Reciprocal Interaction between Vascular Filopodia and Neural Stem Cells Shapes Neurogenesis in the Ventral Telencephalon.

Angiogenesis and neurogenesis are tightly coupled during embryonic brain development. However, little is known about how these two processes interact. We show that nascent blood vessels actively contact dividing neural stem cells by endothelial filopodia in the ventricular zone (VZ) of the murine ventral telencephalon; this association is conserved in the human ventral VZ. Using mouse mutants with altered vascular filopodia density, we show that this interaction leads to prolonged cell cycle of apical neural progenitors (ANPs) and favors early neuronal differentiation. Interestingly, pharmacological experiments reveal that ANPs induce vascular filopodia formation by upregulating vascular endothelial growth factor (VEGF)-A in a cell-cycle-dependent manner. This mutual relationship between vascular filopodia and ANPs works as a self-regulatory system that senses ANP proliferation rates and rapidly adjusts neuronal production levels. Our findings indicate a function of vascular filopodia in fine-tuning neural stem cell behavior, which is the basis for proper brain development.

Telencephalon Cytoarchitecture of tsinling dwarf skinks (Scincella tsinlingensis).

The telencephalon of adult Scincella tsinlingensis was detected by light and electron microscopy, which will be used as the basis for further neurobiological comparative studies. The telencephalon of S. tsinlingensis was consisted of paired olfactory bulbs, paired cerebral hemispheres, and a telencephalon medium or impar. Main-olfactory bulb can be classified into six layers such as olfactory nerve fibers layer, glomerular layer, external plexiform layer, mitral layer, internal plexiform layer, granular layer and ependyma layer. The dorsal part of telencephalon contained the cortex and dorsal ventricular ridge. The cerebral cortex of S. tsinlingens was relatively thin, while the dorsal cortex was the thinnest, but gradually thickened as it extended to the medial and lateral cortex. The neural cells, glial cells and ependymal cells widely distributed in the cerebral cortex represented similar ultrastructural characteristics to those described in other vertebrates. Golgi staining revealed multipolar cell, bitufted cell and monotufted cell in three cortical layers of medial cortex. The results indicated that the cytoarchitectonic characteristics of telencephalon in S. tsinlingensis resembled those found in other lizards.

Characterization of multiciliated ependymal cells that emerge in the neurogenic niche of the aged zebrafish brain.

In mammals, ventricular walls of the developing brain maintain a neurogenic niche, in which radial glial cells act as neural stem cells (NSCs) and generate new neurons in the embryo. In the adult brain, the neurogenic niche is maintained in the ventricular-subventricular zone (V-SVZ) of the lateral wall of lateral ventricles and the hippocampal dentate gyrus. In the neonatal V-SVZ, radial glial cells transform into astrocytic postnatal NSCs and multiciliated ependymal cells. On the other hand, in zebrafish, radial glial cells continue to cover the surface of the adult telencephalic ventricle and maintain a higher neurogenic potential in the adult brain. However, the cell composition of the neurogenic niche of the aged zebrafish brain has not been investigated. Here we show that multiciliated ependymal cells emerge in the neurogenic niche of the aged zebrafish telencephalon. These multiciliated cells appear predominantly in the dorsal part of the ventral telencephalic ventricular zone, which also contains clusters of migrating new neurons. Scanning electron microscopy and live imaging analyses indicated that these multiple cilia beat coordinately and generate constant fluid flow within the ventral telencephalic ventricle. Analysis of the cell composition by transmission electron microscopy revealed that the neurogenic niche in the aged zebrafish contains different types of cells, with ultrastructures similar to those of ependymal cells, transit-amplifying cells, and migrating new neurons in postnatal mice. These data suggest that the transformation capacity of radial glial cells is conserved but that its timing is different between fish and mice. J. Comp. Neurol. 524:2982-2992, 2016. © 2016 Wiley Periodicals, Inc.

Cell Junction Pathology of Neural Stem Cells Is Associated With Ventricular Zone Disruption, Hydrocephalus, and Abnormal Neurogenesis.

