.

Regeneration of severed hypothalamic peptidergic neurosecretory axons into the ventral pia-arachnoid was observed in rats at the light microscopic and fine-structural levels. A temporal increase occurred in the number of neurophysin-positive axons regenerating into the leptomeninges for distances up to 3.3 mm by 40 days post-lesioning. A consistent pattern of parallel, meshed and clustered axons, occurring either singly or in bundles, was present within the connective tissue, while plexus and bundles were observed in association with leptomeningeal blood vessels. Axons were characterized by preterminal and terminal dilatations. Neurosecretory granulated vesicles occurred throughout axons. The presence of microvesicles at contact points with basal lamina suggests the possibility of hormone release. Most axons were arranged as fascicles associated closely with basal lamina-bounded support cells whose thin lamellar processes wrapped single axons or fascicles of axons. We conclude, therefore, that cellular and intercellular leptomeningeal microenvironments support and sustain the growth and regeneration of transected neurosecretory axons.

Transmission and scanning electron-microscopic observations on tanycytes in the mediobasal hypothalamus and the median eminence of adrenalectomized rats.

Tanycytes in the median eminence (ME) of the rat exhibit morphological features suggesting their possible participation in transport phenomena. After adrenalectomy, which modifies the hypothalamo-hypophyseal axis, they undergo morphological changes characterized by an accumulation of lipid droplets, an increased number of bleb-like protrusions at their apex, as well as an increased pinocytosis of intraventricularly injected horseradish peroxidase (HRP). In addition, after adrenalectomy an increased number of vacuoles appears at the level of the tubero-infundibular sulci. Their intracellular location in the tanycytes is demonstrated by an intraventricular injection of HRP. The significance of these vacuoles is discussed in relation to the hydroelectrolytic modifications associated with the state of adrenalectomy.

Neuronal pathways to the margin of the hypothalamic median eminence and to the pituitary stalk of the rat. I. A golgi and degeneration study.

The course and termination of nerve fibres approaching the median eminence from lateral direction were studied in Golgi specimens and by the axon-degeneration technique. Varicose nerve fibres could be traced from an area corresponding to the medial and superficial portion of the medial forebrain bundle. They run immediately underneath the free ventral surface of the hypothalamus. Parasagittal knife-cuts placed at various distances (0.5 to 1.4 mm) from the midline resulted in a large number of degenerated axon fragments along the margin of the median eminence, on both sides of the tuberoinfundibular sulcus. Scattered degenerated fragments were found in the lateral part of the palisade zone as well as in the pituitary stalk. No degeneration could be seen in the abo9ve mentioned areas if the cut was as far as 1.8 mm from the midline. Degenerated axon fragments appeared as soon as 5 hours following the lesion indicating that the time course of ultrastructural degenerative alterations is remarkably fast in this fibre system.

Ipsilateral diminution of CRF-granules after unilateral hypothalamic lesions.

Axons terminating in the outer layer of the median eminence of rats contain light microscopically visible granules. The granules are assumed to represent a corticotropin-releasing factor and, therefore, are called CRF-granules. To find out whether neurons containing CRF-granules originate and run together with the neurons of the hypothalamus-neural lobe system (HNS), the effect of unilateral lesions in the HNS on the amount and distribution of CRF-granules was studied in bilaterally adrenalectomized rats. HNS lesions prevented the adrenalectomy-induced increase in CRF-granules on the side of the lesion. Lesions outside the HNS or sham lesions did not influence the amount and distribution of the granules. The findings suggest that CRF-granules are located in terminals of neurons whose perikarya are situated in magnocellular hypothalamic nuclei. It can also be concluded that the axons of these neurons run within the HNS and do not decussate.

Direct projection from the medial preoptic area to the median eminence of the cat.

The projection from the medial preoptic area to the median eminence of the cat was clarified by electron microscopy. After placing the electrolytic lesion in the preoptic area several kinds of degenerating neuronal processes and terminals were observed in the external layer of the median eminence. The one was dark shrunk terminals containing dense cored vesicles, the other was the dark ones containing myeline figure-like structure. The relationship between catecholamine-containing nerve endings and RH/IH-containing endings in the external layer of the median eminence was discussed.

Ultrastructural studies on the secretory cycle of the neurosecretory cells and the formation of Herring bodies in the paraventricular nucleus of the rat.

