Vascular-derived SPARC and SerpinE1 regulate interneuron tangential migration and accelerate functional maturation of human stem cell-derived interneurons.

Cortical interneurons establish inhibitory microcircuits throughout the neocortex and their dysfunction has been implicated in epilepsy and neuropsychiatric diseases. Developmentally, interneurons migrate from a distal progenitor domain in order to populate the neocortex - a process that occurs at a slower rate in humans than in mice. In this study, we sought to identify factors that regulate the rate of interneuron maturation across the two species. Using embryonic mouse development as a model system, we found that the process of initiating interneuron migration is regulated by blood vessels of the medial ganglionic eminence (MGE), an interneuron progenitor domain. We identified two endothelial cell-derived paracrine factors, SPARC and SerpinE1, that enhance interneuron migration in mouse MGE explants and organotypic cultures. Moreover, pre-treatment of human stem cell-derived interneurons (hSC-interneurons) with SPARC and SerpinE1 prior to transplantation into neonatal mouse cortex enhanced their migration and morphological elaboration in the host cortex. Further, SPARC and SerpinE1-treated hSC-interneurons also exhibited more mature electrophysiological characteristics compared to controls. Overall, our studies suggest a critical role for CNS vasculature in regulating interneuron developmental maturation in both mice and humans.

MCH Neurons Regulate Permeability of the Median Eminence Barrier.

Melanin-concentrating hormone (MCH)-expressing neurons are key regulators of energy and glucose homeostasis. Here, we demonstrate that they provide dense projections to the median eminence (ME) in close proximity to tanycytes and fenestrated vessels. Chemogenetic activation of MCH neurons as well as optogenetic stimulation of their projections in the ME enhance permeability of the ME by increasing fenestrated vascular loops and enhance leptin action in the arcuate nucleus of the hypothalamus (ARC). Unbiased phosphoRiboTrap-based assessment of cell activation upon chemogenetic MCH neuron activation reveals MCH-neuron-dependent regulation of endothelial cells. MCH neurons express the vascular endothelial growth factor A (VEGFA), and blocking VEGF-R signaling attenuates the leptin-sensitizing effect of MCH neuron activation. Our experiments reveal that MCH neurons directly regulate permeability of the ME barrier, linking the activity of energy state and sleep regulatory neurons to the regulation of hormone accessibility to the ARC.

Excitatory effects of the puberty-initiating peptide kisspeptin and group I metabotropic glutamate receptor agonists differentiate two distinct subpopulations of gonadotropin-releasing hormone neurons.

Activation of the G-protein-coupled receptor GPR54 by kisspeptins during normal puberty promotes the central release of gonadotropin-releasing hormone (GnRH) that, in turn, leads to reproductive maturation. In humans and mice, a loss of function mutations of GPR54 prevents the onset of puberty and leads to hypogonadotropic hypogonadism and infertility. Using electrophysiological, morphological, molecular, and retrograde-labeling techniques in brain slices prepared from vGluT2-GFP and GnRH-GFP mice, we demonstrate the existence of two physiologically distinct subpopulations of GnRH neurons. The first subpopulation is comprised of septal GnRH neurons that colocalize vesicular glutamate transporter 2 and green fluorescent protein and is insensitive to metabotropic glutamate receptor agonists, but is exquisitely sensitive to kisspeptin which closes potassium channels to dramatically initiate a long-lasting activation in neurons from prepubertal and postpubertal mice of both sexes. A second subpopulation is insensitive to kisspeptin but is uniquely activated by group I metabotropic glutamate receptor agonists. These two physiologically distinct classes of GnRH cells may subserve different functions in the central control of reproduction and fertility.

Vascular endothelial cells promote acute plasticity in ependymoglial cells of the neuroendocrine brain.

