Light and electron microscopic studies on the influence of stress on prolactin-immunoreactivity in rat anterior pituitary lobe.

An increasing number of evidence suggests an important role of prolactin in the modulation of stress response. However, the mechanisms of its action on the HPA axis are not yet understood. Glucocorticoids, liberated from adrenal cortex due to hormonal signals from pituitary corticotrophs are known to play a key role in systemic stress response. Previously we found evidence that corticosteroid-binding globulin (CBG) is involved in rapid, membrane-mediated actions of adrenal steroids. Here we studied qualitatively immunostainings for prolactin and CBG in pituitaries of male rats that had been subjected to osmotic challenge. We also examined late pregnant, parturient and early lactating rats, assuming that parturition represents a strong physiological stress. We employed double immunofluorescencent staining of semithin sections and immunoelectron microscopy. In stressed males we found increased prolactin immunofluorescence associated with membranes while in controls this staining was predominantly cytoplasmatic. CBG immunofluorescence was found in almost all prolactin cells of stressed males while such double staining was only occasionally observed in controls. Similar observations were made in females: While parturient rats showed intense membrane associated double staining for both antigens, late pregnant and early lactating animals showed patterns similar to that of male controls. Immunoelectron microscopy revealed increased exocytosis of prolactin containing vesicles in lactating rats. CBG was localized on cell membranes and additionally within prolactin vesicles. Our observations suggest prolactin liberation from pituitary lactotrophs along with CBG upon systemic stress response. Membrane effects of glucocorticoids mediated by CBG may be linked to stimulus secretion of prolactin.

Microglia-mediated phagocytosis of apoptotic nuclei is impaired in the adult murine hippocampus after stroke.

Following stroke, neuronal death takes place both in the infarct region and in brain areas distal to the lesion site including the hippocampus. The hippocampus is critically involved in learning and memory processes and continuously generates new neurons. Dysregulation of adult neurogenesis may be associated with cognitive decline after a stroke lesion. In particular, proliferation of precursor cells and the formation of new neurons are increased after lesion. Within the first week, many new precursor cells die during development. How dying precursors are removed from the hippocampus and to what extent phagocytosis takes place after stroke is still not clear. Here, we evaluated the effect of a prefrontal stroke lesion on the phagocytic activity of microglia in the dentate gyrus (DG) of the hippocampus. Three-months-old C57BL/6J mice were injected once with the proliferation marker BrdU (250 mg/kg) 6 hr after a middle cerebral artery occlusion or sham surgery. The number of apoptotic cells and the phagocytic capacity of the microglia were evaluated by means of immunohistochemistry, confocal microscopy, and 3D-reconstructions. We found a transient but significant increase in the number of apoptotic cells in the DG early after stroke, associated with impaired removal by microglia. Interestingly, phagocytosis of newly generated precursor cells was not affected. Our study shows that a prefrontal stroke lesion affects phagocytosis of apoptotic cells in the DG, a region distal to the lesion core. Whether disturbed phagocytosis might contribute to inflammatory- and maladaptive processes including cognitive impairment following stroke needs to be further investigated.

Oxytocin, but not arginine-vasopressin neurons project from the hypothalamus to amygdala in human: DiI-based tracing study in postmortem brain.

The hypothalamic neuropeptides oxytocin (OT) and arginine-vasopressin (AVP) are important factors involved in the control of socio-emotional behaviors via their modulation of amygdala functions. Since anatomical pathways of magnocellular projections to limbic structures in the human brain have not been dissected, we infused ethanol-dissolved tracer DiI into three amygdala nuclei - medial, central and lateral nuclei, and into the mammillary bodies of postmortem fixed human brains. With this modification, lipophilic diffusion of DiI occurred much faster than with conventional DiI crystals. After staining of resliced sections with antibodies against OT or AVP, we detected DiI/OT-positive neurons and their axons, specifically in the supraoptic nucleus (SON), but not in other hypothalamic nuclei producing OT or AVP. DiI fluorescence was found in the lateral portion of the paraventricular nucleus (PVN) and in the fornix columns, together with VP- immunoreactivity, only after DiI injections into the mammillary bodies. Our findings indicate that OT and AVP may have distinct neuronal pathways to the limbic system, and they are different from those previously reported in rodents.