Fetal-onset hydrocephalus affects 1 to 3 per 1,000 live births. It is not only a disorder of cerebrospinal fluid dynamics but also a brain disorder that corrective surgery does not ameliorate. We hypothesized that cell junction abnormalities of neural stem cells (NSCs) lead to the inseparable phenomena of fetal-onset hydrocephalus and abnormal neurogenesis. We used bromodeoxyuridine labeling, immunocytochemistry, electron microscopy, and cell culture to study the telencephalon of hydrocephalic HTx rats and correlated our findings with those in human hydrocephalic and nonhydrocephalic human fetal brains (n = 12 each). Our results suggest that abnormal expression of the intercellular junction proteins N-cadherin and connexin-43 in NSC leads to 1) disruption of the ventricular and subventricular zones, loss of NSCs and neural progenitor cells; and 2) abnormalities in neurogenesis such as periventricular heterotopias and abnormal neuroblast migration. In HTx rats, the disrupted NSC and progenitor cells are shed into the cerebrospinal fluid and can be grown into neurospheres that display intercellular junction abnormalities similar to those of NSC of the disrupted ventricular zone; nevertheless, they maintain their potential for differentiating into neurons and glia. These NSCs can be used to investigate cellular and molecular mechanisms underlying this condition, thereby opening the avenue for stem cell therapy.

Arl13b-regulated cilia activities are essential for polarized radial glial scaffold formation.

The construction of cerebral cortex begins with the formation of radial glia. Once formed, polarized radial glial cells divide either symmetrically or asymmetrically to balance appropriate production of progenitor cells and neurons. Following birth, neurons use the processes of radial glia as scaffolding for oriented migration. Radial glia therefore provide an instructive structural matrix to coordinate the generation and placement of distinct groups of cortical neurons in the developing cerebral cortex. We found that Arl13b, a cilia-enriched small GTPase that is mutated in Joubert syndrome, was critical for the initial formation of the polarized radial progenitor scaffold. Using developmental stage-specific deletion of Arl13b in mouse cortical progenitors, we found that early neuroepithelial deletion of ciliary Arl13b led to a reversal of the apical-basal polarity of radial progenitors and aberrant neuronal placement. Arl13b modulated ciliary signaling necessary for radial glial polarity. Our findings indicate that Arl13b signaling in primary cilia is crucial for the initial formation of a polarized radial glial scaffold and suggest that disruption of this process may contribute to aberrant neurodevelopment and brain abnormalities in Joubert syndrome-related ciliopathies.

BrdU birth dating can produce errors in cell fate specification in chick brain development.

Birth dating neurons with bromodeoxyuridine (BrdU) labeling is an established method widely employed by neurobiologists to study cell proliferation in embryonic, postnatal, and adult brain. Birth dating studies in the chick dorsal telencephalon and the mammalian striatum have suggested that these structures develop in a strikingly similar manner, in which neurons with the same birth date aggregate to form "isochronic clusters." Here we show that isochronic cluster formation in the chick dorsal telencephalon is an artifact. In embryos given standardly employed doses of BrdU, we observed isochronic clusters but found that clusters were absent with BrdU doses close to the limits of detection. In addition, in situ hybridization experiments established that neurons in the clusters display errors in cell type specification: BrdU cell clusters in nidopallium adopted a mesopallial neuronal fate, mesopallial clusters were misspecified as nidopallial cells, and in some instances, the BrdU clusters failed to express neuronal differentiation markers characteristic of the dorsal telencephalon. These results demonstrate that the chick dorsal telencephalon does not develop by isochronic cluster formation and highlight the need to test the integrity of BrdU-treated tissue with gene expression markers of regional and cell type identity.

Avian visual behavior and the organization of the telencephalon.

Birds have excellent visual abilities that are comparable or superior to those of primates, but how the bird brain solves complex visual problems is poorly understood. More specifically, we lack knowledge about how such superb abilities are used in nature and how the brain, especially the telencephalon, is organized to process visual information. Here we review the results of several studies that examine the organization of the avian telencephalon and the relevance of visual abilities to avian social and reproductive behavior. Video playback and photographic stimuli show that birds can detect and evaluate subtle differences in local facial features of potential mates in a fashion similar to that of primates. These techniques have also revealed that birds do not attend well to global configural changes in the face, suggesting a fundamental difference between birds and primates in face perception. The telencephalon plays a major role in the visual and visuo-cognitive abilities of birds and primates, and anatomical data suggest that these animals may share similar organizational characteristics in the visual telencephalon. As is true in the primate cerebral cortex, different visual features are processed separately in the avian telencephalon where separate channels are organized in the anterior-posterior axis roughly parallel to the major laminae. Furthermore, the efferent projections from the primary visual telencephalon form an extensive column-like continuum involving the dorsolateral pallium and the lateral basal ganglia. Such a column-like organization may exist not only for vision, but for other sensory modalities and even for a continuum that links sensory and limbic areas of the avian brain. Behavioral and neural studies must be integrated in order to understand how birds have developed their amazing visual systems through 150 million years of evolution.