Glial and endothelial cells interact throughout the brain to define specific functional domains. Whether endothelial cells convey signals to glia in the mature brain is unknown but is amenable to examination in circumventricular organs. Here we report that purified endothelial cells of one of these organs, the median eminence of the hypothalamus, induce acute actin cytoskeleton remodeling in isolated ependymoglial cells and show that this plasticity is mediated by nitric oxide (NO), a diffusible factor. We found that both soluble guanylyl cyclase and cyclooxygenase products are involved in this endothelial-mediated control of ependymoglia cytoarchitecture. We also demonstrate by electron microscopy that activation of endogenous NO release in the median eminence induces rapid structural changes, allowing a direct access of neurosecretory axons containing gonadotropin-releasing hormone (GnRH) (the neuropeptide controlling reproductive function) to the portal vasculature. Local in vivo inhibition of NO synthesis disrupts reproductive cyclicity, a process that requires a pulsatile, coordinated delivery of GnRH into the hypothalamic-adenohypophyseal portal system. Our results identify a previously unknown function for endothelial cells in inducing neuroglial plasticity and raise the intriguing possibility that endothelial cells throughout the brain may use a similar signaling mechanism to regulate glial-neuronal interactions.

Variation of endothelial nitric oxide synthase synthesis in the median eminence during the rat estrous cycle: an additional argument for the implication of vascular blood vessel in the control of GnRH release.

Recent studies from our laboratory suggested that the vascular endothelium of the median eminence was involved via nitric oxide secretion in the modulation of GnRH release during the estrous cycle. To further investigate that issue, we studied the variations of endothelial nitric oxide synthase protein and mRNA in the median eminence of female rats killed at different time points of the day and/or of the estrous cycle. Endothelial nitric oxide synthase protein levels were measured by Western blot, and endothelial nitric oxide synthase mRNA analysis was performed with semiquantitative RT-PCR (for each time point, n = 4). The results revealed that endothelial nitric oxide synthase synthesis varied markedly across the estrous cycle. Indeed, endothelial nitric oxide synthase protein (n = 20) and mRNA (n = 16) levels increase significantly on 0800 h and 1600 h proestrus compared with 1400 h diestrus II. In a second step, quantification analysis were made in median eminence obtained from ovariectomized and ovariectomized, E2 benzoate primed rat. The results show a significant increase in expression of endothelial nitric oxide synthase protein as well as endothelial nitric oxide synthase mRNA in ovx-E2 primed rat median eminence. Concurrently, the levels of the cav-1 protein, a specific endogenous inhibitor of endothelial nitric oxide synthase, were measured in median eminence during estrous cycle and in ME from ovx and ovx-E2 primed rats. A significant decrease of median eminence cav-1 was noted on 1600 h proestrus and in ovx-E2 primed rats when compared with 1400 h diestrus II and ovx, respectively. Altogether, these results strongly suggest that high NO release from median eminence observed on proestrus may be due to an increase of endothelial nitric oxide synthase expression and a decrease of the cav-1 protein levels. These findings demonstrate that E2 is able to modulate endothelial nitric oxide synthase and cav-1 expression both during the estrous cycle and in experimental conditions and consequently reinforce the idea that nitric oxide acting on GnRH release, is essentially endothelial in origin. These results may also imply that variations of endothelial nitric oxide synthase expression are essential for the pulsatile/cyclic nitric oxide median eminence release observed in a previous study.

A second look at the barriers of the medial basal hypothalamus.

The cell bodies of hypothalamic secretory neurons are localized in areas protected by the blood-brain barrier (BBB), whereas their axon terminals are localized in the median eminence, which lacks a BBB. This implies a complex barrier system, allowing neurons of the central nervous system to secrete into the blood stream without making the BBB leaky. In the present study, three experimental protocols were applied to clarify certain relevant aspects of the barriers operating in the medial basal hypothalamus of the rat. We established that the milieu of the arcuate nucleus is exposed to both the ventricular and the subarachnoidal cerebrospinal fluid (CSF). The median eminence milieu, the perivascular space of the portal vessels, and the subarachnoid space appear to be in open communication; also, beta2-tanycytes establish an efficient barrier between the median eminence milieu and the ventricular CSF. Similarly, beta1-tanycytes establish a lateral barrier, separating the intercellular space of the median eminence from that of the arcuate nucleus. We also found that the glucose transporter I (GLUT I), a BBB marker, is localized throughout the whole plasma membrane of beta1-tanycytes, but is missing from beta2-tanycytes. Expression of GLUT I by tanycytes progressively develops during the first postnatal weeks; while the degree of damage of the arcuate nucleus by administration of monosodium glutamate, at different postnatal intervals, parallels that of the GLUT I immunoreactivity of beta1-tanycytes. An explanation is offered for the selective destruction of the arcuate neurons by the parenteral administration of monosodium glutamate to infant rats.

