SPARC-like 1 regulates the terminal phase of radial glia-guided migration in the cerebral cortex

Differential adhesion between migrating neurons and transient radial glial fibers enables the deployment of neurons into appropriate layers in the developing cerebral cortex. The identity of radial glial signals that regulate the termination of migration remains unclear. Here, we identified a radial glial surface antigen, SPARC (secreted protein acidic and rich in cysteine)-like 1, distributed predominantly in radial glial fibers passing through the upper strata of the cortical plate (CP) where neurons end their migration. Neuronal migration and adhesion assays indicate that SPARC-like 1 functions to terminate neuronal migration by reducing the adhesivity of neurons at the top of the CP. Cortical neurons fail to achieve appropriate positions in the absence of SPARC-like 1 function in vivo. Together, these data suggest that antiadhesive signaling via SPARC-like 1 on radial glial cell surfaces may enable neurons to recognize the end of migration in the developing cerebral cortex.     

免疫组织化学方法检测脑红蛋白在大鼠中枢神经系统的分布

目的 探讨脑红蛋白（NGB）基因在中枢神经系统中的分布。方法 用免疫组织化学ABC法研究了NGB蛋白在成年大鼠脑内的分布和定位。结果 NGB蛋白在成年大鼠脑中有非常广泛的表达。其分布区域包括大脑皮质,海马,丘脑和下丘脑的部分核团,脑桥及小脑,NGB免疫反应阳性物质定位于神经元的细胞质。结论 NGB蛋白在大鼠脑中有非常广泛的表达,提示NGB基因在中枢神经系统的功能活动中可能起重要作用。     

Pros and cons of a prion-like pathogenesis in Parkinson's disease

Background  

Parkinson's disease (PD) is a slowly progressive neurodegenerative disorder which affects widespread areas of the brainstem, basal ganglia and cerebral cortex. A number of proteins are known to accumulate in parkinsonian brains including ubiquitin and α-synuclein. Prion diseases are sporadic, genetic or infectious disorders with various clinical and histopathological features caused by prion proteins as infectious proteinaceous particles transmitting a misfolded protein configuration through brain tissue. The most important form is Creutzfeldt-Jakob disease which is associated with a self-propagating pathological precursor form of the prion protein that is physiologically widely distributed in the central nervous system.     

Neurons with choline acetyltransferase immunoreactivity and mRNA are present in the human cerebral cortex

 We examined the cerebral cortex of five autopsied individuals without neurological and psychiatric diseases by immunohistochemistry using an anti-human recombinant choline acetyltransferase (ChAT) polyclonal antibody and in situ hybridization with ³⁵S-labeled human ChAT riboprobes. The immunohistochemistry detected positive neurons which were medium-sized or large pyramidal neurons located predominantly in layers III and V. The density of such neurons was higher in the motor and secondary sensory areas than in other cortical areas; the immunoreactive neurons in layer V were more densely distributed in the motor area and those in layer III were distributed in the secondary sensory areas. Positively stained, non-pyramidal neurons were observed in the superficial layer of the cingulate gyrus and parahippocampus. No immunoreactive neurons were found in the primary sensory areas. The in situ hybridization detected some neurons with signals for ChAT mRNA in the cerebral cortex, most of which were distributed in layer V of the motor area and in layer III of the secondary visual area. These results indicate that the human cerebral cortex contains cholinergic neurons and displays regional and laminal variations in their distribution. Accepted: 17 November 1998     

Induction of heme oxygenase-1 in the rat brain by kainic acid-mediated excitotoxicity: the dissociation of mRNA and protein expression in hippocampus.

