[Morpho-functional characteristics of conducting airways and the respiratory portion of rat lung under the conditions of intratracheal infection from the positions of the neuroendocrine regulation of reparative histogeneses].

With the use of light and electron microscopy and immunohistochemistry, the morpho-functional changes in the lungs, the hypothalamus and the neurohypophysis were studied in 45 outbred albino male rats 1, 3, 7 and 14 days after the intratracheal infusion of Staphylococcus aureus strains either possessing anti-lactoferrin activity (ALfA(+)) or lacking it (ALfA(-)). After the infusion of ALfA(+) bacteria, the bronchial wall and the respiratory portion of the lungs demonstrated the destructive changes of tissues, sclerosis phenomena, disturbances of regeneration processes (polypoid outgrowth, metaplasia), while in the neurohypophysis a delay in the release of neurosecretion into the blood from the terminals of nonapeptidergic neurosecretory cells took place. These phenomena were not observed after the infection with ALfA(-)bacteria. The results obtained indicate the disturbances of the structural-functional homeostasis of pulmonary tissues associated with bacterial ALfa, taking place together with the limitations of the hypothalamic neurosecretion.

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.

Glutamatergic innervation of the hypothalamic median eminence and posterior pituitary of the rat.

Recent studies have localized the glutamatergic cell marker type-2 vesicular glutamate transporter (VGLUT2) to distinct peptidergic neurosecretory systems that regulate hypophysial functions in rats. The present studies were aimed to map the neuronal sources of VGLUT2 in the median eminence and the posterior pituitary, the main terminal fields of hypothalamic neurosecretory neurons. Neurons innervating these regions were identified by the uptake of the retrograde tract-tracer Fluoro-Gold (FG) from the systemic circulation, whereas glutamatergic perikarya of the hypothalamus were visualized via the radioisotopic in situ hybridization detection of VGLUT2 mRNA. The results of dual-labeling studies established that the majority of neurons accumulating FG and also expressing VGLUT2 mRNA were located within the paraventricular, periventricular and supraoptic nuclei and around the organum vasculosum of the lamina terminalis and the preoptic area. In contrast, only few FG-accumulating cells exhibited VGLUT2 mRNA signal in the arcuate nucleus. Dual-label immunofluorescent studies of the median eminence and posterior pituitary to determine the subcellular location of VGLUT2, revealed the association of VGLUT2 immunoreactivity with SV2 protein, a marker for small clear vesicles in neurosecretory endings. Electron microscopic studies using pre-embedding colloidal gold labeling confirmed the localization of VGLUT2 in small clear synaptic vesicles. These data suggest that neurosecretory neurons located mainly within the paraventricular, anterior periventricular and supraoptic nuclei and around the organum vasculosum of the lamina terminalis and the preoptic area secrete glutamate into the fenestrated vessels of the median eminence and posterior pituitary. The functional aspects of the putative neuropeptide/glutamate co-release from neuroendocrine terminals remain to be elucidated.

Localization of transforming growth factors, TGFbeta1 and TGFbeta3, in hypothalamic magnocellular neurones and the neurohypophysis.

The distribution of transforming growth factor beta (TGFbeta) in the rat and human hypothalamus and neurohypophysis was investigated by immunocytochemical techniques using rabbit polyclonal antisera against TGFbeta(1) and TGFbeta(3). Colocalization of TGFbeta(1) or TGFbeta(3) and arginine vasopressin (AVP) in the rat hypothalamus was studied by double immunolabelling in light microscopy, while their subcellular localization in the rat neurohypophysis was investigated by immunoelectron microscopy. TGFbeta(1) and TGFbeta(3) immunoreactivity was demonstrated in the cell bodies and processes of neurones in the supraoptic nucleus (SON) and paraventricular nucleus (PVN). The TGFbeta-immunoreactive cells were more numerous in the SON compared to the PVN. TGFbeta/AVP double-labelled cells were seen in both nuclei, but some neurones in the SON were labelled for TGFbeta(1) or TGFbeta(3), although not for AVP. In the rat and human neurohypophysis, TGFbeta(3) immunolabelling was more diffuse and stronger than TGFbeta(1) immunolabelling. TGFbeta(1) expression was seen in axonal vesicles and in neurosecretory granules of the axonal endings, while TGFbeta(3) was observed in axonal fibres. Colocalization of TGFbeta(3) or TGFbeta(1) and AVP was observed in some neurosecretory granules, but many were either single-labelled for TGFbeta or AVP or unlabelled. Our results demonstrate, for the first time, the colocalization of TGFbeta and neurohypophysial hormones in magnocellular neurones. We suggest that TGFbeta secreted by the neurohypophysis regulates the proliferation and secretion of certain anterior pituitary cells.

