Genzoh Isomura

Cajal-Retzius neurons identified by GABA immunohistochemistry in layer I of the rat cerebral cortex

By means of immunohistochemistry using anti-gamma-aminobutyric acid (GABA) antibodies, characteristic neurons of pyriform, bipolar or pleomorphic shapes, regarded as the Cajal-Retzius (CR) neurons, were clearly demonstrated in layer I of the rat cerebral cortex at the various experimental periods. On embryonic day 15, ovoid neurons only in the marginal zone indicated immunoreactivity for GABA. They gradually extended thick processes often in parallel with the pial surface and formed a dense GABA fiber network in immature layer I during the early postnatal periods. Some GABA neurons seemed to migrate into the underlying layers to settle as nonpyramidal cells. With the expansion of brain volume, GABA neurons relatively diminished and decreased in number. Nevertheless, a small number of GABA neurons did exist as essential CR neurons in layer I even in the adult rats aged 9 months.

Phenotypic changes of AADC-only immunopositive premammillary neurons in the brain of laboratory shrew Suncus murinus by systemic administration of monoamine precursors

After 5-hydroxy-L-tryptophan (5-HTP) and L-3,4-dihydroxyphenylalanine (L-DOPA) were injected i.p. in the laboratory shrew Suncus murinus, immunocytochemical and immunofluorescence studies were conducted on continuous or same sections of the brain, using specific anti-tyrosine hydroxylase (TH), anti-aromatic L-amino acid decarboxylase (AADC), anti-dopamine (DA) and anti-serotonin (5-HT) antisera which were produced in our laboratory. The results of double-staining by the immunofluorescence method as well as immunoelectron microscopy strongly indicate that the cells of the premammillary nucleus of the laboratory shrew brain (AADC-only-positive neurons) are capable of synthesizing DA and 5-HT simultaneously upon simultaneous administration of L-dopa and 5-HTP.

Increase of glial fibrillary acidic protein fragments in the spinal cord of motor neuron degeneration mutant mouse

We analyzed protein fractions extracted from the spinal cord of the motor neuron degeneration (Mnd) mouse, a mutant that exhibits progressive degeneration of lower spinal motor neurons, by one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) after solubilization of the tissue with medium containing sodium dodecyl sulfate (SDS)-urea during growth of the animal, in comparison with those of age-matched controls (C57BL/6). Several protein spots were detected around a region of pI 5.6-6.0 and molecular mass of 35-50 kDa in Mnd spinal cord tissue on the two-dimensional PAGE separation profile with Coomassie brilliant blue staining, while only a few spots around the same region were found in the control spinal cord. These spots were all immunoreactive with an antibody against glial fibrillary acidic protein (GFAP), a cytoskeleton filamentous protein specific to astroglial cells. The protein spot with molecular mass of 50 kDa showed immunoreactivity with anti-GFAP antibody, had a blocked amino-terminus, and is assumed to be intact GFAP. Several protein spots with slightly smaller molecular masses of 35 to 48 kDa lacked the head domain of the GFAP molecule as a result of cleavage at the 29th and 56th residues from the amino terminus. In Mnd spinal cord tissue, the densities of the immunoreactive GFAP bands with smaller molecular masses increased with development, and became dominant at the time of the appearance of behavioral paralytic gait around 6 to 7 months of age. These results suggest that the increased GFAPs devoid of head domains are related to the degenerative loss of motor neurons in the Mnd spinal cord. Histopathological and GFAP immunohistochemical examination of Mnd spinal cord preparation demonstrated progressive degenerative loss of motor neurons, and considerable increases in number of GFAP-stained astrocytes in the ventral horn at 7 to 9 months of age. These processes of degenerative loss of motor neurons and proliferation of reactive astrocytes with increased levels of fragmented GFAP in the Mnd spinal cord during development seem to be characteristic and preceded the deterioration of motor activities in this animal model of amyotrophic lateral sclerosis.

Production of specific antibody against l-DOPA and its ultrastructual localization of immunoreactivity in the house-shrew (Suncus murinus) lateral habenular nucleus

An antiserum was raised against L-DOPA bound to bovine serum albumin, purified by affinity chromatography, and its specificities were verified by immunoblotting and enzyme-linked immunosorbent assays. The antiserum did not cross-react with dopamine (DA), tyrosine, tyramine, octopamine, norepinephrine or epinephrine. Immunocytochemical studies using the PAP method revealed that tyrosine hydroxylase- and L-DOPA positive but aromatic L-amino acid decarboxylase- and DA-negative neurons were present in the lateral habenular nucleus of the house-shrew (Suncus murinus) brain. Ultrastructurally L-DOPA immunoreactive products were localized in the cytoplasmic matrix and terminals with vesicles.