Morphological aspects of the median eminence--place of accumulation and secretion of regulatory neurohormones and neuropeptides.

The median eminence (ME) is a small brain area forming both the structural and functional bridge between the hypothalamus and the hypophysis. It is supplied by a variety of neurohormones and neuropeptides which are delivered to the ME by different hypothalamic and extrahypothalamic pathways. These biologically active substances may act in the ME locally influencing the activity of secretion of the neighbouring terminals or, after being released from the neuronal endings into the network of fenestrated capillaries and transported to the hypophysis, they may be involved in the regulation of secretion of adenohypophyseal hormones. Recent demonstrations of extensive colocalizations of these biologically active substances in individual axonal endings in the ME with wide spectrum of biological actions further emphasizes the ME as an important place involved in the neuroendocrine regulatory processes.

Dynamic changes in LHRH neurovascular terminals with various endocrine conditions in adults.

Luteinizing hormone-releasing hormone (LHRH) release is required for ovulation in mammals. Although evidence for the direct action of gonadal steroids on LHRH neurons has been minimal, their importance in inducing the preovulatory surge of LHRH is unequivocal. We have identified a subgroup of LHRH neurons with progestin receptors in guinea pigs. Given their central position, these neurons may constitute foci of initial activity, which are amplified throughout the population of LHRH neurons, resulting in increased LHRH neurosecretion on the afternoon of proestrus. Additionally, gonadal steroids may regulate LHRH secretion at the level of the terminals. Using immunoelectron microscopy and image analysis, we have illustrated the dramatic influence of gonadal steroids on individual LHRH terminals in the median eminence of rats. Indirectly, gonadal steroids may modulate LHRH release by modulating glial elements. Using double-label fluorescence confocal microscopy, we illustrate that LHRH terminals in the median eminence are encased by end-feet of tanycytes. Acting on glial elements, gonadal steroids may regulate access of LHRH terminals to the basal lamina and influence the amount of the neuropeptide reaching the portal vessels. We propose that during the preovulatory surge, LHRH release is coordinated by synergistic mechanisms operating at the level of particular subgroups of neuronal perikarya and/or discrete regions of the median eminence. These synergistic actions may ensure that LHRH is released in a precipitous fashion, to induce the surge of LH from the pituitary, required for ovulation.

The vascularization of the pituitary gland of the chicken (Gallus domesticus). A scanning electron microscope study of vascular corrosion casts.

Microvascular corrosion casts of the pituitary gland of one to nine-day-old chickens (Gallus domesticus, white leghorn hybrids) were analysed with the scanning electron microscope. Results show that the chicken median eminence is supplied by branches of the infundibular and the neural-lobe arteries. They form a flat two-dimensional primary capillary plexus, which lacks any capillary loops and is continuous with the plexus in the neural lobe. The capillaries occupy about 60% of the total area of the median eminence. A subependymal plexus is present, showing no contact with the plexus of the median eminence. The chicken neural lobe consists of many hollow buds. These buds are supplied by branches of the neural-lobe artery, which gives rise to a two-dimensional capillary meshwork similar to that of the median eminence. An anterior group of portal vessels, comprising 14-16 vessels with a mean diameter of 37 microns, and a posterior group of portal vessels, comprising 2-4 shorter and slightly thinner vessels, arise from the median eminence are the sole blood supply for the adenohypophysis (distal lobe). Anterior portal vessels supply the cephalic lobe and the most rostral area of the caudal lobe, and posterior portal vessels supply the caudal lobe of the distal lobe. There are no short portal vessels connecting the neural lobe with the distal lobe. The (sinusoidal) capillary bed of the distal lobe is laminated. The chicken hypophysis drains into the cavernous sinus, which empties into the cerebral carotid veins. Within the period studied (days 1-9 after hatching) no age-related changes were found.