Heme oxygenase-1 (HO-1) is induced under various stresses. Here we report the induction and localization of HO-1 in the rat brain by intraperitoneal administration of kainic acid (KA). Both mRNA and protein of HO-1 were markedly induced by KA treatment, and each maximal induction was observed 24 h after KA administration. In situ hybridization analysis showed that HO-1 mRNA appeared predominantly in glial cells, and confined neurons were positive in the cerebral cortex, basal ganglia, and hippocampal pyramidal cell layer. Immunohistochemical analysis showed that the positive cells in the cerebral cortex and hippocampus were mainly astrocytes and microglia, whereas neurons in the basal ganglia showed intense immunoreactivity. We also demonstrate the dissociation between HO-1 mRNA and protein level in the hippocampal pyramidal neurons, which is known to be vulnerable against excitotoxicity, and discuss the correlation between this dissociation and the vulnerability of hippocampal pyramidal neurons.     

The spatial distribution of neurons and glia in human cortex based on the poisson distribution

A method was devised that employs deviations from the Poisson distribution to analyze the spatial arrangement of neurons and glia in human cerebral cortex. A field of randomly distributed points equal in number of a sample field of neuronal or glial cells is generated by computer, and the proportion of cells in the sample field that are closer to the nearest neighboring cells than to the nearest randomly distributed point is determined. We call this proportion the "Poisson ratio." When the cells are randomly distributed, the Poisson ratio is equal to 0.5. If the Poisson ratio is less than 0.5, the cells are farther away from one another than a random distribution would predict (exclusionary pattern); if the Poisson ratio is greater than 0.5, the cells are closer to one another than a random distribution would predict (clustering). A simple nonparametric statistical test is used to determine the significance of differences in the ratios. This method was applied to samples of human cerebral cortex in order to test the hypothesis that patients with schizophrenic psychosis may have an altered pattern of neuronal clustering. The analysis revealed that there is no difference in the nearest-neighbor distribution of either neurons or glia between psychotic patients and controls. It was found, however, that there is a highly significant difference in the spatial distribution of neurons versus glia in human cerebral cortex. Neurons of layers II to VI in the human cortex show greater-than-expected distances among them and are distributed according to an exclusionary pattern, while neurons in layer I show a clustering pattern.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estradiol-induced phosphorylation of ERK1/2 in explants of the mouse cerebral cortex: the roles of heat shock protein 90 (Hsp90) and MEK2.

Confocal laser scanning microscopy was used to identify the cells within organotypic slice cultures of the developing mouse cerebral cortex that respond to estradiol treatment by phosphorylation of ERK1 and ERK2. Estrogen-responsive cells resembled neurons morphologically and expressed the neuronal marker microtubule-associated protein 2B. The intracellular distribution of the phospho-ERK signal was both cytoplasmic and nuclear, but inhibition of protein synthesis abolished the appearance of the nuclear signal. ERK1and ERK2 also coimmunoprecipitated with heat shock protein 90 (Hsp90) in the cerebral cortical explants. Geldanamycin effectively disrupted this association and prevented ERK phosphorylation. Surprisingly, MEK2 but not MEK1 was the principal mediator of estradiol-induced activation of ERK. Our data demonstrate the requirement for Hsp90 in estrogen-induced activation of ERK1 and ERK2 by MEK2 in the developing mouse cerebral cortex and also provide insight into alternative mechanisms by which estradiol may influence cytoplasmic and nuclear events in responsive neurons via the MAP kinase cascade.     

Brain oscillations,medium spiny neurons,and dopamine

1. The striatum is part of a multisynaptic loop involved in translating higher order cognitive activity into action. The main striatal computational unit is the medium spiny neuron, which integrates inputs arriving from widely distributed cortical neurons and provides the sole striatal output.2. The membrane potential of medium spiny neurons' displays shifts between a very negative resting state (down state) and depolarizing plateaus (up states) which are driven by the excitatory cortical inputs.3. Because striatal spiny neurons fire action potentials only during the up state, these plateau depolarizations are perceived as enabling events that allow information processing through cerebral cortex – basal ganglia circuits. In vivo intracellular recording techniques allow to investigate simultaneously the subthreshold behavior of the medium spiny neuron membrane potential (which is a reading of distributed patterns of cortical activity) and medium spiny neuron firing (which is an index of striatal output).4. Recent studies combining intracellular recordings of striatal neurons with field potential recordings of the cerebral cortex illustrate how the analysis of the input–output transformations performed by medium spiny neurons may help to unveil some aspects of information processing in cerebral cortex – basal ganglia circuits, and to understand the origin of the clinical manifestations of Parkinson's disease and other neurologic and neuropsychiatric disorders that result from alterations in dopamine-dependent information processing in the cerebral cortex – basal ganglia circuits.     