The distribution of melanin-concentrating hormone in the lamprey brain.

In addition to its novel, colour-regulating hormonal role in teleosts, the melanin-concentrating hormone (MCH) serves as a neuromodulatory peptide in all vertebrate brains. In gnathostome vertebrates, it is produced in several neuronal cell groups in the hypothalamus. The present work examines the organisation of the MCH system in the brain of lampreys, which separated from gnathostome vertebrates at an early stage in evolution. In all three lamprey genera examined-Petromyzon, Lampetra, and Geotria spp.-MCH perikarya were found in one major anatomical site, the periventricular dorsal hypothalamic nucleus of the posterior hypothalamus. Axons from these cell bodies projected medially into the ventricular cavity, and laterally to the neuropile of the lateral hypothalamus. From here, they extended anteriorly and posteriorly to the fore- and hindbrain. Other fibres extended dorsomedially to the habenular nucleus. In Lampetra, but not in Petromyzon, MCH fibres were seen in the pituitary neurohypophysis, most prominantly above the proximal pars distalis. The hypothalamic region in which the MCH perikarya are found forms part of the paraventricular organ (PVO), which is rich in monoamines and other neuropeptides. The association of MCH neurones with the PVO, which occurs also in many other nonmammalian vertebrates, may reflect the primary location of the MCH system. These MCH neurones were present in ammocoetes, postmetamorphic juveniles, and adults. They were more heavily granulated in adults than in young lampreys but showed no marked change in secretory appearance associated with metamorphosis or experimental osmotic challenge to indicate a role in feeding or osmoregulation. In sexually maturing Lampetra fluviatilis, however, a second group of small MCH neurones became detectable in the telencephalon, suggesting a potential role in reproduction and/or behaviour.

Combined use of immunocytochemistry and lectin histochemistry for the study of the hypothalamic neurosecretory system of the snake Natrix maura (L.).

Natrix maura snakes were processed for immunocytochemistry and lectin histochemistry at both light- and electron-microscopic levels. Antisera against bovine neurophysins, vasotocin and mesotocin were used as well as concanavalin A, wheat germ and Limax flavus agglutinin lectins. The hypothalamic supraoptic and paraventricular nuclei were studied. Vasotocin neurons should contain a glycopeptide and displayed large colloid droplets consisting of large cisternae filled with packed secretory material. Mesotocin was located in different neurons.

[The oxytocinergic neurosecretory system in genetically selected rats differing by their emotionality. Morphometric research]. / Oksitotsinergicheskaia neirosekretornaia sistema u geneticheski selektirovannykh krys, razlichaiushchikhsia po émotsional'nosti. Morfometricheskoe issledovanie.

The rats selectively bred for rapid (KHA) and slow (KLA) acquisition of the avoidance response were subjected to inescapable shock (IS). Synthesis and secretion of oxytocin (OT) were higher in intact KLA rats as compared to KHA ones. Preliminary exposure to IS resulted in opposite changes of the OT synthesis and secretion. The findings suggest a dependence of the stress-reactivity of the OT-ergic system on the copying of the behaviour strategy.

Direct and indirect retinohypothalamic projections to the supraoptic nucleus in the female albino rat.