Characterization of membrane-associated peptidase activities expressed by endothelial cells of the ovine median eminence.

The capillary endothelial cells of the median eminence represent a potential site for the degradation/modification of both circulating and hypothalamic peptides passing through the hypophysial portal system toward the pituitary. This study examines endothelial cell peptidase expression in vitro by monitoring the metabolism of gonadotropin-releasing hormone (GnRH) by cultured endothelial cells from sheep median eminence. Cleavage of GnRH by median eminence endothelial cell membranes generated GnRH1-5 as the primary stable product, which was then degraded to GnRH1-3 and free amino acids. Degradation of GnRH was completely inhibited by TPCK, ZnCl2 and N-ethylmaleimide, and partially inhibited by EDTA and by a specific inhibitor of the metalloendopeptidase EC 3.4.24.15, CFP-AAY-pAB. Interestingly, an increase in GnRH1-9 production was seen with the latter inhibitors, suggesting a two-step mechanism of GnRH degradation involving a primary cleavage at the Pro9-Gly10-NH2 bond, inhibitable by TPCK, ZnCl2, and NEM, followed by cleavage by EC 3.4.24.15 to generate GnRH1-5. Phosphoramidon and angiotensin converting enzyme inhibitors (as well as other non-specific inhibitors) were without effect, indicating that endopeptidase EC 3.4.24.11 and angiotensin converting enzyme are not involved. Neither bovine aortic endothelial cell nor AtT-20 cell membranes exhibited this pattern of peptidase activity. Degradation of GnRH by intact median eminence endothelial cells in culture was also observed, suggesting an extracellular orientation for these enzymes; the potential role of such peptidases in the fine regulation of both pituitary function and local blood flow is currently under investigation.

Light microscopic study of the hypophyseal angioarchitecture in the rabbit.

This study describes the hypophyseal angioarchitecture found in 79 adult New Zealand white rabbits. The pituitary glands and attached hypothalami were removed and carefully processed following routine histological methods, and the vascular organization was studied by light microscopy. Whole mounts of the pituitary median eminence complex were prepared and studied with a binocular dissecting microscope employing transmitted and epi-illumination. Arterial blood was found to be directed primarily to the neurohypophysis by the superior hypophyseal artery (SHA) and the inferior hypophyseal artery (IHA). A direct arterial blood supply was found to the adenohypophysis, but was limited solely to the pars intermedia by branches of the anterior hypophyseal artery (AHA) and the IHA. Capillaries of the pars intermedia were subdivided into an intermediate and a superficial plexus. The superficial plexus was situated between the intermediate plexus and the capillaries of the infundibular process. Capillaries of the superficial plexus did not form anastomoses between themselves, but ramified into the intermediate plexus to form a dense network of anastomosing capillaries that were continuous with capillaries of the pars distalis. A direct arterial blood supply was found only to the superficial plexus.

Some blood vessels in the rat median eminence are surrounded by a dense plexus of vasoactive intestinal polypeptide/peptide histidine isoleucine (VIP/PHI) immunoreactive nerves.

Using immunohistochemistry at the light and electron microscopic level some blood vessels along the median eminence were shown to be surrounded by dense networks of vasoactive intestinal polypeptide/peptide histidine isoleucine (VIP/PHI)-positive fibers. VIP and PHI released from these fibers may contribute to the elevated levels of these two peptides measured in portal blood as compared to peripheral blood by radioimmunoassay. VIP and PHI may also be important in the control of blood flow through the median eminence.

Fine structure and vascular supply of the median eminence (ME) in Acipenser ruthenus (Chondrostei).