Evidence for accessory chemosensory cells in the adult human nasal cavity.

The existence of functionally relevant accessory olfactory organs in humans is still a matter of controversy. A vomeronasal organ (VNO) with sensory and non-sensory epithelia exists only in macrosmatic mammals. A similar structure is regularly observed in humans during fetal development. The postnatal persistence of a VNO like epithelial duct has been described in about 10 %. Here we studied tissue samples of nasal mucosa from adults. In all individuals we found epithelial cells in the lower part of the nasal septum which exhibited morphological features of sensory neurons and which showed immunostaining for olfactory marker protein OMP. These cells were interposed by ciliated cells, goblet cells and small intraepithelial capillaries. Only occasionally we found such cells within a morphologically defined epithelial duct. A clear separation of sensory and non-sensory epithelia could not be observed. In most cases we found OMP positive groups of cells either in epithelial cavities or just embedded in respiratory epithelium. With RT-PCR we could confirm the presence of OMP encoding mRNA thus supporting the idea of intrinsic expression of this protein in the nasal mucosa. We conclude that accessory chemosensory structures are regularly conserved in adult humans in the approximate anatomical location of the VNO of microsmatic animals. Their functional importance is yet to be determined.

Steroidal pheromones and their potential target sites in the vomeronasal organ.

Steroids are important olfactory signals in most mammalian species. The vomeronasal organ has been suspected to be the primary target of pheromones. In rat vomeronasal sensory neurons express steroid binding proteins and nuclear receptors. Some binding globulins were found also in single ciliated cells of the non-sensory vomeronasal epithelium. Immunoelectron microscopy revealed VDR in olfactory microvilli and DPB in apical membrane protrusions of supporting sells within the sensory epithelium. Pilot behavioral studies with dogs showed increased sniffing duration upon exposure to low concentrations of vitamin D while higher concentrations were less effective. It has been shown that vitamin D has pheromone-like properties in lizards. Our histochemical and behavioral observations indicate that the mammalian vomeronasal organ may be a vitamin D target. Olfactory functions of vitamin D involve most likely rapid membrane mediated effects rather than actions through nuclear receptors.

Estrogen receptors and sex hormone binding globulin in neuronal cells and tissue.

Estrogens exert a critical influence on neuronal tissues and cells. As demonstrated in many clinical studies, estrogens are neuroprotective to the extent that they improve prognosis for women with neurodegenerative diseases. Unfortunately, we still do not know exactly how these effects are mediated. Fifty years ago the first estrogen receptor was found, but since then many other new pathways of estrogen action have been identified. This review describes several of these pathways of estrogen effects and provides some conclusions and correlations about these as determined by recent studies with nerve growth factor differentiated rat pheochromocytoma cell line.

Diversity of central oxytocinergic projections.

Localization and distribution of hypothalamic neurons expressing the nonapeptide oxytocin has been extensively studied. Their projections to the neurohypophyseal system release oxytocin into the systemic circulation thus controlling endocrine events associated with reproduction in males and females. Oxytocinergic neurons seem to be confined to the ventral hypothalamus in all mammals. Groups of such cells located outside the supraoptic and the paraventricular nuclei are summarized as "accessory neurons." Although evolutionary probably associated with the classical magocellular nuclei, accessory oxytocin neurons seem to consist of rather heterogenous groups: Periventricular oxytocin neurons may gain contact to the third ventricle to secrete the peptide into the cerebrospinal fluid. Perivascular neurons may be involved in control of cerebral blood flow. They may also gain access to the portal circulation of the anterior pituitary lobe. Central projections of oxytocinergic neurons extend to portions of the limbic system, to the mesencephalon and to the brain stem. Such projections have been associated with control of behaviors, central stress response as well as motor and vegetative functions. Activity of the different oxytocinergic systems seems to be malleable to functional status, strongly influenced by systemic levels of steroid hormones.