Earlier studies have shown that retinohypothalamic projections terminate extensively within the hypothalamus of the rat. Recently, we identified a light retinal projection to the supraoptic nucleus as well as a larger, well-focused projection resulting in a peri-supraoptic nucleus terminal field. In this study, we employed a double labeling method with cholera toxin conjugated to horseradish peroxidase (CT-HRP) and pseudorabies virus, a transsynaptic neural tracer, to evaluate retinorecipient neurons in both the supraoptic nucleus and peri-supraoptic nucleus terminal field. In addition, we looked for evidence that cells in the peri-supraoptic nucleus terminal field project into the supraoptic nucleus. Three strains of pseudorabies virus were compared. A direct retinosupraoptic nucleus circuit was confirmed with all three strains. Retinorecipient neurons in the peri-supraoptic nucleus terminal field were also confirmed. However, there was a strain-based difference in the identification of these neurons. The wild-type Becker strain labeled cells in the peri-supraoptic nucleus terminal field in a manner paralleling the early, intermediate and late stages of infection of the suprachiasmatic nucleus. This parallel time course suggests that retinal ganglion cells terminate directly on cells in the peri-supraoptic nucleus terminal field. Conversely, the Bartha and PRV-91 strains showed appreciable labeling of peri-supraoptic neurons only at long survival times. This longer time course suggests that these mutant strains label neurons in the peri-supraoptic nucleus terminal field indirectly, after passing through additional neurons. In addition, experiments with monocular injection of CT-HRP and posterior pituitary injection of pseudorabies virus showed retrogradely labeled second-order cells in the peri-supraoptic nucleus amidst the CT-HRP labeled terminal field of the retinohypothalamic tract. These results demonstrate a direct projection from the retina to the supraoptic nucleus and provide evidence for an indirect circuit from the retina to the supraoptic nucleus via neurons located in the peri-supraoptic nucleus terminal field. The strain-based differences imply that those retinal ganglion cells that project to the peri-supraoptic nucleus terminal field differ from those that project to the suprachiasmatic nucleus. In addition, these results suggest a neuroanatomic basis for photic effects on physiological mechanisms that are not mediated by the circadian timing system.

Microcirculatory patterns in adult rat cerebral hypophysis: a scanning electron microscope study of replicated specimens.

The blood vascular bed of the cerebral hypophysis in the adult rat was replicated completely or incompletely by arterial injection of different amounts of methacrylate resin, to be observed with a scanning electron microscope. Complete replication confirmed our previous findings (Murakami et al., 1987) on the distribution and structure of the vascular beds in and around the hypophysis of the rat. One long major and several minor portal routes (vide infra) were reproduced sufficiently together with the systemic veins of the posterior lobe. Incomplete replication demonstrated that resin flows: 1) via the long portal vessels from the median eminence and neural stalk to the anterior lobe; 2) via the accessory long portal vessels from the subependyma to the anterior lobe; 3) via the short portal vessels from the posterior lobe to the anterior lobe; 4) via the neuro-intermedial portal vessels from the posterior lobe to the intermediate lobe; 5) via the intermedio-distal portal vessels from the intermediate lobe to the anterior lobe; and 6) via the tuberal portal vessels from the tuberal lobe to the anterior lobe. Incomplete replication also demonstrated that resin in the median eminence and neural stalk is drained preferentially into the anterior lobe via the long portal vessels, and that resin in the posterior lobe is drained mainly into the systemic veins. We were unable to demonstrate a retrograde resin flow from the anterior lobe to the median eminence, subependyma, neural stalk, intermediate lobe and posterior lobe, nor an ascending resin flow from the posterior lobe to the median eminence and subependyma. Also failing to be noted were an ascending resin flow from the hypophysis to the hypothalamus and a descending resin flow from hypothalamus to the hypophysis.

Capturing and quantifying the exocytotic event.

Although exocytosis is now known to be the universal method by which proteins are released from eukaryotic cells, we know surprisingly little of the mechanism by which exocytosis occurs. One reason for this is that it has proved difficult to capture sufficient of these evanescent events to permit their study. The difficulty with which exocytoses can be visualized with standard preparative techniques varies among tissues, but the problem is particularly apparent in the mammalian nervous system. Tannic acid has recently been introduced as an agent by which exocytosed granule cores can be captured and visualized electron-microscopically. Application of tannic acid to the magnocellular neurosecretory system reveals exocytoses from all parts of their terminal arborization within the neural lobe, and also from their dendrites within the hypothalamus. Quantification of the exocytoses in unstimulated tissue and in tissue stimulated by a variety of exogenous and endogenous mechanisms indicates: (a) that exocytosis occurs equally from each unit of membrane of the perivascular nerve endings, and of the axonal swellings that were previously thought to be sites of granule storage, rather than release; (b) that, in the nerve endings, a greater proportion of the stored granules are exocytosed, and thus the endings are specialized for release not by any particular property of their membrane, but by a high surface membrane:volume ratio. Together, the data cast doubt on the hypothesis that exocytosis occurs only at some functionally specialized sites at certain loci in the membrane. Rather, the data favour the hypothesis that magnocellular granules can fuse with any part of the membrane, depending on constraints imposed by the cytoskeleton, and a local increase in cytosolic free calcium level. When applied to hypothalamic central nervous tissue, tannic acid reveals that exocytosis of dense-cored synaptic vesicles occurs preferentially, but not exclusively, at the membrane apposed to the postsynaptic element. However, about half of all exocytoses from synaptic boutons occur at bouton membrane unrelated to the synaptic cleft. In all tissues studied, tannic acid reveals a heterogeneity among secretory cells in the extent of exocytosis that occurs in response to stimulation, and permits an analysis of the degree to which secretion is polarized in any one direction. These results question long-held assumptions concerning the site at which neurones release transmitters and modulators. Tannic acid seems likely to prove a potent tool in the investigation of both the mechanism of exocytosis and the ways in which different types of cells adapt the process to perform their physiol

