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.

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.

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.

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.

Distribution of vitamin D binding protein expressing neurons in the rat hypothalamus.

We observed immunostaining for vitamin D binding protein (DBP) in rat hypothalamus. Part of the supraoptic and of the paraventricular neurons showed DBP immunoreactivity, in part colocalized with Arg-vasopressin. DBP was also observed in widespread axonal projections throughout the lateral hypothalamus, the median eminence and the posterior pituitary lobe. A portion of ependymal cells, the choroids plexus epithelium and some of the endocrine cells in the anterior pituitary lobe contained DBP immunoreactivity. In situ hybridization of semithin sections with a synthetic oligonucleotide probe to DBP mRNA resulted in staining of magnocellular hypothalamic neurons, but not of ependymal cells or anterior lobe cells. Our observations indicate an intrinsic expression of DBP in the rat hypothalamus. DBP may be synthesized and transported along with the classical neurohypophyseal hormones. The multiple locations of DBP-expressing neurons indicate multiple functional properties: DBP may be released from in the posterior lobe, it may act as a hypophyseotropic factor and as a central neuroactive substance.

Expression of corticosteroid-binding globulin in human astrocytoma cell line.

Glial tumor cells are known to be sensitive to glucocorticoids (GC) in vivo and in vitro. Here we studied the expression of corticosteroid-binding globulin (CBG) in the low-grade malignant human astrocytoma cell line 1321N1. CBG was observed in cytoplasm of most of these cells with immunocytochemistry. RT-PCR revealed the presence of the respective mRNA. Only scattered cells contained nuclear immunoreactivity for glucocorticoid receptor as visualized by double immunostaining. Immunoreactive CBG could be recovered from the supernatant of cultures that had been exposed to 10(-5) M cortisol. Our observations indicate the endogenous expression of CBG in 1321N1 cells which may occur independently from classical glucocorticoid receptor pathways. Cortisol seems to facilitate liberation of CBG in a paracrine manner, perhaps through membrane action of the steroid. Effects of adrenal steroids on proliferation and apoptosis of certain glial tumors may in part depend on these mechanisms.

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.

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.

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.

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.

Beacon-like/ubiquitin-5-like immunoreactivity is highly expressed in human hypothalamus and increased in haloperidol-treated schizophrenics and a rat model of schizophrenia.

The beacon gene is involved in the regulation of energy metabolism, food intake, and obesity. We localized its gene product, beacon-/ubiquitin 5-like immunoreactivity in brains of normal-weight, non-psychotic individuals, adipose (BMI over 32), non-psychotic individuals, and haloperidol-treated schizophrenics. The protein was found to be highly expressed in many neurons of the paraventricular and supraoptic hypothalamic nuclei. Besides, it was detected in neurons of other hypothalamic areas (suprachiasmatic, arcuate, and ventromedial nuclei) as well as outside the hypothalamus (Nuc. basalis Meynert, thalamus, hippocampus, and some neocortical areas). A morphometric analysis of beacon-immunoreactive hypothalamic and neocortical neurons revealed that compared to normal-weight controls in haloperidol-treated schizophrenics, there was a significant increase of protein-expressing supraoptic, paraventricular, and orbitofrontal neurons. However, a significant increase in beacon-expressing supraoptic neurons was also seen in adipose, non-psychotic individuals in comparison with normal-weight controls. Haloperidol at different doses has no effect on beacon expression in SHSY5Y neuroblastoma cells, which makes the assumption unlikely that haloperidol per se is responsible for the increased neuronal expression of the peptide in schizophrenics. In rats with a neonatal lesion of the ventral hippocampus (a widely used animal model of schizophrenia), we found an increased neuronal expression of beacon in the paraventricular and supraoptic nuclei. We suppose that elevated hypothalamic expression of beacon-like protein in non-obese schizophrenics is not primarily related to metabolic alterations, but to a certain role in schizophrenia, which is possibly unrelated to aspects of weight gain and obesity. The latter assumption finds some support by data obtained in rats with ventral hippocampus lesion.

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.

Strongly reduced number of parvalbumin-immunoreactive projection neurons in the mammillary bodies in schizophrenia: further evidence for limbic neuropathology.

The mammillary bodies (MB) are important relay nuclei within limbic and extralimbic connections. They are known to play important roles in memory formation and are affected in alcoholism and vitamin B1 deficiency. Their strategic position linking temporo-limbic to cortico-thalamic brain structures make the MB a candidate brain structure for alterations in schizophrenia. We studied 15 postmortem brains of schizophrenics and 15 matched control brains. Brain sections were stained either with Heidenhain-Woelcke, glutamic acid decarboxylase (GAD), calretinin, or parvalbumin. We determined the volumes of the MB and performed cell countings using stereological principles and a computerized image analysis system. The volumes of MB do not differ between schizophrenics and controls. However, in schizophrenia the number of neurons as well as the resulting neuronal densities was significantly reduced on both sides (on left side by 38.9%, on right side by 22%). No changes were seen in the number of GAD-expressing or calretinin-containing neurons, whereas the number of parvalbumin-immunoreactive MB neurons was reduced by more than 50% in schizophrenia. This cell loss (as a result of developmental malformation and/or neurodegeneration) points to a prominent involvement of the MB in the pathomorphology of schizophrenia. Parvalbumin-immunoreactive GABAergic interneurons have been reported to be diminished in schizophrenia. However, in the MB parvalbumin labels a subpopulation of glutamate/aspartate-containing neurons projecting mainly to the anterior thalamus. Thus, our data provide new evidence for impaired limbic circuits in schizophrenia.

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.

Co-expression of vasopressin and androgen-binding protein in the rat hypothalamus.

In previous studies we have observed the expression of androgen binding protein (ABP) in the rat hypothalamo-neurohypophysial system. With immunocytochemical double staining we found partial co-localization with oxytocin. In the present study we used antibodies to the anti-diuretic hormone arginine vasopressin (AVP) for co-localization with ABP in the rat hypothalamus. Both antigens were seen in the magnocellular paraventricular and supraoptic nuclei. Dense fiber networks with varicosities containing both AVP and ABP immunoreactivity were visible throughout the hypothalamus, the median eminence and in the posterior pituitary lobe. Double immunostaining revealed also co-existence in the parvocellular portion of the paraventricular nucleus and in the suprachiasmatic nucleus. ABP immunoreactive neurons in the preoptic region were devoid of AVP staining, AVP neurons in the bed nucleus of the stria terminalis stained only occasionally for ABP. We conclude that both the magnocellular and the parvocellular hypothalamic vasopressin systems are capable of expressing the steroid binding globulin, which is probably subject to axonal transport, along with the peptide hormone. Intrahypothalamic expression of ABP may be among the mechanisms necessary for rapid actions of steroids on hypothalamic neuroendocrine systems.

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.