The fine structure and vascular supply of the median eminence (ME) was studied in 57 specimens of Acipenser ruthenus (Chondrostei) by means of light microscopy (normal histology, horseradish-peroxidase (HRP), formaldehyde induced fluorescence (FIF), scanning electron microscopy (SEM) and transmission electron microscopy (TEM) as well as by SEM of vascular corrosion casts. Light microscopy revealed that the ME increases in thickness from caudal to rostral with capillaries invading the subependymal layers only at the rostral median eminence (rME). At the middle (mME) and caudal median eminence (cME) capillaries are limited to the ME outer surface. Short term HRP-application (systemically as well as intraventricularly) resulted in reaction product in the intercellular space throughout the ME after a survival time of 2 hours. The ME tanycytes were distinctly marked after 7 days survival. FIF revealed aminergic cerebrospinal fluid (CSF-) contacting neurons; also the apical part of tanycytes showed specific fluorescence. SEM of vascular corrosion casts showed that the arterial supply of the whole ME results from branches of the hypothalamic artery and/or from those arteries supplying the saccus vasculosus. The whole 2-dimensional primary capillary plexus of the ME drains via portal vessels into the adenohypophysis. As demonstrated by SEM, the ME ependymal surface is made up by oligociliated tanycytes, abundant crown cells and intraventricular protrusions of CSF-contacting neurons. Supraependymal cells are lacking, supraependymal fibres are very sparse. TEM revealed the ME tanycytes with long ciliary rootlets. Their basal processes split into numerous delicate branches forming many end-feet, which end at the basal lamina opposite the fenestrated capillaries of the primary plexus. Like tanycytes, also the crown cells possess branching processes which may contribute to the outer glial membrane. CSF-contacting neurons of the type I (with dense core vesicles ranging in diameter from 100-120 nm and of presumed aminergic nature) and type II (with dense core vesicles ranging in diameter from 160-180 nm and of presumed peptidergic nature) were found. Both types are most abundant in the cME. Small astrocytes reveal delicate processes which cover parts of the CSF-contacting neurons and of tanycytes. Another glial cell type is situated near the basal lamina and forms processes parallel the latter. There are few axo-somal and axo-axonal synapses pointing rather to a humoral regulation of the ME with signals from the ME blood vascular bed or from the CSF acting on CSF-contacting neurons.

Neurohemal contact in the internal zone of the rabbit median eminence.

The concept of neurosecretion as the mechanism by which neural control of adenohypophyseal function is accomplished was based on the observation that long capillary loops penetrate deeply into the supraopticohypophyseal tract as it passes through the median eminence internal zone. However, neural contact upon these capillary loops has not been demonstrated in the mammalian median eminence. The present transmission electron microscopic investigation of the rabbit median eminence demonstrates neurohemal contact in the median eminence internal zone. Axons containing small lucent vesicles 53.3 +/- 3.28 nm in diameter (mean +/- SEM) or small lucent and large granular vesicles with a mean diameter of 122.4 (+/- 3.28 nm) in their terminals make neurohemal contact with capillary loops in the internal zone and form a cuff about them. These terminals resemble terminals found in the external zone. Intravenous injection of the false neurotransmitter 5-hydroxydopamine (5-OH-DA) renders small lucent vesicles granular in both the external and internal zone. The effect of 5-OH-DA injection is abolished by concurrent reserpine administration. Whereas large granular vesicles in many terminals become lucent after reserpine administration, in others they remained electron dense. Viewed in the light of previous studies our findings suggest that the internal plexus arises from the external plexus and invaginates the neuropil carrying connective tissue and parvicellular axon terminals of aminergic and peptidergic systems from the external zone into the internal zone, that some elements making neurohemal contact with long capillary loops are terminals of the noradrenergic reticular infundibular tract arising outside the hypothalamus in the brainstem, and that long capillary loops form a system of repeating microvascular modules which markedly increase the surface available for neurohemal contact.

Pituitary blood flow.

The direction of pituitary blood flow, the amount of pituitary blood flow, its regional control, and the role of the median eminence microcirculation are the subjects of this review. Present concepts of pituitary blood flow are focused almost entirely on its direction and arouse from studies of pituitary vascular anatomy performed almost 50 years ago. The development of new anatomic techniques has led to a reappraisal of pituitary angioarchitecture, stimulated physiological studies to clarify the pattern of blood flow within the entire gland, and led to a reappraisal of accepted concepts of directional pituitary blood flow. The availability of techniques to accurately measure organ blood flow has permitted study of pituitary blood flow; and, when combined with knowledge of pituitary anatomy, the application of these techniques promises to provide a means to develop insight into control of the mechanisms by which chemical messengers are delivered to the pituitary to control its function. New anatomic techniques promise to develop new understanding of the three-dimensional arrangement of median eminence microvasculature and yield new concepts of blood flow regulation within the median eminence that can be tested by physiological means.