Neurosecretory axon regeneration into intrahypothalamic neural lobe allografts: neurophysin immunohistochemistry and fine structure.

Neural lobe allografts placed stereotactically into the hypothalamo-neurohypophysial tract between the paraventricular and supraoptic nuclei were investigated between 5 and 70 days post-transplantation (dpt). They contained temporally increasing numbers of neurophysin-positive axons. At the fine structural level, endogenous neurosecretory axons had virtually disappeared from successful (vascularized) grafts by 5 dpt. At this time, single host neurosecretory axons and especially numerous growth cones were associated with pituicyte processes and/or scalloped basal lamina scaffolds. Axon terminals containing neurosecretory granulated vesicles and microvesicles were present only occasionally at 5 dpt but became much more numerous subsequently. These terminals were associated with pituicytes and abutted the parenchymal basal lamina of pericapillary connective tissue spaces. In addition, beginning at 10 dpt, neurolemmocyte-like cells were associated with neurosecretory axons. At 70 dpt, the fine structural characteristics of grafted neural lobes were virtually indistinguishable from those of intact controls, except for the presence of occasional areas of more extensive connective tissue, nonfenestrated capillaries and neurolemmocyte-like cells.

Radioautographic study of glycoprotein synthesis and fate in the hypothalamo-neurohypophyseal system of vasopressin-deficient Brattleboro rats.

L-3H-fucose was injected into the lateral cerebral ventricle of vasopressin-deficient Brattleboro and control Long-Evans rats which were subsequently killed at several time intervals after the injection. The hypothalamus and the neurohypophysis were processed for light- and electronmicroscopic radioautography. Other complementary experiments using immunocytochemical and enzyme-histochemical techniques were also undertaken. L-3H-fucose was incorporated into newly synthesized glycoproteins in the Golgi apparatus of supraoptic and paraventricular neurons, and later on labelled glycoproteins migrated to lysosomes and the plasma membrane surrounding the perikaryon. The Golgi apparatus of the vasopressin-deficient neurons remained heavily labelled as long as 3 days after injection, in sharp contrast with the normal neurons in which there was a remarkable decrease of label in the Golgi region between 4 and 24 h after the isotope administration. Labelled glycoproteins also migrated to the neurohypophysis and were mainly found in the axonal plasma membrane, vesicles and axoplasm. The renewal of glycoproteins in the neurohypophysis of Brattleboro rats was faster than in the normal rats and this was attributed to the lack of formation of products which are normally packaged in secretory granules in the perikaryon and released at the axon terminal in the neurohypophysis. Colchicine caused a disturbance in the topography of the organelles of the perikaryon and the most striking features were the displacement of Golgi stacks to the periphery of the perikaryon and an accumulation of mitochondria in this neuronal region. No secretory granules were observed in the vasopressin-deficient neurons of untreated or colchicine-treated Brattleboro rats. By contrast, secretory granules (most of them labelled with 3H-fucose) were concentrated in the perikaryon of colchicine-treated Long-Evans rats. In these rats, colchicine caused a severe block in the migration of 3H-fucose-labelled glycoproteins to the neurohypophysis, but this did not occur in the Brattleboro rats. The results of the experiments were interpreted in the light of the genetic defect known to occur in Brattleboro rats which causes the inability to produce vasopressin and also remarkable morphological and physiological changes in the affected neurons.

Ultrastructural localization of immunoreactive neurophysins using monoclonal antibodies and protein A-gold.