Osmotic stress induces corticosteroid-binding globulin expression in the rat hypothalamo-hypophyseal system.

Corticosteroid-binding globulin CBG is expressed in magnocellular hypothalamic nuclei, in part colocalized with vasopressin (VP) and oxytocin (OT). Here we subjected intact adult male rats to chronic osmotic stress to determine effects on distribution of CBG in VP and OT neurons and in neurons expressing corticotropin- releasing hormone (CRH). Drinking 2% NaCl solution for seven days resulted in increased CBG-immunoreactivity in magnocellular neurons. Triple immunofluorescence revealed increased colocalization with either VP, OT or CRH. Colocalization of CRH with VP was found only in a small portion of parvocellular neurons in the PVN. Most of the CBG-immunostained neurons within the magnocellular nuclei were devoid of CRH-immunoreactivity. Increased numbers of axons with colocalization of CBG and VP or OT were found in the internal zone of the median eminence (ME) of osmotically challenged rats. The external zone of the ME showed numerous CRH-positive neuronal projections. A small portion of them contained also CBG-immunofluorescence in both experimental animals and controls. Immunoassays of cerebrospinal fluid showed increased levels of CBG in osmotically stressed animals. Our observations suggest that hypothalamic CBG expression is malleable to functional status and that coexpression with the magnocellular peptide hormones may be of significance for endocrine stress response.

The oxytocin system of mice and men-Similarities and discrepancies of oxytocinergic modulation in rodents and primates.

Nonapeptides and their respective receptors have been conserved throughout evolution and display astonishing similarities among the animal kingdom. They can be found in worms, birds, fish, amphibians, reptiles and mammals, including rodents, non-human primates and humans. In particular, the neuropeptide oxytocin (OT) has attracted the attention of scientists due to its profound effects on social behavior. However, although both the neuropeptide and its receptor are identical in rodents and primates, the effects of OT vary greatly in the two species. Here, we provide a brief overview about OT's role in the evolution of mammals and provide reasons for the manifold effects of OT within the brain with a particular focus on the discrepancy of OT's effects in rodents and primates. In addition, we suggest new approaches towards improvement of translatability of scientific studies and highlight the most recent advances in animal models for autism spectrum disorder, a disease, in which the normal function of the OT system seems to be impaired.

Oxytocin and Steroid Actions.

Biosynthesis and secretion of the hypothalamic nonapeptide oxytocin largely depends on steroid hormones. Estradiol, corticosterone, and vitamin D seem to be the most prominent actors. Due to their lipophilic nature, systemic steroids are thought to be capable of crossing the blood-brain barrier, thus mediating central functions including neuroendocrine and behavioral control. The actual mode of action of steroids in hypothalamic circuitry is still unknown: Most of the oxytocinergic perikarya lack nuclear steroid receptors but express proteins suspected to be membrane receptors for steroids. Oxytocin expressing neurons contain enzymes important for intrinsic steroid metabolism. Furthermore, they produce and probably liberate specific steroid-binding globulins. Rapid responses to steroid hormones may involve these binding proteins and membrane-associated receptors, rather than classic nuclear receptors and genomic pathways. Neuroendocrine regulation, reproductive behaviors, and stress response seem to depend on these mechanisms.

Magnocellular hypothalamic system and its interaction with the hypothalamo-pituitary-adrenal axis.

