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.

Blood-to-brain communication in the hypothalamus for energy intake regulation.

The arcuate nucleus (Arc) integrates circulating hormonal and metabolic signals to control energy expenditure and intake. One of the most important routes that enables the Arc to sense circulating molecules is through the median eminence (ME), which lacks a typical blood-brain barrier. However, the mechanism by which circulating molecules reach the Arc neurons remains unclear. This review focuses on what is known to date regarding the special structure and permeability of the ME vasculature and active transport of circulating molecules from the ME to the Arc. Recent studies have demonstrated that the ME displays angiogenic behavior that is expected to provide high vascular permeability. Parenchymal diffusion of circulating molecules from the ME vasculature is size-dependent, and tanycytes actively transport circulating molecules from the ME to the Arc. Finally, we highlight structural plasticity of the Arc and ME as playing an important role in maintaining energy balance homeostasis.

Vascular Influence on Ventral Telencephalic Progenitors and Neocortical Interneuron Production.

The neocortex contains glutamatergic excitatory neurons and γ-aminobutyric acid (GABA)ergic inhibitory interneurons. Extensive studies have revealed substantial insights into excitatory neuron production. However, our knowledge of the generation of GABAergic interneurons remains limited. Here we show that periventricular blood vessels selectively influence neocortical interneuron progenitor behavior and neurogenesis. Distinct from those in the dorsal telencephalon, radial glial progenitors (RGPs) in the ventral telencephalon responsible for producing neocortical interneurons progressively grow radial glial fibers anchored to periventricular vessels. This progenitor-vessel association is robust and actively maintained as RGPs undergo interkinetic nuclear migration and divide at the ventricular zone surface. Disruption of this association by selective removal of INTEGRIN ß1 in RGPs leads to a decrease in progenitor division, a loss of PARVALBUMIN and SOMATOSTATIN-expressing interneurons, and defective synaptic inhibition in the neocortex. These results highlight a prominent interaction between RGPs and periventricular vessels important for proper production and function of neocortical interneurons.

Rapid sensing of circulating ghrelin by hypothalamic appetite-modifying neurons.

To maintain homeostasis, hypothalamic neurons in the arcuate nucleus must dynamically sense and integrate a multitude of peripheral signals. Blood-borne molecules must therefore be able to circumvent the tightly sealed vasculature of the blood-brain barrier to rapidly access their target neurons. However, how information encoded by circulating appetite-modifying hormones is conveyed to central hypothalamic neurons remains largely unexplored. Using in vivo multiphoton microscopy together with fluorescently labeled ligands, we demonstrate that circulating ghrelin, a versatile regulator of energy expenditure and feeding behavior, rapidly binds neurons in the vicinity of fenestrated capillaries, and that the number of labeled cell bodies varies with feeding status. Thus, by virtue of its vascular connections, the hypothalamus is able to directly sense peripheral signals, modifying energy status accordingly.

Aquaporin-1 in blood vessels of rat circumventricular organs.

Although the water channel protein aquaporin-1 (AQP1) is widely observed outside the rat brain in continuous, but not fenestrated, vascular endothelia, it has not previously been observed in any endothelia within the normal rat brain and only to a limited extent in the human brain. In this immunohistochemical study of rat brain, AQP1 has also been found in microvessel endothelia, probably of the fenestrated type, in all circumventricular organs (except the subcommissural organ and the vascular organ of the lamina terminalis): in the median eminence, pineal, subfornical organ, area postrema and choroid plexus. The majority of microvessels in the median eminence, pineal and choroid plexus, known to be exclusively fenestrated, are shown to be AQP1-immunoreactive. In the subfornical organ and area postrema in which many, but not all, microvessels are fenestrated, not all microvessels are AQP1-immunoreactive. In the AQP1-immunoreactive microvessels, the AQP1 probably facilitates water movement between blood and interstitium as one component of the normal fluxes that occur in these specialised sensory and secretory areas. AQP1-immunoreactive endothelia have also been seen in a small population of blood vessels in the cerebral parenchyma outside the circumventricular organs, similar to other observations in human brain. The proposed development of AQP1 modulators to treat various brain pathologies in which AQP1 plays a deleterious role will necessitate further work to determine the effect of such modulators on the normal function of the circumventricular organs.

