Miyuki Yamamoto

Identification of radial glial cells within the developing murine central nervous system:studies based upon a new immunohistochemical marker

The monoclonal antibody RC2 was generated in mouse by conventional hybridoma methodology. The antigen recognized by RC2 is robust, allowing aldehyde fixation appropriate to high resolution light and electron microscopic analyses. From the neural tube stage of fetal development the antibody delineates throughout the central nervous system a subpopulation of neuroepithelial cells which have a radial bipolar morphology. A descending process extends to the ventricular margin, and an ascending process contacts the glial limiting membrane by one or more endfeet varicosities. The persistence of these cells through the neurogenetic period allows their identification as radial glial. From as early as E9-10 the fibers appear to be organized in simple straight fascicles. Later in fetal development these fascicles show marked region-specific transformations in density and trajectory, particularly in association with cerebral corticogenesis and with cerebellar and basal ganglia development. The bipolar forms continue to stain with RC2 until they disappear in the postnatal period. Concurrently with a progressive perinatal loss of stained bipolar radial glia, RC2 identifies multipolar cell forms at various levels of the brain wall, as consistent with the transformation of radial glia into astrocytes. RC2 also recognizes monopolar cell forms in the spinal cord and the cerebellum as early as E15, and in the dentate gyrus of the hippocampal formation from the day of birth. Monopolar forms in the cerebellum are inferred to be progenitors of Bergmann glia. Although Bergmann glia are known to persist in adult life, these cells do not stain with RC2 beyond the 2nd postnatal week. The robustness of the antigen recognized by RC2 makes this probe a valuable tool to study the morphological transformations of the bipolar radial glia during their mitotic turnover. It also provides a sensitive stain for the study of the organization and the histogenetic role of the overall radial fiber system.

Organization of radial glia and related cells in the developing murine CNS. An analysis based upon a new monoclonal antibody marker

A monoclonal antibody, RC1, has been generated which provides a selective and sensitive immunohistochemical marker of radial glial cells and related cell forms during development of the mouse CNS. Beginning on embryonic day E10, immunocytochemistry performed on cryostat sections stains throughout the CNS a subpopulation of cells in the ventricular zone with radial processes that terminate with endfeet at the pial surface. These processes become fasciculated and attain maximal densities by E12-14 in the spinal cord and lower brainstem and by E14-16 in the midbrain, cerebellum and forebrain. Fasciculation is especially prominent for a subclass of these cells at the midline of the brainstem and spinal cord. As nuclear and cortical structures develop, the trajectories of the radial fiber fascicles undergo systematic and region-specific distortions in their initially simple linear configuration, in the process maintaining a consistent spatial registration of germinal ventricular zones with distal sites of assembly of post-migratory neurons. In the late fetal period, radial glial progressively disappear and scattered immature astrocytes bearing multiple fine processes appear in most regions of the CNS. In the spinal cord, a transitional unipolar radial form is identified in the emerging ventral and lateral funiculi between E13 and E17. In the cerebellum, precursors to the unipolar Bergmann glial cell are identified by E15, and in the retina, precursors of the bipolar Müller cell are identified by E16. Postnatally, RC1-stained radial glia become sparse, and after one week, immunoreactive cells include only ependymal cells, hypothalamic tanycytes, Bergmann glia, Müller cells, a unipolar radial form in the dentate gyrus, and a subpopulation of white matter astrocytes. These results suggest that radial cells of astroglial lineage comprise a diverse set of cell classes which subserve multiple functions in the developing and adult brain.

mitotic cycling of radial glial cells of the fetal murine cerebral wall: a combined autoradiographic and immunohistochemical study.