Using three different monoclonal antibodies against rat neurophysins (5), with protein A-gold as immunocytochemical marker (27), the murid hypothalamoneurohy-pophysial system was studied at the ultrastructural level. Postembedding staining was done on epoxy-embedded sections of supraoptic nuclei and posterior pituitaries. Specific immunolabeling of vasopressinergic and oxytocinergic neurosecretory granules was observed in tissues fixed with glutaraldehyde or glutaraldehyde mixtures (containing paraformaldehyde and picric acid), with or without osmium tetroxide postfixation and with or without sodium metaperiodate oxidation. Some autophagic vacuoles containing lysed neurosecretory granules were also neurophysin immunoreactive. Nonspecific background staining was extremely low. An attempt was made to appraise labeling intensities semiquantitatively by counting gold particles in relation to number of secretory granules per axonal varicosity. Immunoreactivity was measurably influenced by the mode of fixation, sodium metaperiodate oxidation, and titer and affinity of the antibody. The protein A-gold technique using monoclonal antibodies against neurophysins provides a superior means of ultrastructural analysis of the hypothalamoneurohypophysial system, both visually and morphometrically.

Thiamine pyrophosphatase activity in the axonal smooth endoplasmic reticulum of neurosecretory neurons.

Neurosecretory cells of the supraoptic-neurohypophysial system of normal mice were investigated with the use of the cytochemical reaction for thiamine pyrophosphatase (TPPase) at the ultrastructural level. In the hypothalamic perikarya dense lead percipitates occur within the cisterns of the mature face of the Golgi apparatus, these being the cisterns that give rise to neurosecretory granules (NSG). Smooth endoplasmic reticulum is occasionally confluent with TPPase-positive Golgi cisterns. Along axons, within swellings, and within terminals distinct profiles of TPPase-positive tubules and cisterns are revealed, apparently part of a network of axonal smooth endoplasmic reticulum (AER). Some NSG appear to be confluent with AER. NSG with TPPase-positive tubular protrusions (likely vestiges of AER) are seen. Apart from reaction product (lead precipitate), the AER often contains an electron dense substance optically similar to that of NSG. TPPase-containing AER is often associated with mitochondria. Profiles of electron-lucent, precipitate-free tubules and cisterns are occasionally seen alongside reactive AER. Optimal TPPase activity in the AER occurs at pH 7.0--7.4, whereas in the Golgi complex intense marking is in the range of pH 6.0--8.5. A faint peppering of precipitate occasionally appears in the AER in controls (incubation medium without substrate), but neither in density nor in extent is this comparable to the reaction product seen after incubation in the presence of TPP. Preliminary comparison has been made between the AER revealed by the TPPase reaction, and that visualized after heavy metal impregnation according to the method of Alonso and Assenmacher (1978a). The nature of the close association between NSG and AER, and the possible roles of this membrane system in neurosecretory cells is discussed.

Electron microscopic immunocytochemical investigation on the postnatal development of the vasopressin system in the rat.

The present ultrastructural results indicate that, in the rat, the vasopressin-synthesizing perikarya of the supraoptic nucleus (NSO) attain a certain degree of maturity earlier than those of the paraventricular nucleus (NPV). In the neonate rat, the stainability of the nuclear areas is very weak; in the perikarya of the NSO a few labeled granules can be found, whereas the perikarya of the NPV often display only a labeled Golgi area, the cytoplasm being devoid of granules. At the end of the first (NSO) and the second (NPV) postnatal weeks, the filling of the neurosecretory granules with vasopressin is inhomogeneous with irregular spots of reaction product distributed on the granules. This feature is less obvious during the following week and has nearly disappeared after the third and fourth postnatal weeks. Already in the neonate two types of vasopressin-positive fibers are observed in the median eminence, characterized by the different diameters of their granules and by their typical location in the internal and the external pericapillary contact zone. Especially in one and two week-old animals, in the internal zone of the median eminence and, to a lesser degree in the neural lobe, the immuncytochemical reaction product is deposited on an axonal tubular network. Judging from the presence of very few vasopressin-negative fibers in the neural lobe of the neonate, the development of the oxytocin system appears to be delayed. A characteristic relationship between pituicytes and the neurosecretory fibers can be observed during the first two postnatal weeks. After the third postnatal week the immunocytochemical features of the vasopressin system correspond approximately to that in adult rats.