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.

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.

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.

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.