Radial glial cells of the embryonic murine cerebral wall are selectively labeled by staining with antibody RC1. In order to study the mitotic cycling of these cells, we combined RC1 immunohistochemistry and autoradiographic analysis following [3H]thymidine injection at 1, 2, 6, 48 h prior to sacrifice. Many radial glial cells, i.e. RC1-positive cells, incorporate the DNA tracer and hence must be mitotically active. Other proliferative cells of the ventricular zone do not stain with RC1. With the transition from S to M phase, the nuclei of the radial glial cells participate in the interkinetic to-and-fro nuclear translocation characteristic of the non-radial glial cells of the ventricular zone. The density of radioactive grains over nuclei of both RC1-positive and negative cells of the ventricular zone becomes similarly reduced in the 48 h following the [3H]thymidine incorporation. Thus, the subpopulation of radial glia with nuclei within the ventricular zone which have incorporated the DNA tracer does not appear to become arrested in a prolonged G1 phase. The results suggest that the ventricular zone includes at least two subpopulations of stem cells, neuronal and radial glial. Radial glial cells, i.e. RC1-positive cells, are inferred to serve initially as a progenitor population for new radial glial cells. Later in development, they probably become a source of other cells of astroglial lineage.

Architectonic and hodological organization of the cerebellum in reeler mutant mice

The architectonic and hodologic organization of the reeler cerebellum has been studied by means of immunohistochemistry, general cell and fiber stains and by horseradish peroxidase and autoradiographic tracing methods. Malposition of Purkinje cells, which varies in degree, is the most salient architectonic anomaly of the mutant cerebellum. Mapping the distribution of Purkinje cells is facilitated by a monoclonal antibody which selectively stains neurons of this class in the cerebellum. Although some Purkinje cells form a normal monolayer, most lie in heterotopic positions within or below the granule cell layer. The major contingent is segregated in subcortical masses in the depths of the cerebellum. Fiber bundles continuous with the cerebellar peduncles run in septa between the subcortical Purkinje cell masses. The distribution of Purkinje cell masses as well as the roof nuclei and areas of normal cortex and fiber bundles are identical from animal to animal. These consistent architectonic variations serve to partition the reeler cerebellum into 7 sagittally oriented compartments: one medial, two intermediate, two lateral and two additional lateral lobular appendages which may correspond to paraflocculus and/or flocculus of the normal cerebellum. The topography of the reeler olivocerebellar, or climbing fiber, system is normal in that the caudal-to-rostral axis of the olivary complex maps onto the medial-to-lateral axis of the contralateral hemicerebellum. The climbing fiber projection in reeler, like that of the normal animal, appears to be organized in parasagittal strips. In the mutant, mossy fibers from the pons and spinal cord project respectively to the lateral and medial cerebellar fields, and overlap in the intermediate compartment. They thus invest different and to a large extent complementary cerebellar territories, which approximate the architectonic divisions. This segregation of the two principal mossy fiber systems is not so marked in the normal cerebellum. In terms of laminar distribution, the pontine projection is distributed principally to the granule cell stratum in the mutant. The reeler spinocerebellar afferents, by contrast, project not only to the granule cell layer but also to the heterotopic Purkinje cells. The present observations suggest that the primary defect in the reeler cerebellum is malposition of Purkinje cells. As appears to be the case during development of the forebrain in reeler, the mutation may affect the terminal phase of migration of Purkinje cells in the cerebellum.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of glucuronic acid-and-sulfate-containing glycoproteins in the central nervous system of the adult mouse

The distribution of glucuronic acid-and-sulfate-containing carbohydrate (GSC) epitope recognized by two monoclonal antibodies, HNK-1 and 4F4, was studied by immunocytochemistry in adult mouse brain. Both antibodies recognized proteins ranging in molecular weight from 60 to above 250 kDa in Western blot but no glycolipid was recognized in the adult brain. With both light and electron microscopic study, two patterns of staining are observed: diffuse neuropil staining, and individual neuronal somata staining. The diffuse neuropil staining is concentrated in discrete anatomically defined areas. At the EM level, this immunoreactivity is associated with numerous dendrites or astrocytic processes. At cell somata, most of neurons are stained only at Golgi apparatus (type 2); however, a distinct population of cells showed membranous staining (type 1) as well. Type 1 membranous immunoreactivity is observed only in membrane adjacent to astrocytic processes. In the cerebral cortex, type 1 neurons are found in layers III and V-VIa of somatosensory cortex, but only in layers V-VIa in most other cortical fields. Other areas containing type 1 neurons include the globus pallidus, the thalamic reticular nucleus, the hippocampus, the deep cerebellar nuclei, and a majority of the primary sensory and motor nuclei in the brainstem. The subpopulation of type 1 neurons show an overlap in distribution and morphology with some GABA-containing cells.