The hypothalamo-neurohypophyseal system plays a key role in maintaining homeostasis and in regulation of numerous adaptive reactions, e.g., endocrine stress response. Nonapeptides vasopressin and oxytocin are the major hormones of this system. They are synthesized by magnocellular neurons of the paraventricular and supraoptic hypothalamic nuclei. Magnocellular vasopressin is known to be one of the main physiological regulators of water-electrolyte balance. Its importance for control of the hypothalamo-pituitary-adrenal axis has been widely described. Magnocellular oxytocin is secreted predominantly during lactation and parturition. The complex actions of oxytocin within the brain include control of reproductive behavior and its involvement in central stress response to different stimuli. It's neuroendocrine basis is activation of the hypothalamo-pituitary-adrenal axis: corticotropin-releasing hormone is synthesized in parvocellular neurons of the paraventricular hypothalamic nuclei. The transitory coexpression of vasopressin in these cells upon stress has been described. Glucocorticoids, the end products of the hypothalamo-pituitary-adrenal axis have both central and peripheral actions. Their availability to target tissues is mainly dependent on systemic levels of corticosteroid-binding globulin. Intrinsic expression of this protein in different brain regions in neurons and glial cells has been recently demonstrated. Regulation of the hypothalamo-pituitary-adrenal axis and hypothalamo-neurohypophyseal system is highly complex. The role of both systems in the pathogenesis of various chronic ailments in humans has extensively been studied. Their disturbed functioning seems to be linked to various psychiatric, autoimmune and cardiovascular pathologies.

Estrogen dependent expression of sex hormone binding globulin in PC 12 cells.

Rat pheochromocytoma PC 12 cells are known to develop features of dopaminergic neurons upon treatment with nerve growth factor. They express in part estrogen receptors α and ß, and G-protein coupled receptor 30. Estrogens promote development of these cells and exert neuroprotective effects. Here we treated differentiated PC 12 cells with physiological concentrations of 17-ß-estradiol. We observed with immunocytochemistry cytoplasmic staining for SHBG in a portion of these cells Double immunostaining for estrogen receptor-ß revealed that some PC 12 cells contained both antigens. Numbers of estrogen receptor-ß positive cells were significantly higher after estradiol treatment; an effect that was not altered by pretreatment of cultures with tamoxifen. With reverse transcriptase polymerase chain reaction we observed sex hormone binding globulin encoding transcripts indicating intrinsic expression of the steroid binding globulin. We conclude that estrogen treatment induces SHBG expression in differentiated PC12.

Sex hormone binding globulin and corticosteroid binding globulin as major effectors of steroid action.

Contrary to the long-held postulate of steroid-hormone binding globulin action, these protein carriers of steroids are major players in steroid actions in the body. This manuscript will focus on our work with sex hormone binding globulin (SHBG) and corticosteroid binding globulin (CBG) and demonstrate how they are actively involved in the uptake, intracellular transport, and possibly release of steroids from cells. This manuscript will also discuss our own findings that the steroid estradiol is taken up into the cell, as demonstrated by uptake of fluorescence labeled estradiol into Chinese hamster ovary (CHO) cells, and into the cytoplasm where it may have multiple actions that do not seem to involve the cell nucleus. This manuscript will focus mainly on events in two compartments of the cell, the plasma membrane and the cytoplasm.

Adrenal steroids in the brain: role of the intrinsic expression of corticosteroid-binding globulin (CBG) in the stress response.

