Pituicyte Cues Regulate the Development of Permeable Neuro-Vascular Interfaces.

The hypothalamo-neurohypophyseal system (HNS) regulates homeostasis through the passage of neurohormones and blood-borne proteins via permeable blood capillaries that lack the blood-brain barrier (BBB). Why neurohypophyseal capillaries become permeable while the neighboring vasculature of the brain forms BBB remains unclear. We show that pituicytes, the resident astroglial cells of the neurohypophysis, express genes that are associated with BBB breakdown during neuroinflammation. Pituicyte-enriched factors provide a local microenvironment that instructs a permeable neurovascular conduit. Thus, genetic and pharmacological perturbations of Vegfa and Tgfß3 affected HNS vascular morphogenesis and permeability and impaired the expression of the fenestral marker plvap. The anti-inflammatory agent dexamethasone decreased HNS permeability and downregulated the pituicyte-specific cyp26b gene, encoding a retinoic acid catabolic enzyme. Inhibition of Cyp26b activity led to upregulation of tight junction protein Claudin-5 and decreased permeability. We conclude that pituicyte-derived factors regulate the "decision" of endothelial cells to adopt a permeable endothelial fate instead of forming a BBB.

Two-Photon Excitation of Fluorescent Voltage-Sensitive Dyes: Monitoring Membrane Potential in the Infrared.

Functional imaging microscopy based on voltage-sensitive dyes (VSDs) has proven effective for revealing spatio-temporal patterns of activity in vivo and in vitro. Microscopy based on two-photon excitation of fluorescent VSDs offers the possibility of recording sub-millisecond membrane potential changes on micron length scales in cells that lie upwards of one millimeter below the brain's surface. Here we describe progress in monitoring membrane voltage using two-photon excitation (TPE) of VSD fluorescence, and detail an application of this emerging technology in which action potentials were recorded in single trials from individual mammalian nerve terminals in situ. Prospects for, and limitations of this method are reviewed.

60 YEARS OF NEUROENDOCRINOLOGY: The posterior pituitary, from Geoffrey Harris to our present understanding.

Geoffrey Harris pioneered our understanding of the posterior pituitary, mainly with experiments that involved the electrical stimulation of the supraoptico-hypophysial tract. In the present essay, we explain how his observations included clues to the pulsatile nature of the oxytocin signal - clues that were followed up by subsequent workers, including his students and their students. These studies ultimately led to our present understanding of the milk-ejection reflex and of the role of oxytocin in parturition. Discoveries of wide significance followed, including: the recognition of the importance of pulsatile hormone secretion; the recognition of the importance of stimulus-secretion coupling mechanisms in interpreting the patterned electrical activity of neurons; the physiological importance of peptide release in the brain; the recognition that peptide release comes substantially from dendrites and can be regulated independently of nerve terminal secretion; and the importance of dynamic morphological changes to neuronal function in the hypothalamus. All of these discoveries followed from the drive to understand the milk-ejection reflex. We also reflect on Harris's observations on vasopressin secretion, on the effects of stress, and on oxytocin secretion during sexual activity.

VEGF-dependent and PDGF-dependent dynamic neurovascular reconstruction in the neurohypophysis of adult mice.