Neuronal localization of amyloid beta protein precursor mRNA in normal human brain and in Alzheimer''s disease.

Clones for the amyloid beta protein precursor gene were isolated from a cDNA library prepared from the frontal cortex of a patient who had died with a histologically confirmed diagnosis of Alzheimer's disease; they were used to investigate the tissue and cellular distribution of amyloid beta protein precursor mRNA in brain tissues from control patients and from Alzheimer's disease patients. Amyloid beta protein precursor mRNA was expressed in similar amounts in all control human brain regions examined, but a reduction of the mRNA level was observed in the frontal cortex from patients with Alzheimer's disease. By in situ hybridization amyloid beta protein precursor mRNA was present in granule and pyramidal cell bodies in the hippocampal formation and in pyramidal cell bodies in the cerebral cortex. No specific labelling of glial cells or endothelial cells was found. The same qualitative distribution was observed in tissues from control patients and from patients with Alzheimer's disease. Senile plaque amyloid thus probably derives from neurones. The tissue distribution of amyloid beta protein precursor mRNA and its cellular localization demonstrate that its expression is not confined to the brain regions and cells that exhibit the selective neuronal death characteristic of Alzheimer's disease.     

Amyloid Precursor Protein Metabolism in Primary Cell Cultures of Neurons, Astrocytes, and Microglia

Abstract: Amyloid precursor protein (APP) gives rise by proteolytic processing to the amyloid β peptide (Aβ) found abundantly in cerebral senile plaques of individuals with Alzheimer's disease. APP is highly expressed in the brain. To assess the source of cerebral Aβ, the metabolism of APP was investigated in the major cell types of the newborn rat cerebral cortex by pulse/chase labeling and immunoprecipitation of the APP and APP metabolic fragments. We describe a novel C-terminally truncated APP isoform that appears to be made only in neurons. The synthesis, degradation, and metabolism of APP were quantified by phosphorimaging in neurons, astrocytes, and microglia. The results show that although little APP is metabolized through the amyloidogenic pathways in each of the three cultures, neurons appear to generate more Aβ than astrocytes or microglia.     

Distribution and origins of VIP-immunoreactive nerves in the cephalic circulation of the cat

VIP-immunoreactive (IR) nerves were visualized in whole mounts and sections of cephalic arteries and cranial nerves of cats with indirect immunofluorescence. Perivascular VIP-IR nerves were very widely distributed in arteries and arterioles supplying glands, muscles and mucous membranes of the face. Within the cerebral circulation, perivascular VIP-IR nerves were most abundant in the Circle of Willis and the proximal portions of the major cerebral arteries and their proximal branches supplying the rostral brain stem and ventral areas of the cerebral cortex. VIP-IR nerves were absent from arterial branches supplying the posterior brain stem, cerebellum and dorsal cerebral cortex. Cerebral perivascular VIP-IR nerves probably arise from VIP-IR perikarya within microganglia found in the cavernous plexus and external rete. Extracerebral perivascular VIP-IR nerves probably arise from VIP-IR perikarya in microganglia associated with the tympanic plexus, chorda tympani, lingual nerve and Vidian nerve as well as from cells in the otic, sphenopalatine, submandibular and sublingual ganglia. It seems likely, therefore, that each major segment of the cephalic circulation is supplied by local VIP-IR neurons.     