Brain-endocrine interactions: a microvascular route in the mediobasal hypothalamus.

Blood-borne hormones acting in the mediobasal hypothalamus, like those controlling food intake, require relatively direct access to target chemosensory neurons of the arcuate nucleus (ARC). An anatomical substrate for this is a permeable microvasculature with fenestrated endothelial cells in the ARC, a system that has awaited comprehensive documentation. Here, the immunofluorescent detection of endothelial fenestral diaphragms in the rat ARC allowed us to quantitate permeable microvessels throughout its rostrocaudal extent. We have determined that permeable microvessels are part of the subependymal plexus irrigating exclusively the ventromedial (vm) ARC from the subadjacent neuroendocrine median eminence. Unexpectedly, permeable microvessels were concentrated proximal to the pituitary stalk. This marked topography strongly supports the functional importance of retrograde blood flow from the pituitary to the vmARC, therefore making a functional relationship between peripheral long-loop, pituitary short-loop, and neuroendocrine ultra-short loop feedback, altogether converging for integration in the vmARC (formerly known as the hypophysiotrophic area), thereby so pivotal as a multicompetent brain endocrinostat.

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.

Protein components of the blood-brain barrier (BBB) in the mediobasal hypothalamus.

The blood-brain barrier (BBB) plays an important role in controlling the access of substances to the brain. Of the circumventricular organs (CVO), i.e. areas that lack a BBB, the median eminence and its close relationship with the hypothalamic arcuate nucleus plays an important role in controlling the entry of blood-borne substances to neurons of the mediobasal hypothalamus. In order to clarify the nature of the BBB in the median eminence-arcuate nucleus complex, we have used immunohistochemistry and antisera to protein components of the BBB-(1) tight junctions, claudin-5 and zona occludens-1 (ZO-1); (2) endothelial cells: (a) all endothelial cells: rat endothelial cell antigen-1 (RECA-1), (b) endothelial cells at BBB: endothelial barrier antigen (EBA), glucose transporter 1 (GLUT1) and transferrin receptor (TfR), and (c) endothelial cells at CVOs: dysferlin; (3) basal lamina: laminin; (4) vascular smooth muscle cells: smooth muscle actin (SMA); (5) pericytes: chondroitin sulfate proteoglycan (NG2); (6) glial cells: (a) astrocytes: glial fibrillary acidic protein (GFAP), (b) tanycytes: dopamine- and cAMP-regulated phosphoprotein of 32kDA (DARPP-32), (c) microglia: CD11b. Neuronal cell bodies located in the ventromedial aspect of the arcuate nucleus were visualized by antiserum to agouti-related protein (AgRP). The study provides a detailed analysis on the cellular localization of BBB components in the mediobasal hypothalamus. Some vessels in the ventromedial aspect of the arcuate nucleus lacked the BBB markers EBA and TfR, suggesting an absence of an intact BBB. These vessels may represent a route of entry for circulating substances to a subpopulation of arcuate nucleus neurons.

Glutamatergic innervation of the hypothalamic median eminence and posterior pituitary of the rat.