Hypothalamo-neurohypophysial system (HNS) releases arginine vasopressin (AVP) and oxytocin (OXT) from axonal terminals of the neurohypophysis (NH) into blood circulation for controlling body fluid homeostasis and lactation. Chronic osmotic and suckling stimulations have been shown to cause neurovascular and neuroglial reconstruction in the NH of adult mammals and no study has been reported for vascular dynamics. The aim of this study was to elucidate the occurrence of continuous angiogenesis and growth factor-dependent neurovascular reconstruction in the NH of adult mice. Active proliferation of endothelial cells and oligodendrocyte progenitor cells (OPCs) was observed using the immunohistochemistry of bromodeoxyuridine and Ki-67. Vascular endothelial growth factor A (VEGFA) and VEGF receptor 2 (VEGFR2 (KDR)) were highly expressed at pituicytes and endothelial cells respectively. Moreover, prominent expression of platelet-derived growth factor B (PDGFB) and PDGF receptor beta was observed at OXT-containing axonal terminals and pericytes respectively. Administration of the selective tyrosine kinase inhibitor AZD2171 for VEGFRs and STI571 for PDGFRs significantly decreased proliferation of endothelial cells and OPCs. Moreover, AZD2171 treatment decreased vascular density by facilitating apoptosis of endothelial cells and the withdrawal of its treatment led to remarkable rebound proliferation of endothelial cells, so that vascular density rapidly returned to normal levels. AZD2171 decreased the density of both AVP- and OXT-containing axonal terminals, whereas STI571 selectively decreased the density of AVP-containing ones. Thus, this study demonstrates that the signaling pathways of VEGF and PDGF are crucial mediators for determining proliferation of endothelial cells and OPCs and the density of AVP- and OXT-containing axonal terminals in the HNS.

Endonasal endoscopic pituitary adenoma resection: preservation of neurohypophyseal function.

OBJECTIVES: In the past 10 years, endoscopic resection of pituitary adenomas has become an alternative to microsurgical resection with the additional advantage of increasing the patient's postoperative comfort. This analysis explored whether endoscopic resection can reduce the risk of postoperative neurohypophyseal dysfunction. DESIGN: We rated and compared the need to administer desmopressin during the first four postoperative days and with the need after a follow-up of at least 3 months (chronic administration). SETTING: Three groups of patients were compared: Patients in group 1 were operated on microscopically. Patients in group 2 were operated on endoscopically. Patients in group 3 were operated on endoscopically with intraoperative magnetic resonance imaging (iMRI). PARTICIPANTS: Group 1 was made up of 50 patients treated in 1999; group 2 comprised 50 patients operated on from 2006 to 2007; and Group 3 comprised 50 patients operated on in 2008. MAIN OUTCOME MEASURES: In group 1 the need to use desmopressin postoperatively occurred in eight patients; three needed chronic treatment. In group 2 the need for postoperative application of desmopressin occurred in four patients; none required chronic treatment. In group 3 desmopressin had to be administered postoperatively in five patients but only temporarily. RESULTS AND CONCLUSIONS: Endoscopic surgery is a safe and effective method for the resection of pituitary adenomas. The rate of chronic desmopressin application was reduced. In conjunction with iMRI and navigation, the endoscopic technique allows increased radicality together with fewer adverse effects.

Development of the medial hypothalamus: forming a functional hypothalamic-neurohypophyseal interface.

The medial hypothalamus is composed of nuclei of the tuberal hypothalamus, the paraventricular nucleus of the anterior hypothalamus, and the neurohypophysis. Its arrangement, around the third ventricle of the brain, above the adenohypophysis, and in direct contact with the vasculature, means that it serves as an interface with circulating systems, providing a key conduit through which the brain can sample, and control, peripheral body systems. Through these interfaces, and interactions with other parts of the brain, the medial hypothalamus centrally governs diverse homeostatic processes, including energy and fluid balance, stress responses, growth, and reproductive behaviors. Here, we summarize recent studies that reveal how the diverse cell types within the medial hypothalamus are assembled in an integrated manner to enable its later function. In particular, we discuss how the temporally protracted operation of signaling pathways and transcription factors governs the appearance and regionalization of the hypothalamic primordium from the prosencephalic territory, the specification and differentiation of progenitors into neurons in organized nuclei, and the establishment of interfaces. Through analyses of mouse, chick, and zebrafish, a picture emerges of an evolutionarily conserved and highly coordinated developmental program. Early indications suggest that deregulation of this program may underlie complex human pathological conditions and dysfunctional behaviors, including stress and eating disorders.

Molecular tolerance of voltage-gated calcium channels is evident after short exposures to alcohol in vasopressin-releasing nerve terminals.