Distribution of cholesterol 24-hydroxylase in the monkey brain.

OBJECTIVE: Cholesterol 24-hydroxylase catalyzes the conversion of cholesterol to 24-hydroxycholesterol, which is a major pathway for cholesterol elimination from the brain, since 24-hydroxycholesterol can readily cross the blood brain barrier. The present study aimed to elucidate the distribution of cholesterol 24-hydroxylase in the monkey brain. METHODS: The distribution of cholesterol 24-hydroxylase in the monkey brain was examined using Western blot and immunohistochemistry methods, and was observed under light microscopy and electron microscopy. RESULTS: High levels of cholesterol 24-hydroxylase were observed in projection neurons and neuropil in structures derived from telencephalon, including the cerebral neocortex, hippocampus, amygdala, nucleus basalis of Meynert, and striatum. Electron microscopy revealed that the enzyme was localized in the axon terminals. One the other hand, cholesterol 24-hydroxylase was expressed at a lower level in the thalamus, globus pallidus and brainstem. CONCLUSION: The high level of cholesterol 24-hydroxylase in the telencephalon possibly reflects a high rate of cholesterol turnover in this part of brain.

[Change of composition of intermediate filaments in cells of rat telencephalon at early period of postnatal ontogenesis].

The goal of the present work was to study composition and spatial-temporal distribution of cells containing various proteins of intermediate filaments (nestin, vimentin, GFAP) in various brain parts at the early postnatal period of rat ontogenesis. By using methods of immunochemical determination of proteins of intermediate filaments it has been established that at they early period of ontogenesis, in the course of maturation of the nervous tissue, in cells of cortex, hippocampus, and subventricular area there occurs a change ofimmunochemical profile of intermediate filaments: the nestin+/vimentin+/GFAP- -cells become the nestin-/vimentin-/GFAP- ones.

Zinc at glutamatergic synapses.

It has long been known that the mammalian forebrain contains a subset of glutamatergic neurons that sequester zinc in their synaptic vesicles. This zinc may be released into the synaptic cleft upon neuronal activity. Extracellular zinc has the potential to interact with and modulate many different synaptic targets, including glutamate receptors and transporters. Among these targets, NMDA receptors appear particularly interesting because certain NMDA receptor subtypes (those containing the NR2A subunit) contain allosteric sites exquisitely sensitive to extracellular zinc. The existence of these high-affinity zinc binding sites raises the possibility that zinc may act both in a phasic and tonic mode. Changes in zinc concentration and subcellular zinc distribution have also been described in several pathological conditions linked to glutamatergic transmission dysfunctions. However, despite intense investigation, the functional significance of vesicular zinc remains largely a mystery. In this review, we present the anatomy and the physiology of the glutamatergic zinc-containing synapse. Particular emphasis is put on the molecular and cellular mechanisms underlying the putative roles of zinc as a messenger involved in excitatory synaptic transmission and plasticity. We also highlight the many controversial issues and unanswered questions. Finally, we present and compare two widely used zinc chelators, CaEDTA and tricine, and show why tricine should be preferred to CaEDTA when studying fast transient zinc elevations as may occur during synaptic activity.

Predominant expression of phospholipase Cbeta1 in telencephalic principal neurons and cerebellar interneurons, and its close association with related signaling molecules in somatodendritic neuronal elements.