The complex interaction between hypothalamus, pituitary and adrenal glands is a key component of the neuroendocrine stress response. The major stress hormones--glucocorticoids--have both central and peripheral effects. Among the factors regulating their availability to target tissues are levels of corticosteroid-binding globulin, as the major transport protein for glucocorticoids in systemic circulation. Our recent findings demonstrated expression of corticosteroid-binding globulin in various brain regions and in different cell populations (neurons and glial cells). We showed at the cellular level the presence of corticosteroid-binding globulin in the human hypothalamus, where it was co-localized with the classical neurohypophyseal neurohormones--vasopressin and oxytocin. For the first time we demonstrated in mouse that the same gene encodes brain and liver corticosteroid-binding globulin. The full-length sequencing of hypothalamic corticosteroid-binding globulin revealed a full homology with liver corticosteroid-binding globulin cDNA. Thus, we confirmed that corticosteroid-binding globulin mRNA is produced locally within various cerebral regions and thus not transported from blood. However, the amounts of mRNA encoding corticosteroid-binding globulin are in liver about 200 times higher than in brain. The wide distribution of corticosteroid-binding globulin, distinct from the localization of glucocorticoid receptors, observed in our comparative study in rodents, led us to propose two possibilities: (1) corticosteroid-binding globulin is made in certain neurons to deliver glucocorticoids into the cell and within the cell in the absence of cytoplasmic glucocorticoid receptors or (2) is internalized into neurons specifically to deliver glucocorticoids to classical glucocorticoid receptors. Brain corticosteroid-binding globulin may be involved in the response to changing systemic glucocorticoid levels either additionally to known nuclear and membrane corticosteroid receptors or in glucocorticoid responsive brain regions devoid of these receptors. Clearly the multiple locations of corticosteroid-binding globulin within the central nervous system of humans and rodents imply multiple functional properties in normal and/or pathological conditions, which are yet to be determined. Most likely, the importance of brain corticosteroid-binding globulin exceeds the function of a mere steroid transporter.

Differences in colocalization of corticosteroid-binding globulin and glucocorticoid receptor immunoreactivity in the rat brain.

Endocrine regulation of central and systemic stress response as well as learning and memory are in part controlled by systemic glucocorticoid levels. So far steroids have been thought to act on the brain predominantly through nuclear receptors. However, some brain systems known to respond to glucocorticoids seem to be devoid of the respective receptor proteins (GR). It is likely that known central actions of adrenal steroids may also be mediated by non-genomic actions involving intrinsic binding globulins. In recent studies we described the intrinsic expression of corticosteroid-binding globulin (CBG) in rat, mouse and human brains. Here we report an immunohistochemical mapping study on the colocalization of CBG and of GR in the rat brain. In the nucleus accumbens, septum, hippocampus, globus pallidus, medial and basolateral amygdale nuclei, magnocellular preoptic nuclei, diagonal band of Broca high intensity of CBG immunoreactivity was accompanied by weak or moderate GR staining, and vice versa. In the caudate putamen, bed nucleus of stria terminalis, septohypothalamic nucleus and parvocellular subdivision of the paraventricular nucleus strong GR immunoreactivity was observed, but CBG was almost undetectable. In contrast, throughout the supraoptic nucleus and magnocellular subdivision of the paraventricular nucleus numerous strongly CBG-positive cells were observed, devoid of specific GR immunoreactivity. It is most likely that CBG in the brain may be involved in the response to changing systemic glucocorticoid levels in addition to known nuclear and membrane corticosteroid receptors, or in glucocorticoid responsive regions devoid of these receptors.

Intrinsic expression of transcortin in neural cells of the mouse brain: a histochemical and molecular study.

Corticosteroid binding globulin (CBG, transcortin) has been shown to be expressed in the brain of rat and human species. In this study, we examined the CBG brain expression and cDNA structure in mice, comparing wild-type (Cbg(+/+)) and Cbg knockout mice (Cbg(-/-), obtained by genetic disruption of the SerpinA6 alias Cbg gene). We used double immunofluorescence labeling with specific neuronal and glial markers to analyze the cellular localization of CBG in various regions of the mouse brain. In wild-type (Cbg(+/+)) mice, we found CBG immunoreactivity in neuronal perikarya of the magnocellular hypothalamic nuclei, amygdala, hippocampus, cerebral cortex, cerebellum and pituitary. A portion of glial cells (astrocytes, oligodendrocytes) contained CBG immunoreactivity, including some of the ependymal cells and choroid plexus cells. No CBG immunoreactivity was detected in Cbg(-/-) brain tissues. Using RT-PCR, we showed that the full-length Cbg mRNA is present in those regions, indicating an intrinsic expression of the steroid-binding globulin. Furthermore, sequencing analysis showed that Cbg cDNA obtained from the mouse hypothalamus was homologous to Cbg cDNA obtained from the liver. Finally, we have evaluated the relative levels of CBG expression in various brain regions and in the liver by quantitative PCR. We found that brain levels of Cbg mRNA are low compared with the liver but significantly higher than in CBG-deficient mice. Although derived from the same gene as liver CBG, brain CBG protein may play a specific or complementary role that requires the production and analysis of brain-specific Cbg knockout models.