Hyperinnervation of arrested granule cells produced by the transplantation of monoamine-containing neurons into the fourth ventricle of rat

SummaryAn attempt to learn whether chemically specific neurons affect the sequence of cerebellar development was made by transplanting ectopic tissues rich in monoamines adjacent to the early developing cerebellum of neonatal rat. Three types of brainstem grafts were used: (1) ventral midline raphe region, (2) inferior olivary region, (3) locus coeruleus region. When transplanted into the fourth ventricle of host animals, neurons from the transplant sprout axons into the host cerebellar parenchyma producing changes in cerebellar cytoarchitecture. The changes produced by the three types of brain grafts were investigated with conventional light and electron microscopy. Autoradiography with tritiated serotonin (3H-5HT) and norepinephrine (3H-NE) and immunocytochemistry using antibodies raised against serotonin allowed identification of the chemical specificity of the process. The three fundamental changes caused by the transplants were folial malformation, arrest of migration of external granule cells, and disruption of the Purkinje cell monolayer. By intraventricular infusion of 3H-5HT and immunocytochemistry with antibodies raised against serotonin, an extraordinarily rich serotonin innervation was detected within or around the foci of arrested granule cells after transplantation with raphe-rich tissue. In addition to an increase in the number of parallel fibers that accumulate 3H-5HT, numerous glomerulus-like structures were observed within the foci. After transplantation with locus coeruleus fragments, intraventricular infusion of 3H-NE demonstrated some increase of labeled fibers inside the foci of arrested granule cells, but the extent of the increase of NE fibers was less marked than the increase in 5-HT fibers. Conventional electron microscopic study revealed numerous synaptic formations within the arrested granule cell foci. Terminals containing large granular vesicles were seen, which resemble serotonin nerve terminals previously described (Chan-Palay, 1975, 1977).Thus ectopic neuronal tissues rich in monoamine neurons survive after transplantation into the fourth ventricle of neonatal rats, can disrupt cerebellar development, and sprout axons that hyperinnervate foci of neurons in disarray, in a pattern reminiscent of the normal innervation.

Effects of nervous mutation on purkinje cell compartments defined by Zebrin II and 9-O-acetylated gangliosides expression

The cerebellum is organized into a series of parasagittally aligned bands which are well delineated in the adult mouse by the largely complementary immunostaining of Purkinje cell groups with the monoclonal antibodies Zebrin II (ZII; antigen: aldolase C) and P-path (antigen: 9-O-acetyl gangliosides). We examined the effect of nervous mutation on compartmental organization using these markers and an antibody to calbindin. In nervous mutant, up to 90% of Purkinje cells die in late postnatal development. The size of the cerebellum is about half that of normal, and caudal lobules appear to decrease in size more than anterior ones. Surviving Purkinje cells corresponded to P-path positive ones that were concentrated in two bilateral bands in the vermis and in medial portions of the hemispheres. Only small numbers of ZII positive cells remained, confirming the report by Wassef et al. with Zebrin I antibody. They were primarily located in caudal lobules IX, X and a portion of lobule IV, paraflocculus and flocculus, and their immunoreactivity was weak compared to that of normal. ZII positive cells are dominant in these caudal lobules, while P-path positive cells dominate in rostral lobules in normal mice, and the similar tendency remains in mutant. Thus, the nervous gene action respects not only sagittal compartments delineated by two antibodies, but also rostro-caudal gradient. The cause of the dominant survival of P-path positive cells awaits future study.