Wnt/��-catenin signaling in cerebral cortical development

The cerebral cortex is the multilayered sheet of neurons that underlies our highest cognitive abilities. Canonical Wnt/β-catenin signaling has well-known activities in tissue patterning in regulating rostral-caudal and medial-lateral patterning in the developing cortex. In addition, recent studies suggest that Wnt/β-catenin signaling also plays important roles in establishing the radial inside to outside organization of the cerebral cortex. Different Wnts, Wnt receptors and inhibitors are expressed in overlapping radial compartments of the cerebral cortex, and in vivo functional studies have provided evidence for Wnt/β-catenin regulation of neural precursor self-renewal, laminar fate determination and establishing or stabilizing the patterns of neuronal communication of cortical neurons. Wnt/β-catenin alterations have been observed in human brain tumors, and understanding its many diverse functions during normal neural development may provide greater insight into the mechanisms underlying the development and progression of neural tumors.Key words: cerebral cortex, neural stem cell, neural precursor, ventricular zone, laminar fate, regional specification, radial patterning     

Comparative analysis of the subventricular zone in rat, ferret and macaque: evidence for an outer subventricular zone in rodents

The mammalian cerebral cortex arises from precursor cells that reside in a proliferative region surrounding the lateral ventricles of the developing brain. Recent work has shown that precursor cells in the subventricular zone (SVZ) provide a major contribution to prenatal cortical neurogenesis, and that the SVZ is significantly thicker in gyrencephalic mammals such as primates than it is in lissencephalic mammals including rodents. Identifying characteristics that are shared by or that distinguish cortical precursor cells across mammalian species will shed light on factors that regulate cortical neurogenesis and may point toward mechanisms that underlie the evolutionary expansion of the neocortex in gyrencephalic mammals. We immunostained sections of the developing cerebral cortex from lissencephalic rats, and from gyrencephalic ferrets and macaques to compare the distribution of precursor cell types in each species. We also performed time-lapse imaging of precursor cells in the developing rat neocortex. We show that the distribution of Pax6+ and Tbr2+ precursor cells is similar in lissencephalic rat and gyrencephalic ferret, and different in the gyrencephalic cortex of macaque. We show that mitotic Pax6+ translocating radial glial cells (tRG) are present in the cerebral cortex of each species during and after neurogenesis, demonstrating that the function of Pax6+ tRG cells is not restricted to neurogenesis. Furthermore, we show that Olig2 expression distinguishes two distinct subtypes of Pax6+ tRG cells. Finally we present a novel method for discriminating the inner and outer SVZ across mammalian species and show that the key cytoarchitectural features and cell types that define the outer SVZ in developing primates are present in the developing rat neocortex. Our data demonstrate that the developing rat cerebral cortex possesses an outer subventricular zone during late stages of cortical neurogenesis and that the developing rodent cortex shares important features with that of primates.     

Widespread distribution of the major polypeptide component of MAP 1 (microtubule-associated protein 1) in the nervous system