Ultrastructural and ultraimmunohistochemical changes upon partial nephrectomy and uranyl intoxication in the rat kidney.

We studied kidneys of rats intoxicated with uranylnitrate (UN) or subjected to 5/6 nephrectomy (NX) or after a combination of both procedures (NX-UN). Our observations indicate that UN causes impressive changes of ultrastructure (partial loss of brush border, appearance of intercellular clefts in the epithelial barrier) and altered protein expression (α-SMA, collagen I and III) in proximal tubule cells. Renal parameters (creatinine clearance, proteinuria) seemed to be unaffected. Blood pressure recovered to normal values within 12 months. However ultrastructural and functional restoration of modified proximal tubules was not complete. We conclude that changed proximal tubules may induce progression of interstitial fibrosis causing renal failure. NX animals and more pronounced NX-UN animals showed dramatic changes in renal function. We observed increased levels of proteinuria, blood pressure and decreased creatinine clearance. Progressive glomerular reorganization includes loss of filtration gaps and enhanced thickness of glomerular basement membranes (GBM) with increased immunoreactivity for collagen IV. Cells in vicinity of Bowman's capsule contained high amounts of immunoreactive α-smooth muscle actin. The NX-UN group showed more dramatic changes in ultrastructure of proximal tubules including apoptosis. Enhanced expression and secretion of extracellular matrix proteins (ECM e.g. collagens I, III, fibronectin) indicate progressive epithelial-mesenchymal transition (EMT) leading to permanent impairment of renal function.

Polarization optical-histochemical characterization and supramolecular structure of carbohydrate fibrils.

Topo-optical staining reactions were used to investigate the structures of bacterial cellulose, insect chitosan and alginic acid from brown algae. Polysaccharide complexes, glycosaminoglycans and sulfate groups were presented and demonstrated selectively. Chitosan and alginic acid are structurally similar to glycosaminoglycans (GAGs), which are constituents of human amyloid fibrils. The staining sequences shown can be used as reliable methods for histochemistry with light and polarization microscopy. They will help to clarify the complex protein-polysaccharide structure of amyloid fibrils.

Expression of corticosteroid binding globulin in the rat olfactory system.

Glucocorticoids are known to act on the olfactory system although their mode of action is still unclear since nuclear glucocorticoid receptors are mostly absent in the olfactory mucosa. In this study we used immunocytochemistry, in situ hybridization, and RT-PCR to study the expression and distribution of corticosteroid binding globulin (CBG) in the rat olfactory system. Mucosal goblet cells could be immunostained for CBG. Nasal secretion contained measurable amounts of CBG suggesting that CBG is liberated. CBG immunoreactivity was localized in many of the basal cells of the olfactory mucosa, while mature sensory cells contained CBG only in processes as determined by double immunostaining with the olfactory marker protein OMP. This staining was most pronounced in the vomeronasal organ (VNO). The appearance of CBG in the non-sensory and sensory parts of the VNO and in nerve terminals in the accessory bulb indicated axonal transport. Portions of the periglomerular cells, the mitral cells and the tufted cells were also CBG positive. CBG encoding transcripts were confirmed by RT-PCR in homogenates of the olfactory mucosa and VNO. Olfactory CBG may be significant for uptake, accumulation and transport of glucocorticoids, including aerosolic cortisol.