Upon activation of receptors coupled to the Gq subclass of G proteins, phospholipase C (PLC)beta hydrolyses membrane phospholipid to yield a pair of second messengers, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. Of four PLCbeta isoforms, PLCbeta1 is transcribed predominantly in the telencephalon and its gene inactivation in mice impairs metabotropic glutamate receptor- and muscarinic acetylcholine receptor-dependent hippocampal oscillations, endocannabinoid production in the hippocampus and barrel formation in the somatosensory cortex. Here we examined cellular and subcellular distributions of PLCbeta1 in adult mouse brains. In the telencephalon, high levels of PLCbeta1 were observed in principal neurons, including pyramidal cells in the cortex and hippocampus, granule cells and mossy cells in the dentate gyrus, and medium spiny neurons in the caudate-putamen, whereas most interneurons had low levels of or were negative for PLCbeta1 and, instead, expressed PLCbeta4. By immunofluorescence, tiny clusters of PLCbeta1 were distributed in somatodendritic compartments of principal neurons and positioned close to those of metabotropic glutamate receptor 5, muscarinic acetylcholine receptor M1 and diacylglycerol lipase-alpha, respectively. Immunoelectron microscopy revealed that PLCbeta1 was often associated with the smooth endoplasmic reticulum, cell membrane or postsynaptic density. In particular, it was highly accumulated at the perisynapse of dendritic spines forming asymmetrical synapses. In the cerebellum, PLCbeta1 was generally low but was enriched in axons and dendrites of basket cells. These results suggest that PLCbeta1 is the key effector in telencephalic principal neurons and cerebellar interneurons. Furthermore, the well-orchestrated molecular arrangement appears to be the anatomical basis for the specificity, efficiency and convergence of the neuronal phosphoinositide signaling system.

Eomes::GFP-a tool for live imaging cells of the trophoblast, primitive streak, and telencephalon in the mouse embryo.

Expression of T-box family member Eomesodermin (Tbr2) is spatiotemporally restricted in the mouse embryo; initially expressed in extraembryonic lineages in the sequential progression from the trophectoderm of the blastocyst, its derivatives the extraembryonic ectoderm, and thereafter the chorion, in addition to the visceral endoderm and primitive streak at gastrula stages, and the telencephalon at later stages. We describe the spatiotemporal expression of GFP in embryos of a Tg(Eomes::GFP) BAC transgenic strain, and have compared it with the localization of endogenous Eomes transcripts and protein. Our analysis reveals the following: (1) robust easily visualized reporter expression in live hemizygous transgenic embryos, (2) increased levels of expression in live homozygous transgenic embryos that are compatible with embryo viability, and (3) a close correlation between endogenous Eomes and GFP reporter expression in BAC transgenic embryos. These features establish the Tg(Eomes::GFP) BAC transgenic strain as a novel reagent for both live imaging and the isolation of Eomes expressing cells from specific locations within the embryo.

Subcellular compartmentalization of aromatase is sexually dimorphic in the adult zebra finch brain.

The vertebrate brain is a source of estrogen (E) via the expression of aromatase (E-synthase). In the zebra finch (Taeniopygia guttata), despite documented dimorphisms in E-action, no differences are detectable in circulating E, or the neural levels of aromatase transcription, activity, or somal protein expression. Studies of aromatase expression at the light- and electron-microscope levels reveal greater numbers of fibers and presynaptic boutons in adult males relative to females. We assayed aromatase activity and content in synaptosomes and microsomes from the anterior [containing lMAN and Area X (males)] and posterior telencephalon (containing HVC and RA) of adult birds. In contrast to non-song birds and mammals, both cell fractions contain abundant aromatase measurable in terms of activity (enzyme assays) and content (Western blots) with minimal enrichment in microsomes. From brain homogenates of identical concentration, aromatase activity was higher in the synaptosomal relative to the microsomal fraction, in males relative to females, and in the posterior compared to anterior telencephalon. These effects were driven by high levels of synaptosomal aromatase in the male posterior telencephalon. These data suggest that males possess more aromatase per presynaptic bouton, or a greater number of aromatase-containing presynaptic boutons than females in the posterior telencephalon. Further, the present report reveals synaptic aromatization as a considerable source of E in the zebra finch brain, and supports the idea that telencephalic synapses in and around the adult male song production nuclei may be exposed to higher levels of E compared to the female brain.

An intimate interplay between precocious, migrating pericytes and endothelial cells governs human fetal brain angiogenesis.