We prepared a monoclonal antibody to microtubule-associated protein 1 (MAP 1), one of the two major high molecular weight MAP found in microtubules isolated from brain tissue. We found that MAP 1 can be resolved by SDS PAGE into three electrophoretic bands, which we have designated MAP 1A, MAP 1B, and MAP 1C in order of increasing electrophoretic mobility. Our antibody recognized exclusively MAP 1A, the most abundant and largest MAP 1 polypeptide. To determine the distribution of MAP 1A in nervous system tissues and cells, we examined tissue sections from rat brain and spinal cord, as well as primary cultures of newborn rat brain by immunofluorescence microscopy. Anti-MAP 1A stained white matter and gray matter regions, while a polyclonal anti-MAP 2 antibody previously prepared in this laboratory stained only gray matter. This confirmed our earlier biochemical results, which indicated that MAP 1 is more uniformly distributed in brain tissue than MAP 2 (Vallee, R.B., 1982, J. Cell Biol., 92:435-442). To determine the identity of cells and cellular processes immunoreactive with anti-MAP 1A, we examined a variety of brain and spinal cord regions. Fibrous staining of white matter by anti-MAP 1A was generally observed. This was due in part to immunoreactivity of axons, as judged by examination of axonal fiber tracts in the cerebral cortex and of large myelinated axons in the spinal cord and in spinal nerve roots. Cells with the morphology of oligodendrocytes were brightly labeled in white matter. Intense staining of Purkinje cell dendrites in the cerebellar cortex and of the apical dendrites of pyramidal cells in the cerebral cortex was observed. By double-labeling with antibodies to MAP 1A and MAP 2, the presence of both MAP in identical dendrites and neuronal perikarya was found. In primary brain cell cultures anti-MAP 2 stained predominantly cells of neuronal morphology. In contrast, anti-MAP 1A stained nearly all cells. Included among these were neurons, oligodendrocytes and astrocytes as determined by double-labeling with anti-MAP 1A in combination with antibody to MAP 2, myelin basic protein or glial fibrillary acidic protein, respectively. These results indicate that in contrast to MAP 2, which is specifically enriched in dendrites and perikarya of neurons, MAP 1A is widely distributed in the nervous system.     

A monoclonal antibody against a neuron-specific 65-kDa protein with laminar expression in the developing cerebral cortex

The extracellular matrix component chondroitin sulfate supports the survival of neocortical neurons and influences their differentiation in culture. During development of the rat forebrain expression of chondroitin sulfate peaks at around birth. To elucidate functional partners of this important player of neurogenesis, a monoclonal antibody, termed MAb-9, was generated after immunization with chondroitin sulfate-binding proteins from neonatal rat brain. In western blots of neonatal tissue, MAb-9 recognized a major brain-specific 65-kDa protein band. While most of the 65-kDa protein was present in the soluble compartment, a significant fraction was membrane-associated. Prolonged extraction of brain membranes with CHAPS revealed three additional minor protein bands of approximately 48, 50, and 58 kDa. Of these, the 50-kDa protein specifically bound to chondroitin sulfate C-Sepharose. Immunocytochemical studies and western blot analyses of cultures of neocortical neurons and astrocytes demonstrated that MAb-9 recognizes a neuron-specific cytosolic protein. In the developing cerebral cortex the protein was detectable by immunohistochemistry in the preplate and ventricular zones as early as embryonic day 15. On embryonic day 18, the protein was found in the marginal zone and the subplate, but it was not present in the emerging cortical plate. At the neonatal stage the immunoreactivity was distributed throughout the cerebral cortex with prominent staining of the marginal zone. A similar pattern was observed in the adult animal. Notably, the laminar distribution of MAb-9 immunoreactivity in the cerebral cortex during the prenatal period closely resembled the expression pattern reported for chondroitin sulfate. Thus, MAb-9 recognizes a neuronal cytosolic protein which might participate in neurotrophic signaling events triggered by chondroitin sulfate.     

A radial glia-specific role of RhoA in double cortex formation

The positioning of neurons in the cerebral cortex is of crucial importance for its function as highlighted by the severe consequences of migrational disorders in patients. Here we show that genetic deletion of the small GTPase RhoA in the developing cerebral cortex results in two migrational disorders: subcortical band heterotopia (SBH), a heterotopic cortex underlying the normotopic cortex, and cobblestone lissencephaly, in which neurons protrude beyond layer I at the pial surface of the brain. Surprisingly, RhoA(-/-) neurons migrated normally when transplanted into wild-type cerebral cortex, whereas the converse was not the case. Alterations in the radial glia scaffold are demonstrated to cause these migrational defects through destabilization of both the actin and the microtubules cytoskeleton. These data not only demonstrate that RhoA is largely dispensable for migration in neurons but also showed that defects in radial glial cells, rather than neurons, can be sufficient to produce SBH.     

