Yozo Nishimura

Determination of the developmental pattern of retinal ganglion cells in chick embryos by Golgi impregnation and other methods.

SummaryThe differentiation of retinal ganglion cells was investigated in chick embryos, with special attention to accurate incubation times from stage 20 (about 3 days of incubation) to after hatching (after 20 days of incubation). The morphological differentiation of these cells comprised four main events:1.Shortening of the bipolar processes attached to the inner and outer limiting membranes and the sprouting of an axon from part of the cell body. These were recognized at stage 25 (about 4.5 days of incubation) by Golgi impregnation methods.2.Beginning of differentiation of the dedrites, which was recognized at stage 29 to 31 (about 6 or 7 days of incubation).3.The rapid development of the dendrites. This occurred at stage 34 (about 8 days of incubation).4.The essential completion of the retinal ganglion cells, which was recognized at stage 39 (about 13 days of incubation).

Morphological development of retinal ganglion cells in the chick embryo.

Abstract The morphological changes of the external shapes of the developing ganglion cells in the chicken retina were investigated by light microscopy and chiefly with Golgi methods. At stage 20, the neural retina revealed an almost pseudostratified type of arrangement of epithelial cells on routine staining. In silver-impregnation images, most cells in the neural retina showed bipolarly elongated shapes. The inner process of this cell extended toward the vitreous side and attached to the inner limiting membrane, and the outer process extended toward the scleral side and attached to the outer limiting membrane. At stage 25, the ganglion cells sprouted an axon from the inner process or sent an axon directly from the perikaryon of the vitreous side. Next, the original processes, which had attached at first to the inner and outer limiting membranes, disappeared. At stage 29, spine-like processes appeared, extending in every direction from the cell body. At stage 31, some of these spine-like processes grew more extensively to form the dendrites, but the others disappeared. At stage 34, the cells began to form the ganglion cell layer, and their dendrites extended into tertiary branches. At stage 39, the cells could be classified into six types according to the branching pattern of the dendrites, which was identical with the ganglion cells after hatching. This indicated that the fundamental differentiation of ganglion cells could be regarded as essentially completed at stage 39.

Immunohistochemical Localization of 2′,3′‐Cyclic Nucleotide 3′‐Phosphodiesterase and Myelin Basic Protein in the Chick Retina

Abstract: Antisera against bovine 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNPase) and against chick myelin basic protein (MBP) were raised in New Zealand white rabbits. The specificity of CNPase antiserum was examined by Ouchterlony double‐immunodiffusion test and immunoadsorption assay. With use of the specific antiserum, immunohistochemical localizations of CNPase and MBP were investigated in the chick retina. Light microscopic immunohistochemical studies have shown that MBP is localized in the optic nerve fiber layer and that CNPase is localized in the optic nerve fiber and photo‐receptor layers. Electron microscopic immunohistochemical examinations demonstrated that the myelin‐like neural sheaths in the optic nerve fiber layer were clearly stained by both antisera, whereas the membranes of the Müller cell were not stained. In the photoreceptor layer, membranes of the inner and outer segments of rod and cone photoreceptor cells were intensely stained by CNPase antiserum. However, these portions were not stained by MBP antiserum. Membranes of bipolar cells, amacrine cells, horizontal cells, and ganglion cells were not stained by either antiserum.

Neurochemical Characteristics of Myelin‐like Structure in the Chick Retina

Abstract: Certain characteristics of myelin‐like structures in the chick retina were examined morphologically and biochemically. Developmental changes of 2′, 3′‐cyclic nucleotide 3′‐phosphohydrolase (CNPase) in the chick retina and optic nerve were examined. The measurable activity in the retina was first detected at 16 days of incubation and thereafter, it increased rapidly until 4 weeks post‐hatching. By contrast, CNPase activity in the optic nerve reached the maximum level at 4 days post‐hatching and maintained a constant level thereafter. The purifed myelin fraction from the chick retina showed higher activity of CNPase, whereas its activity in the retinal homogenate was very low. Hence, it was considered that the myelin fraction from the chick retina is similar to that of CNS myelin with respect to CNPase. Protein profiles of the purified myelin fractions isolated from the chick optic tectum, optic nerve, retina and sciatic nerve were analysed by SDS‐polyacrylamide gel elec‐trophoresis. Myelin fractions from the chick optic tectum and optic nerve contained basic protein (BP) and Folch‐Lees proteolipid protein (PLP). Myelin fraction from the chick sciatic nerve contained BP, P2 and two glycoproteins (PO and 23K). In contrast, retinal myelin fraction contained only BP. PLP, PO, 23K and P2 proteins were definitely undetectable. Electron micrographs revealed that some axons in the optic nerve fiber layer of the chick retina were wrapped by a spiral‐structured myelin‐like sheath, which showed some differences from those of CNS and PNS myelin sheaths. It was suggested that the origin of the myelin‐like structure in the chick retina is other than from oligodendroglia or Schwann cells.