In order to better understand the process of angiogenesis in the developing human brain, we have examined the spatial relationship and relative contributions of endothelial cells and pericytes, the two primary cell types involved in vessel growth, together with their relation with the vascular basement membrane. Pericytes were immunolocalized through use of the specific markers nerve/glial antigen 2 (NG2) proteoglycan, endosialin (CD248) and the platelet-derived growth factor receptor beta (PDGFR-beta), while endothelial cells were identified by the pan-endothelial marker CD31 and the blood brain barrier (BBB)-specific markers claudin-5 and glucose transporter isoform 1 (GLUT-1). The quantitative analysis demonstrates that microvessels of the fetal human telencephalon are characterized by a continuous layer of activated/angiogenic NG2 pericytes, which tightly invest endothelial cells and participate in the earliest stages of vessel growth. Immunolabelling with anti-active matrix metalloproteinase-2 (aMMP-2) and anti-collagen type IV antibodies revealed that aMMP-2 producing endothelial cells and pericytes are both associated with the vascular basement membrane during vessel sprouting. Detailed localization of the two vascular cell types during angiogenesis suggests that growing microvessels of the human telencephalon are formed by a pericyte-driven angiogenic process in which the endothelial cells are preceded and guided by migrating pericytes during organization of the growing vessel wall.

Apoptotic cell death in the central nervous system of Bufo arenarum tadpoles induced by cypermethrin.

Tadpoles of the toad Bufo arenarum treated with cypermethrin (CY) at concentrations above 39 mug CY/L showed dose-dependent apoptotic cell death in immature cells of the central nervous system as demonstrated by morphometric analysis, the TUNEL method, and DNA fragmentation assay. Light-and electron-microscopic studies showed structural alterations in the intermediate and marginal layers of the brain. Immature cerebral tissue showed cellular shrinkage, nuclear fragmentation and increase of intercellular spaces. In this study we demonstrated high toxicity of CY to larval stages of Bufo arenarum. Our results show that doses lower than those used in routine insecticide applications can cause massive apoptosis in the immature cells of the central nervous system. These results coincide with our previous studies in Physalaemus biligonigerus, confirming the severe toxic effects of CY to the central nervous system of anuran species from Argentina. This may increase the mortality index in wild animals and contribute to the loss of biodiversity in our agroecosystems. We postulate that CY induces apoptosis in central nervous system cells of Bufo arenarum tadpoles by specific neurotoxic mechanisms.

Intermediate filament immunohistochemistry of astroglial cells in the leopard gecko, Eublepharis macularius.

The distribution of intermediate filament molecular markers, glial fibrillary acidic protein (GFAP) and vimentin, has been studied in the central nervous system (CNS) of the adult leopard gecko, Eublepharis macularius. This immunohistochemical study points out the presence of different astroglial cell types. The main pattern is constituted by ependymal radial glia, which have their cell bodies located in the ependymal layer throughout the brain ventricular system. Radial glia proper or radial astrocytes show their cell bodies displaced from the ependymal layer into a periependymal zone and are observed only in the spinal cord. Star-shaped astrocytes are scarce. They are detected in the ventral and lateral regions of the diencephalon and mesencephalon, in the superficial layer of the optic tectum, in the ventral medulla oblongata, and in the ventral and lateral spinal cord. In the different regions of the CNS, the staining intensity appears not to be identical even in the same cellular type. The results reported in the present study show an heterogeneous feature of the astroglial pattern in E. macularius.

Specific characteristic of radial glia in the human fetal telencephalon.

Phenotypic characteristics of cells in the developing human telencephalic wall were analyzed using electron microscopy and immunocytochemistry with various glial and neuronal cell markers. The results suggest that multiple defined cell types emerge in the neocortical proliferative zones and are differentially regulated during embryonic development. At 5-6 weeks gestation, three major cell types are observed. Most proliferating ventricular zone (VZ) cells are labeled with radial glial (RG) markers such as vimentin, glial fibrillary acidic protein (GFAP), and glutamate astrocyte-specific transporter (GLAST) antibodies. A subpopulation of these RG cells also express the neuronal markers beta III-tubulin, MAP-2, and phosphorylated neurofilament SMI-31, in addition to the stem cell marker nestin, indicating their multipotential capacity. In addition, the presence of VZ cells that immunoreact only with neuronal markers indicates the emergence of restricted neuronal progenitors. The number of multipotential progenitors in the VZ gradually decreases, whereas the number of more restricted progenitors increases systematically during the 3-month course of human corticogenesis. These results suggest that multipotential progenitors coexist with restricted neuronal progenitors and RG cells during initial corticogenesis in the human telencephalon. Since the multipotential VZ cells disappear during the major wave of neocortical neurogenesis, the RG and restricted neuronal progenitors appear to serve as the main sources of cortical neurons. Thus, the diversification of cells in human VZ and overlying subventricular zone (SVZ) begins earlier and is more pronounced than in rodents.