CIP98, a novel PDZ domain protein,is expressed in the central nervous system and interacts with calmodulin-dependent serine kinase

Receptors and various molecules in neurons are localized at precise locations to perform their respective functions, especially in synaptic sites. Among synaptic molecules, PDZ domain proteins play major roles in scaffolding and anchoring membrane proteins for efficient synaptic transmission. In the present study, we isolated CIP98, a novel protein (98 kDa) consisting of three PDZ domains and a proline-rich region, which is widely expressed in the central nervous system. In situ hybridization and immunohistochemical staining patterns demonstrate that CIP98 is expressed strongly in certain types of neurons, i.e. pyramidal cells in layers III-V of the cerebral cortex, projecting neurons in the thalamus and interneurons in the cerebellum. The results of immunocytochemical staining and electron microscopy revealed that CIP98 is localized both in dendrites and axons. Interestingly, CIP98 interacts with CASK (calmodulin-dependent serine kinase), a member of the membrane-associated guanylate kinase (MAGUK) family that plays important roles in the molecular organization of proteins at synapses. CIP98 was shown to co-localize with CASK along the dendritic processes of neurons. In view of its direct association with CASK, CIP98 may be involved in the formation of CASK scaffolding proteins complex to facilitate synaptic transmission in the CNS.     

Estradiol‐induced phosphorylation of ERK1/2 in explants of the mouse cerebral cortex: The roles of heat shock protein 90 (Hsp90) and MEK2

Confocal laser scanning microscopy was used to identify the cells within organotypic slice cultures of the developing mouse cerebral cortex that respond to estradiol treatment by phosphorylation of ERK1 and ERK2. Estrogen‐responsive cells resembled neurons morphologically and expressed the neuronal marker microtubule‐associated protein 2B. The intracellular distribution of the phospho‐ERK signal was both cytoplasmic and nuclear, but inhibition of protein synthesis abolished the appearance of the nuclear signal. ERK1and ERK2 also coimmunoprecipitated with heat shock protein 90 (Hsp90) in the cerebral cortical explants. Geldanamycin effectively disrupted this association and prevented ERK phosphorylation. Surprisingly, MEK2 but not MEK1 was the principal mediator of estradiol‐induced activation of ERK. Our data demonstrate the requirement for Hsp90 in estrogen‐induced activation of ERK1 and ERK2 by MEK2 in the developing mouse cerebral cortex and also provide insight into alternative mechanisms by which estradiol may influence cytoplasmic and nuclear events in responsive neurons via the MAP kinase cascade. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 1–12, 2002     

Expression of cyclin E in postmitotic neurons during development and in the adult mouse brain

Cyclin E, a member of the G1 cyclins, is essential for the G1/S transition of the cell cycle in cultured cells, but its roles in vivo are not fully defined. The present study characterized the spatiotemporal expression profile of cyclin E in two representative brain regions in the mouse, the cerebral and cerebellar cortices. Western blotting showed that the levels of cyclin E increased towards adulthood. In situ hybridization and immunohistochemistry showed the distributions of cyclin E mRNA and protein were comparable in the cerebral cortex and the cerebellum. Immunohistochemistry for the proliferating cell marker, proliferating cell nuclear antigen (PCNA) revealed that cyclin E was expressed by both proliferating and non-proliferating cells in the cerebral cortex at embryonic day 12.5 (E12.5) and in the cerebellum at postnatal day 1 (P1). Subcellular localization in neurons was examined using immunofluorescence and western blotting. Cyclin E expression was nuclear in proliferating neuronal precursor cells but cytoplasmic in postmitotic neurons during embryonic development. Nuclear cyclin E expression in neurons remained faint in newborns, increased during postnatal development and was markedly decreased in adults. In various adult brain regions, cyclin E staining was more intense in the cytoplasm than in the nucleus in most neurons. These data suggest a role for cyclin E in the development and function of the mammalian central nervous system and that its subcellular localization in neurons is important. Our report presents the first detailed analysis of cyclin E expression in postmitotic neurons during development and in the adult mouse brain.