A monoclonal antibody, WCC4, recognizes a developmentally regulated ganglioside containing α-galactose and α-fucose present in the rat nervous system

Abstract A monoclonal antibody, WCC4, raised against PC12 cells, recognizes a ganglioside which is present in low concentrations in the postnatal rat nervous system. The antigen is also present in the adrenal and kidney, as determined immunohistochemically, but is not detectable in liver or spleen. A neutral glycosphingolipid is also immunoreactive. In the present report, the chemical characterization of this ganglioside, isolated from PC12 cells, and the anatomical distribution of the antigens recognized by the WCC4 antibody are described. By enzymatic cleavage of terminal saccharide moieties, the ganglioside is identified as α-galactosyl, (α-fucosyl) G MI . The ganglioside increases in concentration postnatally to day 35 (P35) and is present in a slightly diminished concentration in the adult. Immunohistochemical studies revealed that this glycolipid is also present on neuronal cell soma throughout the cerebrum, cerebellum and spinal cord. It is expressed in highest concentration in the molecular layer of the dentate gyrus and is also present in the olfactory bulb, the molecular layer of the hippocampus, the piriform cortex, the olfactory tubercle and the entorhinal cortex. The dentate molecular layer receives most of its innervation from neurons in the entorhinal cortex, and gangliosides are known to have an effect on plasticity following entorhinal cortical lesions. Therefore, the WCC4 antibody should prove to be a useful tool for the study of the role of endogenous gangliosides in this region of the nervous system.

Autoradiographic experiments to examine uptake, anterograde and retrograde transport of tritiated serotonin in the mammalian brain

SummaryIn an attempt to define the potential application of neurotransmitter-specific transport as a method of tracing fiber connections, we have examined the uptake and subsequent ortho- and retrograde transport of tritiumlabeled serotonin (3H-5HT) in the cerebellum-raphe pallidus system. Injection of various concentrations of 3H-5HT followed by different post-injection survival times revealed different labeling patterns in the injected sites and different patterns of transport. The most striking feature is that nonserotonin neurons as well as serotonin cells were able to take up and transport the tritium label in both ortho- and retrograde fashion. The non-sertonin-specific nature of this uptake and transport is more obvious at higher concentrations of 3H-5HT (more than 9x10-5 M), with longer survival times and following pretreatment with monoamine oxidase inhibitors. At a concentration of 9x10-6 M 3H-5HT, only specific uptake seems to take place as evidenced by label in known serotonin cells and fiber systems; however, it was impossible to detect by autoradiography any ortho- or retrograde transport at this low concentration. Non-specific uptake and transport were observed following injection into the vestibular nuclei and oculomotor complex. This suggests that non-specific uptake and the transport of 3H-5HT or metabolites may also occur in other regions of the central nervous system.

Early axonal differentiation in mouse CNS delineated by an antibody recognizing extracted neurofilaments.

A monoclonal antibody, C2, raised against chick embryo spinal cord, is shown by a solid phase immunoabsorbent assay to recognize a molecular species associated with neurofilaments extracted from adult mouse and rat brain. As immunoreactivity is lost following pre-treatment with alkaline phosphatase, the antibody probably recognizes a phosphorylated protein. Immunocytochemical staining in fetal mouse indicates that this antigen is expressed selectively in axons from the earliest stages of their development. Neuronal somata tend to show only weak immunoreactivity. The C2 antibody allowed visualization of the spatiotemporal pattern of axonal growth in the retina, neocortex and cerebellum with greater resolution than in previous light microscopic descriptions. The concept that the leading process of some classes of migratory neurons becomes transformed into an axon is supported by the expression of C2 immunoreactivity in radially ascending processes from principle neuron classes in the fetal retina and cerebellum.