Malignant astrocytoma with binucleated granular cells in a Sprague-Dawley rat.

A 2-year-old Sprague-Dawley rat with hindlimb paralysis was diagnosed with a cerebral malignant astrocytoma. The distinctive feature of this astrocytoma was the presence of scattered binucleated cells that contained hypereosinophilic, 1-2 micro m in diameter, cytoplasmic granules. The neoplastic astrocytes stained positively for vimentin (VIM), lysozyme, and phosphotungstic acid hematoxylin (PTAH). Within the binucleated cells, granules stained with PTAH and periodic acid-Schiff (PAS) before and after diastase digestion. Ultrastructurally, neoplastic astrocytes were characterized by cytoplasmic aggregates of electron-dense intermediate filaments consistent with VIM and desmin. The cytoplasm of binucleated cells contained numerous phagolysosomes enlarged by myelin figures and glycoprotein or glycolipid. Intermediate filaments were not present. This is the first description, in the rat, of a neoplasm with features resembling the human granular cell astrocytoma. Our findings suggest that an astrocytic origin should be considered for the binucleated cells in this neoplasm.

Characterization of Oita virus 296/1972 of Rhabdoviridae isolated from a horseshoe bat bearing characteristics of both lyssavirus and vesiculovirus.

Oita virus 296/1972 was isolated from the blood of a wild horseshoe bat, Rhinolophus cornutus (Temminck) in 1972. We investigated the pathogenicity of this virus in mice in relation to its histological, immunohistochemical and ultrastructural characteristics and the entire sequence of nucleoprotein gene. This virus caused lethal encephalitis in mice through intracerebral route. This susceptibility of mice was until 3 weeks of age. Immunohistochemical analysis using the convalescent sera obtained from survived adult mice after intracerebral inoculation revealed that many neurons were positive in the cytoplasm, besides no cross reactivity with normal and rabies virus-infected mouse brain tissues to this anti-sera. Ultrastructural analysis disclosed many bullet-shaped and enveloped virions in neurons. These morphological characteristics of the virions are consistent of that of viruses in the family Rhabdoviridae. Budding from endoplasmic membrane suggests that this virus has a similarity with lyssaviruses. Molecular analysis of cDNA coding a tentative nucleoprotein sequence revealed homology with those of viruses in the family Rhabdoviridae. Distance matrix analysis of this gene sequence with those of other rhabdoviruses isolated from mammals disclosed the discrete position of this virus in the phylogenic tree of rhabdoviridae infecting mammals and we renamed this virus as Oita rhabdovirus.

An ultrastructural study of the brain in fatal Plasmodium falciparum malaria.

Cerebral malaria (CM) is a major cause of death in severe Plasmodium falciparum malaria. We present quantitative electron microscopic findings of the neuropathologic features in a prospective clinicopathologic study of 65 patients who died of severe malaria in Thailand and Vietnam. Sequestration of parasitized red blood cells (PRBCs) in cerebral microvessels was significantly higher in the brains of patients with CM compared with those with non-cerebral malaria (NCM) in all parts of the brain (cerebrum, cerebellum, and medulla oblongata). There was a hierarchy of sequestration with more in the cerebrum and cerebellum than the brain stem. When cerebral sequestration was compared with the peripheral parasitemia pre mortem, there were 26.6 times more PRBCs in the brain microvasculature than in the peripheral blood. The sequestration index was significantly higher in CM patients (median = 50.7) than in NCM patients (median = 6.9) (P = 0.042). The degree of sequestration of P. falciparum-infected erythrocytes in cerebral microvessels is quantitatively associated with pre-mortem coma.