BACKGROUND: Voltage-gated calcium channels (VGCCs) in rat neurohypophysial terminals exhibit molecular tolerance to alcohol, including desensitization to the drug and increased current density, after 3 weeks of alcohol drinking. Moreover, after this time, terminals from drinking rats exhibit diminished alcohol inhibition of vasopressin (AVP) release. METHODS: We took advantage of organotypic cultures (explants) of the hypothalamo-neurohypophysial system (HNS) to extend our analysis of molecular tolerance to 2 classes of the VGCC. The isolated HNS explant allows much finer temporal resolution of molecular tolerance than do voluntary drinking paradigms. After exposure of the HNS explant to alcohol, terminals are isolated by mechanical treatment and plated in a dish. Patch clamp recording techniques are used to obtain VGCC currents, and immunohistochemistry is used to determine VGCC distribution. A release assay is used to provide functional readout of AVP release. RESULTS: We show that even a brief, 1-hour exposure to a clinically relevant concentration of alcohol is sufficient to evoke similar changes to those observed after several weeks of exposure. Acute ethanol (EtOH) exposure inhibits high K(+) -induced AVP release from naïve terminals. However, terminals pre-exposed to 20 mM EtOH for 1 hour become tolerant to EtOH, and subsequent exposure has significantly less effect on high K(+) -induced AVP release. Electrophysiological recordings indicate that among different types of VGCCs present in the neuronal terminal, the L-type is the most affected by alcohol. The current density of L-type current is significantly increased (approximately 50%), while its responsiveness to alcohol is significantly diminished (approximately 50%), after brief alcohol exposure. Fluorescent imaging results were consistent with the electrophysiology and suggest that the increased current density of VGCCs after brief exposure is attributable to combined synthesis of 1.2 and 1.3 subtypes of the L-type VGCC and redistribution of channel protein into terminal plasma membrane. CONCLUSIONS: These data indicate that a brief alcohol exposure affects subsequent alcohol sensitivity of VGCCs and neuropeptide release from presynaptic terminals.

Measuring intrinsic optical signals from Mammalian nerve terminals.

Intrinsic optical changes (light scattering signals) occur in mammalian nerve terminals during and immediately following the arrival of the action potential. In the neurohypophysis (posterior pituitary gland), the action potential is coupled to calcium-mediated secretion of the neuropeptides oxytocin and vasopressin. This excitation-secretion coupling is intimately related to extremely rapid changes in light scattering. These optical signals provide a millisecond-time-resolved monitor of events in the terminals that follow the arrival of the action potential and the entry of calcium. Light scattering procedures are designed to measure intrinsic optical signals from mammalian nerve terminals. In practice, these signals are remarkably simple to record from any of the mammalian neurohypophyses that have been studied. To date, this approach has been used successfully in mouse, rat, and guinea pig. This protocol provides instrumentation requirements and a method for preparation of the neurohypophysis so that intrinsic optical signals can be measured from nerve terminals. It also includes a discussion of the interpretation of the signals that are obtained.

Adaptation of Camelus dromedarius pars nervosa of the hypophysis to winter and summer living conditions.

The aim of this work is to study the characteristics of the dromedary nervous lobe and determine how the seasons condition its organization. To this end, electron microscopy was performed and examined quantitatively on animals from winter and summer periods. The results show a higher number of cells in the nervous lobe in summer than in winter. The most abundant glial elements in winter are light pituicytes engulfing neurosecretory nerve fibers making neuroglial contact, and dark pituicytes containing numerous heterogeneous light bodies. In summer, the most distinctive glial cells may be pituicytes in a phagocytic state making contact with characteristic large light bodies that could represent a degenerative process of large neuropeptide storage. Granular pituicytes were also observed in contact with glial and neuronal components. However, lipid droplets, described in pituicytes of other mammals, were not observed in our samples. Quantitative analysis of neurovascular contacts revealed that the number of nerve terminals contacting the basal lamina did not differ between summer and winter, but the mean number of glial processes increased in winter. Our data provides evidence that the storage of neuropeptides is very marked in summer and that, associated with an autophagic and phagocytic phenomenon, this suggests an adaptation to anticipate any situation that would cause dehydration of the dromedary. Thus, in its tough environment, the animal remains permanently prepared to avoid any large water loss.

Vasopressin secretion control: central neural pathways, neurotransmitters and effects of drugs.

