Hiroshi Nakayasu

Three-dimensional distribution of astrocytes in zebrafish spinal cord

We prepared a monoclonal antibody (A‐22) that recognizes a 60‐kDa protein in the zebrafish brain. The antigen is distributed throughout the brain but is not found outside it. The antibody recognizes star‐shaped cells with long processes in the spinal cord. All A‐22‐positive cells are also GFAP‐immunopositive, but there are GFAP‐positive cells that are A‐22‐negative. The cells are connected to small veins and to the surface of the spinal cord. Immunopositive cells are generally homogeneous in size and shape and are found not only in the spinal cord but also in several areas of the brain. These results indicate that the stained cell is an astrocyte. Most of these cells (88%) are distributed in the gray matter of the spinal cord; the remainder (12%) are found in the white matter. Most of the cells in the gray matter are found in the ventral and dorsal horns, but some are also present in the central area along the ventricle. Glial cell bodies form an array along the longitudinal axis and are connected to each other by thick projections. The cellular array is not visible in coronal sections. In contrast, thin processes from the cells extend to the surfaces of veins, to neurons, and to the periphery of the spinal cord. We estimate that there are about 13,500 A‐22‐positive astrocytes in the spinal cord; however, this represents only 26% of the total number of astrocytes in the spinal cord (∼52,000). GLIA 36:406–413, 2001.

A monoclonal antibody stains radial glia in the adult zebrafish (Danio rerio) CNS

We have prepared a monoclonal antibody, denoted as C-4, which specifically recognizes astroglia with radial forms in the adult zebrafish brain. This report suggests that there are at least two different types of astroglia in the mature teleost brain, only one of which is recognized by C-4. Further, we have found that the C-4-binding astroglia are comprised of three morphologically distinct cellular types. Immunoblot analysis revealed that the antibody recognized only one protein band of approximately 30 kDa in the membrane fraction of the adult zebrafish brain. In the spinal cord, stained glial cells appeared to occur in the same location as ependymocytes. The processes and cell bodies of extra-ependymal cells, many adjacent to ependymocytes and a few near the pial surface, were also stained by the antibody. In the cerebellum, long processes stained by C-4 were found in the molecular layer, and these connected the cerebellum surface to the Purkinje-like cell layer. Long processes were also stained in the mesencephalon, but these were thicker than those in the spinal cord and they linked the two ventricles. The optic tectum, olfactory bulb and cranial nerves, including the optic nerves, were, however, completely devoid of the C-4 antigen. Double-immunofluorescence with antibodies against glial fibrillary acidic protein (GFAP) and C-4 demonstrated that C-4-positive cells were also GFAP-positive, although there was also a subset of GFAP-positive cells which were C-4-negative. The C-4 antibody is thus a useful tool for studying subtypes of GFAP-positive astroglia.

Postembryonic Neurogenesis in Zebrafish (Danio rerio) Brain: Presence of Two Different Systems

Abstract We analyzed postembryonic neurogenesis in zebrafish brain using the thymidine analogue 5-bromo-2′-deoxyuridine (BrdU). At 1 and 3 weeks after fertilization, BrdU-labeled cells were detected at the brain midline, especially in the telencephalon, optic tectum, hypothalamus, and cerebellum. Cell replication ceased in the telencephalon and hypothalamus by 6 weeks after fertilization, but continued in the optic tectum and cerebellum even in adult fish. Although the area of replication was widely distributed in the tectum and cerebellum of young fish, it seemed to gradually become restricted to the medial and lateral margin of the tectum and the dorsal region of the cerebellum. These areas of replication showed common morphological features, with small, tightly packed spherical cells which were stained very densely by toluidine blue. In the optic tectum and cerebellum, the areas which were BrdU-positive (or densely stained by toluidine blue) gradually decreased in size relative to the growing brain, but the total volume of these areas in each individual remained unchanged. These results indicate that there are two different neurogenic systems in teleost fish. One system, found in the telencephalon and the hypothalamus, stops replication at an early stage of development; dividing cells in these areas are repressed or removed. The other system, found in the tectum and cerebellum, retains its replicative activity even in adult fish; the number of dividing cells in these areas seems to be maintained during development of the brain.

Ex vivo culture of isolated zebrafish whole brain

We have succeeded in culturing whole zebrafish brains ex vivo for 1 week. While isolated cells and tissue slices have previously been employed for neurobiological studies, these techniques are limited, because while local networks may be preserved, their original context in the whole brain is lost. Culture of the whole brain would facilitate the study of cells and systems within an intact brain infrastructure. Our culture method entailed isolating the whole brain and placing it on a sterile and porous membrane, after which it was maintained with a conditioned medium in a six-well plate in a CO2 incubator at 28.5 degrees C. Whole brains cultured by this simple method were relatively unaltered in terms of their morphology, cytoarchitecture, immunohistochemistry and ability to transport horse radish peroxidase (HRP). This method of cultivation may be very useful for neurobiological research.