Chemical features of monoaminergic and non-monoaminergic neurons in the brain of laboratory shrew (Suncus murinus) are changed by systemic administration of monoamine precursors

5-Hydroxy-L-tryptophan (5-HTP) and L-3,4-dihydroxyphenylalanine (L-DOPA) were injected intraperitoneally (i.p.) into the laboratory shrew (Suncus murinus). Immunocytochemical and immunofluorescence studies were carried out on serial or same sections of the brain, which were reacted with specific antisera to dopamine (DA) or serotonin (5-HT) produced in our laboratory. We observed that cell bodies and nerve terminals of many catecholaminergic (CAnergic) neurons exhibited 5-HTP uptake and conversion of the precursor into 5-HT. However, the locus ceruleus showed scarcely any 5-HT immunoreactivity. This suggests that the precursor uptake mechanism may be different among various CAnergic groups. In contrast to these findings on CAnergic neurons, all serotoninergic (5-HTnergic) neurons after L-DOPA administration showed DA immunopositive reaction in their cell bodies and nerve terminals, suggesting that 5-HTnergic neurons may have the same capacity for precursor uptake. On the other hand, we observed that all aromatic L-amino acid decarboxylase (AADC)-only-positive neuron groups showed both DA and 5-HT immunoreactions after L-DOPA and 5-HTP administration, respectively, in the double-staining immunofluorescence method. From these results, AADC-only-positive neurons may be considered to belong to the amine precursor uptake and decarboxylation (APUD) system.

Three types of neurons in the medial cuneate nucleus of the cat

A combined study utilizing Golgi-EM and gamma-aminobutyrate (GABA) immunocytochemistry revealed the presence of 3 types of neurons in the cat medial cuneate nucleus. The 2 GABA-immunonegative neuron types were characterized by numerous and richly arborized dendritic processes; they were abundant in the rough endoplasmic reticulum and other cell organelles, and the perikarya were heavily covered by axosomatic terminal boutons. Their large neuron type (35 +/- 3 micron diameter) exhibited thick and bifurcating dendrites, whereas the dendrites of the smaller (28 +/- 3 micron) immunonegative nerve cell were more or less radially oriented. The third and smallest neuron type (24 +/- 4 micron), shape not demonstrated by Golgi stain, indicated strong GABA-immunopositivity. The soma of this type had relatively little rough endoplasmic reticulum and other cell organelles, and received only few or slight axosomatic synapses, in contrast to the GABA-positive cells. A quantitative estimation of immunostained and immunonegative nerve cells in the 50 micron thick sections showed that 11-45% of nerve cells in the medial cuneate nucleus are GABA-positive.

Coexistence of tyrosine hydroxylase and serotonin in the raphe nucleus of the laboratory shrew (Suncus murinus) during postnatal life.

Immunoreactivity (IR) of tyrosine hydoroxylase (TH), which is the rate-limiting enzyme of catecholamine (CA) synthesis, was observed in the serotonergic neurons of the raphe nucleus (RN) of the newborn laboratory shrew from postnatal day (P) 0 to P14. Using an immunohistochemical method involving highly specific antibodies produced in our laboratory, we found that these RN neurons were TH-, GTP cyclohydrolase I-, aromatic L-amino acid decarboxylase-immunoreactive, but DOPA- and dopamine-immunonegative. In addition, they were tryptophan-, 5-hydroxytryptophan- and serotonin-immunoreactive. These results suggest that TH in serotonergic neurons of RN of laboratory shrew has no function as a CA-synthesizing enzyme but may play some role as a regulator or a subsidiary factor in the postnatal development of serotonergic neurons.

Blood supply to the retina in the laboratory shrew (Suncus murinus).

The blood supply to both retinae was studied light microscopically and by scanning electron microscopy in 48 adult laboratory shrews (Suncus murinus) of both sexes. Thirty-eight of the animals were injected into the left ventricle with Neoprene latex (Du Pont. 601A) or with Mercox (Dai Nippon Ink Ltd., CL-2R) to elucidate the blood supply to the retina from the ophthalmic artery. The remaining animals were kept for histological study of the retina. The central retinal artery, originating from the ophthalmic artery in the muscular part of the orbit, enters the optic nerve, passes through the optic disk together with the central retinal vein and penetrates the vitreous space (cavity of the eye) between the lens and the inner limiting membrane of the retina, where it divides into the dorsal, ventral, and caudal branches. Each branch, moreover, bifurcates into nasal and temporal arterioles and is distributed throughout the retina on the inner limiting membrane as far as the ciliary body and the lens. On the way they obliquely send small vessels through the inner limiting membrane into the outer plexiform layer of the retina. Their vascularization appears to correspond to the membrana vasculosa retinae found in teleosts, amphibia and reptiles.

Measurement of DOPA and immunolocalization of l-DOPA-positive nerve fibers in rat dental pulp

dopa, norepinephrine, and traces of dopamine, epinephrine were present in in rat dental pulp. L-dopa was localized in nerve fibers in dental pulp. The results suggest that L-dopa-positive nerve fibers are present in dental pulp as well as classical adrenergic fibers.