Vasopressin (AVP) secretion and release are regulated by a number of central nervous system sites that receive peripheral signals from the osmoreceptors and baroreceptors. Aim of this paper is to review anatomical pathways and neurotransmitters involved as well as drugs affecting AVP secretion.

The neurohypophysis: fishing for new insights.

The neurohypophysis is a neurovascular interface through which the brain regulates peripheral organs to maintain homeostasis. The molecular mechanisms underlying its formation are poorly understood, although the emergence of new genetic and imaging tools has begun to yield new insights. In a recent study, researchers discovered that, in embryonic zebrafish, oxytocin secreted from hypophyseal axons serves as a local angiogenic cue that pulls in nearby blood vessels.

Posterior pituitary functions are not altered after growth hormone replacement therapy in hypopituitarism due to Sheehan's syndrome.

OBJECTIVE: The aim of the present study was to investigate the effects of growth hormone (GH) replacement on posterior pituitary functions of GH-deficient Sheehan's syndrome (SS) patients. DESIGN: Ten patients with SS and 14 healthy control women were included in this prospective study. All patients were given appropriate hormone replacement therapy other than GH, according to present hormone deficiencies. Patients were euthyroid and eucortisolemic at the time of baseline evaluation. Patients and the control group were evaluated with water-deprivation and saline-infusion tests at baseline and the tests were repeated in patients with SS after 3 months of GH replacement therapy. RESULTS: According to the water deprivation test, 3 patients had partial central DI at baseline. Urine osmolalities of the patients were slightly lower and plasma osmolalities were significantly higher than the control group at baseline, after water deprivation and following DDAVP injection and after hypertonic saline infusion. The osmotic threshold of serum for thirst perception was found to be significantly higher in SS patients than the control group, GH replacement therapy did not influence the results of water deprivation and saline infusion tests in SS patients. CONCLUSION: Patients with SS have subtle abnormalities in posterior pituitary functions and the threshold for thirst perception is increased. However GH replacement therapy does not seem to reverse or adversely affect the mildly deteriorated posterior pituitary functions of SS patients.

Modulation/physiology of calcium channel sub-types in neurosecretory terminals.

The hypothalamic-neurohypophysial system (HNS) controls diuresis and parturition through the release of arginine-vasopressin (AVP) and oxytocin (OT). These neuropeptides are chiefly synthesized in hypothalamic magnocellular somata in the supraoptic and paraventricular nuclei and are released into the blood stream from terminals in the neurohypophysis. These HNS neurons develop specific electrical activity (bursts) in response to various physiological stimuli. The release of AVP and OT at the level of neurohypophysis is directly linked not only to their different burst patterns, but is also regulated by the activity of a number of voltage-dependent channels present in the HNS nerve terminals and by feedback modulators. We found that there is a different complement of voltage-gated Ca(2+) channels (VGCC) in the two types of HNS terminals: L, N, and Q in vasopressinergic terminals vs. L, N, and R in oxytocinergic terminals. These channels, however, do not have sufficiently distinct properties to explain the differences in release efficacy of the specific burst patterns. However, feedback by both opioids and ATP specifically modulate different types of VGCC and hence the amount of AVP and/or OT being released. Opioid receptors have been identified in both AVP and OT terminals. In OT terminals, µ-receptor agonists inhibit all VGCC (particularly R-type), whereas, they induce a limited block of L-, and P/Q-type channels, coupled to an unusual potentiation of the N-type Ca(2+) current in the AVP terminals. In contrast, the N-type Ca(2+) current can be inhibited by adenosine via A(1) receptors leading to the decreased release of both AVP and OT. Furthermore, ATP evokes an inactivating Ca(2+)/Na(+)-current in HNS terminals able to potentiate AVP release through the activation of P2X2, P2X3, P2X4 and P2X7 receptors. In OT terminals, however, only the latter receptor type is probably present. We conclude by proposing a model that can explain how purinergic and/or opioid feedback modulation during bursts can mediate differences in the control of neurohypophysial AVP vs. OT release.