Junction-like structure appearing at apposing membranes in the double cone of chick retina

SummaryNo type of junction has yet been recognized between the two entities of the retinal double cone. In the present study, a junction-like structure was observed in serial sections of the double cone of the chick retina. It is recognized from the slightly outer part of the double cone to the outer limiting membrane. The apposing membranes are virtually parallel and separated by 5 to 7 nm of extracellular space. Dense material is associated on the cytoplasmic side of the opposing membranes. The structure resembles a gap junction, but there are no cross striations between the two membranes. Further experiments are required to establish this as a new type of junction.

Physiological and morphological studies of signal pathways in the carp retina

In the vertebrate retina, visual information received at the receptor mosaic is processed in the neuronal network, and the final output is sent out to higher visual centers through the optic nerve fibers. AS a result of signal processing, retinal ganglion cells have precise receptive fields that enable them lo respond to specific stimuli, such as the shape, color or movement of images. In the retinas of many vertebrate% the most commonly found receptive fields of ganglion cells consist of concentrically arranged center and surround areas which are antagonistic lo each other (Kuffler, 1953). These receptive fields are thought to enhance the contrast of an image. Visual signals generated in photoreceptors are passed on first lo bipolar cells and then to ganglion cells, and along this course are modified by lateral input from horizontal and amacrine cells. Since the receptive fields of ganglion cells are the end products of retinal network function. it is important to know the arrangement of the preceding neural connections to understand the behavior of various ganglion cells. Our studies on the carp retina are aimed to answer this question by physiological and morphological methods.

Absence of bundle structure in the neocortex of the reeler mouse at the embryonic stage: studies by scanning electron microscopic fractography

The reeler mutant mouse is characterized by a derangement of the cerebral cortical structure due to abnormalities during the migration step at the embryonic stage. We have analyzed both the control and reeler cerebral cortex by means of scanning electron microscopic fractography. In the control cerebral cortex, the bundle formation was composed of fine fibers on which the migrating neuroblasts were attached perpendicular to the pial surface, whereas no bundle formation was observed in the reeler; instead, there was a fine meshwork of fibers surrounding the neuroblasts. The possible role of bundle formation in the normal cerebral cortex and the correlation between the inability of cells to migrate and the absence of bundle formation in the reeler is discussed.

A monoclonal antibody 5E5 recognizes an intranuclear antigen selectively present in a subpopulation of the neurons

Monoclonal antibody 5E5 labeled the nuclear antigen of the neurons in the guinea pig and rat central nervous systems including the cerebrum, cerebellum, spinal cord and retina. This antibody could discriminate neurons even among the same cell class. In in vitro study, only 10% of dividing PC12 cells was labeled with this antibody. An electron microscopic immunohistochemical study also revealed that this antibody selectively labeled heterochromatins in the neurons. Although we could not obtain any positive result by an immunoblot study, the antigenicity was remarkably diminished by the DNase I or S1 nuclease treatment on the tissue sections whereas RNase and trypsin was ineffective. These results suggested that this antigen might be a single-stranded DNA-protein complex resistant to proteolytic procedures, and possibly related to cell function or state of differentiation.

Mosaic Expression of the Reeler and Normal Phenotypes in the Cerebral Cortex in Reeler-Normal Chimeras at a Late Embryonic Stage

The cerebral cortex of reeler‐normal chimera embryos was studied by hematoxylin‐eosin staining and fractographic scanning electron microscopy in comparison with the cortices of normal and reeler mutant mice. The cerebral cortex of normal mice had a plexiform layer, which was composed of a fine meshwork of matrix cell processes. Spindle shaped neuroblasts formed a radial lining columnar structure, which was formed by attachment of migrating neuroblasts to the radial bundles. The cerebral cortex of reeler mutants did not show a plexiform layer and the cells were round with no radially columnar structures, and no radial bundles. In reeler‐normal chimera embryos, the thickness of the plexiform layer varied in different parts of the cerebral cortex. In parts where the plexiform layer was present, neuroblasts were spindle‐shaped and had a radially oriented columnar structure (normal type). But where the plexiform layer was absent, the neuroblasts were round with a radial architecture (reeler type). Intermediates between the reeler and control types were also observed. Since mosaic expression of the two phenotypes, was observed in chimeras, the reeler abnormality is apparently not caused by humoral factors. The possible mechanism of cell migration is discussed.

Clasping structure in the double cone of the retina of the turtle (Gyoclemys reevesii).

SummaryThe double cone of the turtle retina was reconstructed three-dimensionally from electron micrographs of 400 serial sections. Cytoplasm of the accessory cone extends bilaterally to surround the principal cone incompletely. The cytoplasmic extensions are 0.1 μm in width and 2 μm in length at their longest portions. They arise at approximately the median portion of the ellipsoid (consisting of mitochondria) and terminate at the level of the outer limiting membrane. The possible function of these extensions as a clasping structure is discussed.