Recent studies have localized the glutamatergic cell marker type-2 vesicular glutamate transporter (VGLUT2) to distinct peptidergic neurosecretory systems that regulate hypophysial functions in rats. The present studies were aimed to map the neuronal sources of VGLUT2 in the median eminence and the posterior pituitary, the main terminal fields of hypothalamic neurosecretory neurons. Neurons innervating these regions were identified by the uptake of the retrograde tract-tracer Fluoro-Gold (FG) from the systemic circulation, whereas glutamatergic perikarya of the hypothalamus were visualized via the radioisotopic in situ hybridization detection of VGLUT2 mRNA. The results of dual-labeling studies established that the majority of neurons accumulating FG and also expressing VGLUT2 mRNA were located within the paraventricular, periventricular and supraoptic nuclei and around the organum vasculosum of the lamina terminalis and the preoptic area. In contrast, only few FG-accumulating cells exhibited VGLUT2 mRNA signal in the arcuate nucleus. Dual-label immunofluorescent studies of the median eminence and posterior pituitary to determine the subcellular location of VGLUT2, revealed the association of VGLUT2 immunoreactivity with SV2 protein, a marker for small clear vesicles in neurosecretory endings. Electron microscopic studies using pre-embedding colloidal gold labeling confirmed the localization of VGLUT2 in small clear synaptic vesicles. These data suggest that neurosecretory neurons located mainly within the paraventricular, anterior periventricular and supraoptic nuclei and around the organum vasculosum of the lamina terminalis and the preoptic area secrete glutamate into the fenestrated vessels of the median eminence and posterior pituitary. The functional aspects of the putative neuropeptide/glutamate co-release from neuroendocrine terminals remain to be elucidated.

Astrocytes in the arcuate nucleus and median eminence that take up a fluorescent dye from the circulation express leptin receptors and neuropeptide Y Y1 receptors.

Following systemic injection, several different dyes and markers are found to accumulate rapidly in cells in the arcuate nucleus and median eminence, and the capillaries in this region appear specialised for exchange of molecules. The present study used hydroxystilbamidine (FluoroGold equivalent) to identify cells that take up molecules from the circulation in these regions; 2-6 h following injection, uptake was seen in the external and intermediate zones of the median eminence and the adjacent ventral part of the arcuate nucleus, but not in other regions of the hypothalamus. The labelled cells were small; double-labelling experiments revealed that they expressed glial fibrillary acid protein (GFAP), but not NeuN, Agouti-related protein (AgRP) or beta-endorphin. They had the morphology of astrocytes and were readily distinguished from tanycytes by staining for vimentin. Many of these labelled astrocytes also expressed leptin receptors and neuropeptide Y Y1 receptors. The surrounding neurons that expressed these receptors did not take up this dye. This demonstrates that astrocytes take up molecules from the circulation in the median eminence and adjacent arcuate nucleus, and may have a significant signalling role in regulation of food intake.

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.

Expression of brain-derived neurotrophic factor and its receptors in the median eminence cells with sensitivity to stress.

The median eminence (ME) is considered as the final common pathway connecting the nervous and endocrine systems. In this neurohemal structure, dynamic interactions among nerve terminals, tanycytes, and astrocytes determine through plastic processes the neurohormones access to the portal blood. Because brain-derived neurotrophic factor (BDNF) is involved in plastic changes, we investigated its presence and that of its receptors (TrkB) in the different cellular types described in the ME. Using in situ hybridization and immunohistochemical techniques, we demonstrated that BDNF immunoreactivity was essentially located in the astrocytes and to a lesser extent in tanycytes. By contrast, BDNF was not detected in nerve terminals reaching the external layer of the ME. TrkB antibodies recognizing the extracellular receptor domain labeled all of these different cell types, suggesting an autocrine or paracrine action of BDNF at this level. More selective antibodies showed that TrkB.FL immunostaining was found in tanycytes and nerve endings, whereas TrkB.T1 immunostaining was localized in all cellular types. Immobilization stress increased BDNF mRNA and BDNF immunoreactivity patterns and induced biphasic BDNF release from the ME, as analyzed by push-pull perfusion. In addition, we observed that 60-min stress intensified BDNF immunoreactivity in the internal layer and also its colocalization with glial fibrillary acidic protein. Stress also accentuated BDNF immunostaining in the perivascular space in elements that were not labeled with antibodies recognizing fibroblast or endothelial cells. These data disclosed a novel location of BDNF and its receptors in the ME, which are presumably involved in dynamic processes such as hormone release.

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.