Socially modulated cell proliferation is independent of gonadal steroid hormones in the brain of the adult green treefrog (Hyla cinerea).

Gonadal steroid hormones have been shown to influence adult neurogenesis in addition to their well-defined role in regulating social behavior. Adult neurogenesis consists of several processes including cell proliferation, which can be studied via 5-bromo-2'-deoxyuridine (BrdU) labeling. In a previous study we found that social stimulation altered both cell proliferation and levels of circulating gonadal steroids, leaving the issue of cause/effect unclear. In this study, we sought to determine whether socially modulated BrdU-labeling depends on gonadal hormone changes. We investigated this using a gonadectomy-implant paradigm and by exposing male and female green treefrogs (Hyla cinerea) to their conspecific chorus or control stimuli (i.e. random tones). Our results indicate that socially modulated cell proliferation occurred independently of gonadal hormone levels; furthermore, neither androgens in males nor estrogen in females increased cell proliferation in the preoptic area (POA) and infundibular hypothalamus, brain regions involved in endocrine regulation and acoustic communication. In fact, elevated estrogen levels decreased cell proliferation in those brain regions in the implanted female. In male frogs, evoked calling behavior was positively correlated with BrdU-labeling in the POA; however, statistical analysis showed that this behavior did not mediate socially induced cell proliferation. These results show that the social modulation of cell proliferation can occur without gonadal hormone involvement in either male or female adult anuran amphibians, and confirms that it is independent of a behavioral response in males.

Ghrelin immunoexpression in the human hypophysis.

The aim of this study was to immunohistochemically localize ghrelin in autopsy-obtained, nontumoral human pituitaries. Double immunostaining was also undertaken to determine the pituitary cell type expressing both adenohypophysial hormones and ghrelin. Results showed that ghrelin is present in the adenohypophysis, its immunoexpression being cytoplasmic, weak-to-moderate, and localized to a subset of cells. Double immunostaining showed that ghrelin is present in 51% to 90% of growth hormone-producing, luteinizing-producing, and α-subunit-producing cells. Ghrelin immunoexpression was less frequently observed in other adenohypophysial cell types, being seen in 30% of adrenocorticotropin and follicle-stimulating hormones, 15% of thyrotropin, and 10% of prolactin-immunoreactive cells. Ghrelin immunopositivity was also seen in nerve fibers and Herring bodies of the neurohypophysis and pituitary stalk. More work is needed to elucidate the role of ghrelin in adenohypophysial and neurohypophysial endocrine activity. It may well be that ghrelin exerts an autocrine/paracrine effect and can modulate hormone synthesis and release.

Distribution of osmoregulatory peptides and neuronal-glial configuration in the hypothalamic magnocellular nuclei of desert rodents.

The desert rodents Psammomys obesus and Gerbillus tarabuli live under extreme conditions and overcome food and water shortage by modes of food and fluid intake specific to each species. Using immunohistochemistry and electron microscopy, we found that the hypothalamic magnocellular nuclei, and in particular, their vasopressinergic component, is highly and similarly developed in Psammomys and Gerbillus. In comparison to other rodents, the hypothalamus in both species contains more magnocellular VP neurons that, together with oxytocin neurons, accumulate in distinct and extensive nuclei. As in dehydrated rodents, many magnocellular neurons contained both neuropeptides. A striking feature of the hypothalamic magnocellular system of Psammomys and Gerbillus was its display of ultrastructural properties related to heightened neurosecretion, namely, a significant reduction in glial coverage of neuronal somata and dendrites in the hypothalamic nuclei. There were many neuronal elements whose surfaces were directly juxtaposed and shared the same synapses. Their magnocellular nuclei also showed a high level of sialylated isoform of the Neural Cell Adhesion Molecule (PSA-NCAM) that underlies their capacity for neuronal and glial plasticity. These species thus offer striking models of structural neuronal and glial plasticity linked to natural conditions of heightened neurosecretion.

Changes in angiotensin II receptor bindings in the hen neurohypophysis before and after oviposition.