A novel monoclonal antibody recognizes a previously unknown subdivision of the habenulo-interpeduncular system in zebrafish

The habenulo-interpeduncular system is an evolutionarily conserved structure found in the brain of almost all vertebrates. We prepared a monoclonal antibody (6G11) which very specifically recognizes only a part of this system. 6G11 is a monoclonal antibody prepared from a neuronal membrane protein in adult zebrafish brain. In western blot analysis of the adult zebrafish brain, the antibody recognized a 95 kDa protein, and the class of the antibody was determined to be IgM. The 6G11 antigen was not detected in zebrafish muscle, intestine, testis or ovary. A group of neurons stained by the 6G11 antibody was located in the caudomedial part of the zebrafish habenula. The 6G11-immunopositive neurons extended their axons into the fasciculus retroflexus (FR). One group of immunopositive neurons projected toward the interpeduncular nucleus (IPN), especially to the intermediate and the central subnucleus (type 1 neuron). The other group projected to the ventral midline at the level of the raphe nucleus; these axons passed ipsilaterally beside the IPN and converged in the ventral midline under the raphe nucleus (type 2 neuron). Both type 1 and type 2 fibers are relatively minor components of the FR. Little has previously been known about this topological pattern in any species. The 6G11 monoclonal antibody could be a useful tool for expanding knowledge of the habenulo-interpeduncular system.

Monoclonal antibody stains oligodendrocytes and Schwann cells in zebrafish (Danio rerio)

We prepared a monoclonal antibody that recognizes oligodendrocytes and Schwann cells in zebrafish. On immunoblots, the antibody mainly recognized three protein bands of 34 kDa in a membrane fraction from adult zebrafish brain. Medaka fish (Oryzias latipes) also possessed the same protein bands in a membrane fraction. The antibody did not stain neurons, but stained cells in fiber tracts and cranial and spinal nerves. In order to determine the nature of these cells, the staining pattern of the monoclonal antibody was compared with that of a myelin basic protein antiserum. Both antibodies stained oligodendrocytes and Schwann cells in fixed sections from the adult zebrafish. Both antigens were also co-localized in cultured glial cells. Taken together, these results indicate that the new monoclonal antibody recognizes myelinating glial cells in zebrafish and will be useful for the analysis of piscine glia.

A periaxonal net in the zebrafish central nervous system

We produced a monoclonal antibody, named A20, which specifically recognizes a 35 kDa protein and stains myelinated axons in zebrafish brain. The A20 antigen is located at the outside of the myelin layer of large axons, and comprises a fine meshwork composed of thin unit fibers about 1-2 microm in length and about 100-200 nm in thickness. The unit fibers form pentagonal and hexagonal structures, which further polymerize into an envelope structure on the axons. The A20 monoclonal antibody did not stain neuronal cell bodies nor synapses. Instead, the distribution of the A20 antigen was along axons, practically coincident with the distribution of myelin basic protein. The monoclonal antibody stained only axons in the central nervous system (CNS), and not the extracellular matrix surrounding Schwann cells. These results suggest that this antigenic meshwork (which we call the periaxonal net) is synthesized by oligodendrocytes. During the development of the zebrafish brain, the periaxonal net appeared after the formation of myelin on the axons. The periaxonal net developed first at the brain stem, then gradually appeared at the caudal end of the spinal cord. The thickness of the periaxonal net around the Mauthner axon changed during development. Although the thickness of the Mauthner axon continues to grow throughout life, the thickness of periaxonal net stopped growing at 6 months after fertilization.

Visualization of erythrocytes in the zebrafish brain.

Abstract We found that erythrocytes of zebrafish have cytoplasmic peroxidase activity. Blood in the zebrafish brain was visualized using a standard peroxidase staining method after formaldehyde fixation. The erythrocytes in the brain were heavily stained, but neurons and glias were not stained at all. This easy method enables the distribution of erythrocytes in the whole brain to be determined, and enables the actual number of erythrocytes in each area in the brain to be calculated. The paths of major, thick blood vessels in zebrafish brain are similar to those in higher vertebrates, however, the distribution of thin blood vessels is different. We also found that the erythrocytes were unevenly distributed in the brain. For example, the density of erythrocytes in the surface layer of the tectum was more than 30-fold higher than in the deeper granular layer. Very few erythrocytes were found in bundles of axons like cranial nerves and the medial longitudinal fascicle. In general, fewer erythrocytes were found in areas near the ventricle, whereas many more were found closer to the surface of the brain. The distribution of erythrocytes in the brains of sleeping, awake and actively moving fish were compared. In the brains of sleeping fish, most of the erythrocytes were present in large vessels. This was not observed in brains of awake or actively moving fish. We found that the blood supply to motor neurons in the ventral horn of the spinal cord increased during active movement compared to that in awake or sleeping fish.

Distribution of a brain-specific extracellular matrix protein in developing and adult zebrafish

A monoclonal antibody (IgG) that recognizes a 53-kDa zebrafish brain protein was isolated and used to characterize the distribution of this protein in zebrafish. (1) The antigen was found only in the brain and not in any other tissues such as muscle, dermis and cartilage. Within the brain, the antibody recognized extracellular matrix (ECM) outside neuronal cells. (2) Digestion by hyaluronidase released the antigen from brain tissue, and the monoclonal antibody staining was also decreased by the digestion by hyaluronidase. (3) The pattern of antigen distribution is not perineuronal, as the density of the antigen at the periphery of the cells was practically identical to that of the empty intercellular spaces. Therefore, this monoclonal antibody does not recognize the perineuronal glycocortex. (4) The antigen is distributed only in limited areas of the brain, namely in the periphery of the forebrain, the hypothalamus, the optic tectum, the interpeduncular nucleus, the cerebellum and the ventricular rim of the medulla. In the optic tectum, the antibody strongly stained the most superficial layer, and in the cerebellum, it stained the molecular but not the granular layer. These patterns of distribution are very different from those of other typical brain ECM proteins and suggest that this protein may play quite distinct roles in brain development and maintenance.