The ultrastructure of the neurosecretory cells in the paraventricular nucleus of the normal male rat was studied by electron microscopy during various functional states. Four morphologically distinct types of neurosecretory cells were observed. It appears that they do not represent different classes of cells but different phases of secretory activity of a single cell type. The perikarya of the neurosecretory cells show a definite cycle of formation and transportation of secretory granules. We have designated the phases of this cycle as: (1) phase of synthesis, (2) phase of granule production, (3) phase of granule storage and (4) phase of granule transport. The neurosecretory granules appear to be moved in bulk into the axons, forming a large axonal swelling filled with granules as a result of one cycle in the neurosecretory process. Thus it may be postulated that a secretory cycle in the perikaryon of the neurosecretory cell seems to result in the formation of a Herring body in its axon, and that its content is then conveyed to the posterior pituitary.

Cytological evidence for different types of cerebrospinal fluid-contacting subependymal cells in the preoptic and infundibular recesses of the frog.

Blue-green fluorescent subependymal cells with intraventricular processes were shown by the fluorescent histochemical method to be distributed from the preoptic recess to the infundibular recess of the frog hypothalamus. Electron microscopy revealed at least two types of CSF-contacting subependymal cells, type 1 containing large dense granules (about 100-200 nm in diameter) and type 2 containing small dense core vesicles (about 60-100 nm in diameter). Subsequent to fixation in permanganate solution, the small dense core vesicles in type 2 cells reacted with the fixative and consistently showed a dense content. However, the large granules in type 1 cells were mostly pale or less dense after this fixation. Two hours after intraventricular injection of 3H-dopamine, a large number of silver grains appeared only in the cytoplasm of intraventricular processes possessing dense core vesicles (type 2 cells). A few grains were also found in the perikarya. It is concluded that type 2 cells are catecholamine-storing cells. It is suggested that type 1 cells in the infundibular recess are peptidergic neurons which may secrete some hypothalamic regulating hormones of the anterior pituitary. Most of these cells in the preoptic recess belong to the neurosecretory cells of the preoptic nucleus, while some cells probably function similarly to those in the infundibular recess.

Monosodium glutamate induced lesions of the arcuate nucleus. I. Endocrine deficiency and ultrastructure of the median eminence.

Monosodium glutamate was administered daily on days 5 through 10 postnatally at a dose of 2.5 mg/gm body weight. Counts of remaining perikarya in the arcuate nucleus of adult mice indicated approximately an 80% decrease in the number of perikarya. The arcuate lesion resulted in endocrine deficits; reporductive capacity was reduced, animals were smaller in stature and obese, and the weights of the anterior pituitary, ovaries and testes were significantly decreased while adrenals were unaffected. Light microscopic studies revealed no significant changes in thickness or general histological appearance of the median eminence. At the electron microscope level, there were no alterations in the number of nerve terminals or dense core vesicles per unit area in the contact zone. These observations suggest that afferents to the median eminence from the arcuate nucleus may form a relatively small portion of its total nerve terminal population.

Ultrastructure of the organum vasculosum of the lamina terminalis of the Japanese quail, Coturnix coturnix japonica.

The structure of the organum vasculosum of the lamina terminalis (OVLT) of the Japanese quail has been studied by light and electron microscopy. The OVLT has a palisade arrangement. It forms a part of the terminal plate extending from the optic chiasma to the anterior commissure and is characterized by a special vascular arrangement. The organ consists of ependymal, internal and external zones. The ventricular surface of the organ is covered by non-ciliated ependymal cells characterized by the presence of raspberry-like ventricular protrusions. The ependymal zone is composed of two types of ependymal cells, one being a large, cuboidal cell with columinous cytoplasmic protrusions suggesting secretion into ventricle, and the other a slender tanycyte with long balar plexus of the external surface. The internal zone contains two types of secretory neurons, parvocellular neurons containing a few dense-cored granules 1,000 A in diameter, and mediocellular neurons containing in their perikarya many larger granules 1,300-1,500 A in diameter and well developed granular endoplasmic reticulum and Golgi apparatus. The granular formations are usually observed in the Golgi area of both types of cells, thus indicating of their secretory activities. There are three types of nerve terminals in the neuropil: (1) nerve endings containing spherical, dense-cored granules 800 A in diameter, which display axosomatic synapses with perikarya of the neurons, (2) nerve endings containing dense-cored granules 1,000 A in diameter and clear vesicles 400 A in diameter, and (3) nerve endings containing dense granules 1,300 A in diameter and clear vesicles. Types 2 and 3 do not display axo-xomatic synapses but often show axo-dendritic synaptic contacts with dendrites in the neuropil; thus they are probably axons originating from the parvocellular and mediocellular neurons of this organ. The functional significance of the neurons and axons of the OVLT is discussed and it is suggested that two kinds of neurohormones may be secreted from the OVLT of the Japanese quail.