The present study was performed to elucidate whether the angiotensin II (ANG II) receptor exists in the plasma membrane fraction of the neurohypophysis in hens, to estimate the time of action of ANG II on the neurohypophysis before and after oviposition, and to examine relationships between the action of ANG II on the neurohypophysis and those of estrogen and prostaglandin F(2α) (PGF(2α)) in relation to arginine vasotocin (AVT) release. The specific binding had a binding specificity to chicken ANG II (cANG II), reversibility, and saturation in the [(125)I]cANG II binding assay. Scatchard analysis revealed that the binding sites are of a single class. The equilibrium dissociation constant (K(d)) obtained by kinetic analysis and Scatchard analysis suggested a high affinity, and the maximum binding capacity (B(max)) obtained by Scatchard analysis suggested a limited capacity. These results suggest that an ANG II receptor exists in the neurohypophysis of hens. The K(d) and the B(max) value was significantly smaller in laying hens than in nonlaying hens, which suggests that bindings of the cANG II receptor change, depending on the difference in laying condition. Values of the K(d) and the B(max) decreased approximately 15 min before oviposition in laying hens, and decreased 1 h after an intramuscular injection of estradiol-17ß and 5 min after an intravenous injection of cANG II in nonlaying hens. The amount of specific binding of PGF(2α) receptor in the neurohypophysis also decreased and AVT concentration in blood increased after the cANG II injection. It seems likely that the action of cANG II in the neurohypophysis increases due to the effect of estrogen approximately 15 min before oviposition, and the cANG II action stimulates AVT release through the increase in the PGF(2α) action in this tissue.

Effect of estradiol-17ß on calcitonin receptor bindings in the hen neurohypophysis.

The present study was performed to elucidate whether estradiol-17ß (E2) would affect calcitonin (CT) receptor binding in the hen neurohypophysis. The equilibrium dissociation constant (K(d)) and the maximum binding capacity (B(max)) of the CT receptor in the plasma membrane fraction of the hen neurohypophysis were examined by Scatchard analysis of specific binding of (125)I-labeled chicken CT. A single i.m. injection of E2 into nonlaying hens caused a decrease in K(d) and B(max) values of the CT receptor. The K(d) and B(max) values of the CT receptor were smaller in laying hens than in nonlaying hens. The present study suggests that E2 may increase the action of CT on the neurohypophysis in hens.

Potassium accumulation as dynamic modulator of neurohypophysial excitability.

Activity-dependent modulation of excitable responses from neurohypophysial axons and their secretory swellings has long been recognized as an important regulator of arginine vasopressin and oxytocin release during patterned stimulation. Various activity-dependent mechanisms, including action potential broadening, potassium accumulation, and autocrine or paracrine feedback, have been proposed as underlying mechanisms. However, the relevance of any specific mechanism on net excitability in the intact preparation, during different levels of overall activation, and during realistic stimulation with trains of action potentials has remained largely undetermined. Using high-speed optical recordings and potentiometric dyes, we have quantified the dynamics of global excitability under physiologically more realistic conditions, that is in the intact neurohypophysis during trains of stimuli at varying frequencies and levels of overall activity. Net excitability facilitated during stimulation at low frequencies or at low activity. During persistent high-intensity or high-frequency stimulation, net excitability became severely depressed. Depression of excitable responses was strongly affected by manipulations of extracellular potassium levels, including changes to resting [K(+)](out), increases of interstitial spaces with hypertonic solutions and inhibition of Na(+)/K(+) ATPase activity. Application of the GABA(A) receptor blocker bicuculline or manipulations of Ca(2+) influx showed little effect. Numerical simulation of K(+) accumulation on action potentials of individual axons reproduced optically recorded population responses, including the overall depression of action potential (AP) amplitudes, modest AP broadening and the prominent loss of hyperpolarizing undershoots. Hence, extracellular potassium accumulation dominates activity-dependent depression of neurohypophysial excitability under elevated stimulation conditions. The intricate dependence on the short-term stimulation history and its resulting feedback on neurohypophysial excitability renders [K(+)](out) accumulation a surprisingly complex mechanism for regulating axonal excitability and subsequent neuroendocrine release.