The vascular architecture of the developing pituitary-median eminence complex in the rat.

The development of the pituitary gland and its blood vessels is described in rat embryos (gestational day 12 through day 21) injected with India-ink via the umbilical vessels. The vascularization of all components of the pituitary gland develops from the surface network covering the prosencephalic vesicle. However, vascular connections exist between the prospective median eminence and the anterior pituitary gland in the earliest stages examined (day 12) but are not augmented by vessels from the stomatodeal roof until day 13. Primary portal veins appear initially on day 13, the vascularization of the pars distalis is visible on day 15. The "Mantelplexus" covering the floor of the diencephalon is discernible on day 16. Large-caliber portal veins appear immediately before birth, but otherwise there is no significant change in the vascular pattern during the last five days of gestation. The pars intermedia and the median eminence-pituitary stalk region remain avascular throughout embryonic life.

Observations on the hypothalamo-hypophyseal portal vasculature in the developing human fetus.

Human fetuses, 11.5 to 16.8 weeks' gestational age, were perfused with silicone rubber compound to study the hypothalamo-hypophyseal portal system. Impregnated vessels were observed throughout the median eminence, infundibular stalk, pars tuberalis and both lobes of the pituitary gland in all specimens. The data suggest that an intact portal system for the transport of hypothalamic releasing factors to the pituitary gland is established as early as 11.5 weeks' gestation.

Neural control of gonadotropin secretion in primates.

In the rhesus monkey, there is abundant evidence to indicate that ovarian secretions, mainly estradiol-17beta, control "tonic" as well as "cyclic" secretion of gonadotropins during the menstrual cycle. This mechanism of control ensures coordination of ovarian morphology and anterior pituitary secretory patterns. The primary site of action of estradiol in controlling both "tonic" and "cyclic" secretion of gonadotropins has been circumscribed to the medial basal hypothalamic-pituitary unit. A modulatory role in "cyclic" secretion by neural structures situated within the anterior hypothalamic-preoptic area or by efferent fibers in passage through this region also has been postulated. However, the accrued evidence indicates that in the primate, contrary to the rodent, the role of these rostral neural structures is not essential for menstrual cyclicity. Strong evidence also indicates that the isolated pituitary gland can respond to estrogen signals as well. Secretion of gonadotropin-releasing hormone, the hypothalamic decapeptide, into the long portal vessels is, however, essential to maintain function of the gonadotroph. Further, pulsatile release of luteinizing hormone is distinctly under the control of a similarly paced hypothalamic clock.

The neurohypophyseal capillary bed. II. Specializations within median eminence.

Vascular casts of the pituitary gland, median eminence and hypothalamus from several mammalian species were examined by scanning electron microscopy. These observations were correlated with light microscopic studies of injected, cleared median eminence-pituitary specimens and with light microscopic examination of serial sections of injected hypothalamic, median eminence, and pituitary specimens employing reflected lighting or epi-illumination. Transmission electron microscopy was employed to study long portal vessels on the ventral surface of the rat median eminence. In each of the species examined, the median eminence (infundibular) capillary bed is subdivided into an external and an internal plexus. The external plexus (the neurohemal contact zone) receives an arterial supply from the superior hypophyseal arteries and is continuous with the capillary bed of the infundibular stem and process. Egress from the external plexus is possible via three vascular routes: (a) by fenestrated portal vessels and capillaries to the adenohypophysis, (b) by capillary connections to the medial basilar hypothalamus and, (c) by internal plexus capillaries to the ependyma of the median eminence. Median eminence vasculature is structurally organized to deliver (1) hypothalamic and neurohypophyseal peptides to the glandular pituitary via portal vessels, (2) hypothalamic and pituitary secretions to the medial basilar hypothalamus via capillaries, and (3) hypothalamic and pituitary secretions to distant brain sites through cerebrospinal fluid via ventricular and subarachnoid routes.