Nobuo Shimizu

Noradrenaline innervation of the spinal cord studied by the horseradish peroxidase method combined with monoamine oxidase staining

SummaryThe origin of the spinal cord noradrenaline (NA) has been investigated by means of the horseradish peroxidase (HRP) method, combined with monoamine oxidase staining (Glenner) to identify the NA neurons. Following the injection of HRP to the various levels of rat spinal cord, cervical to sacral cord, A1–3, 5–7 NA neuron groups were labeled with HRP. They showed almost the same distribution pattern regardless of difference in the injected segment. Labeled NA neurons in A6 were concentrated in the ventral division of the locus coeruleus, which continued to the labeled NA neurons in the subcoeruleus area. The HRP positive neurons in the pons outnumbered those of the medulla oblongata. As the NA neurons described above were considered to be the source of NA in the forebrain, such as the hypothalamus and preoptic area, the possibility that the same NA neurons might innervate both the forebrain and spinal cord has been presented.

Afferent fiber connections from lower brain stem to hypothalamus studied by the horseradish peroxidase method with special reference to noradrenaline innervation

SummaryAttempts were made to determine the afferent projections to the anterior hypothalamus including the preoptic area from the lower brain stem by means of the horseradish peroxidase method combined with monoamine oxidase staining to identify noradrenaline (NA) neurons. In addition to this technique, a histofluorescence analysis was performed. NA fibers in the medial part of the anterior hypothalamus were mainly supplied by A1 and A2 NA neuron groups, while the lateral part and periventricular zone received NA terminals from both pontine and medulla oblongata NA neuron groups. Furthermore, the present study indicated that there were direct projections to the anterior hypothalamus from non-noradrenergic neurons in the lower brain stem: nuclei raphe dorsalis, centralis superior, cells in the mesencephalic and pontine central gray matter, nuclei parabrachialis lateralis and medialis, cells around fasciculus longitudinalis medialis.

Histochemical studies of monoamine oxidase of the brain of rodents

SummaryMAO of the brain was investigated histochemically in mice, rats, guinea pigs and rabbits. Fresh frozen sections were subjected to the tryptamine-tetrazolium method by Glenner, Burtner and Brown (1957).MAO activity of the brain of 4 animal species is generally similar with respect to its pattern of distribution. However, the intensity of enzyme action of the brain as a whole differs somewhat in animal species, being highest in guinea pigs, intermediate in rats and lowest in mice and rabbits. The enzyme action occurs mainly in the neuropil of the cerebral grey matter, while weak or negative activity is generally observed in the white matter excepting the tractus retroflexus of Meynert.The marked activity is encountered in the interpeduncular nucleus, locus coeruleus, area postrema, dorsal nucleus of the vagus nerve, hypothalamus, habenular nuclei and midline nuclear group of the thalamus, nucleus of the brachium conjunctivum, and central grey matter. The enzyme activity is weak or negative in the neocortex, striatum, mamillary body, thalamic nuclei (excepting the habenula and midline nuclear group), subthalamic nucleus, substantia nigra, red nucleus and nuclei of the somatic cranial nerves.The possible function and significance of MAO in the brain were discussed particularly by comparing the sites of this enzyme with those of succinic dehydrogenase and cytochrome oxidase, and the inverse relation between these enzymes was suggested.

Histochemical studies of succinic dehydrogenase of the brain of mice, rats, guinea pigs and rabbits.

Succinic dehydrogenase activity of the brain was histochemically investigated in mice, rats, guinea pigs and rabbits. Fresh frozen sections cut at 20 µ were stained with the Seligman-Rutenburg method as modified by Rosa and Velardo. The pattern of enzyme distribution is remarkably similar but the intensity of staining differs somewhat in the animal species, being highest in mice and rats, intermediate in rabbits and lowest in guinea pigs. The enzyme activity is found exclusively in the grey matter and ocurs both in the perikaryon and neuropil. The activity in the grey matter exhibits the characteristic local variations which are ascribed to the varying participation of the nerve cell bodies and surrounding neuropils in the reaction. Nerve cell bodies contain variable amounts of dye particles of different sizes, and the neuropil possesses variable numbers of coarse granules. An intense reaction is given in the caudate nucleus, putamen, anterior nucleus of the thalamus, interpeduncular nucleus and cerebellar cortex. An intermediate activity occurs in the neocortex, rhinencephalon, optic tectum, most of the cranial nerve nuclei, superior and inferior olivary nuclei, and the Goll and Burdach nuclei. A low activity is present in the paraventricular structures, such as supraoptic crest, subfornical and subcommissural bodies, and area postrema, as well as the paraventricular and supraoptic nuclei of the hypothalamus, and dorsal nuclei of the vagus nerve.

Histochemical method for demonstrating aldolase

SummaryBy combing the indirect method of aldolase activity of Warburg and Christian, which consisted in the measurement of reduction of DPN in the presence of glyceraldehyde-3-phosphate dehydrogenase and arsenate, with nitro-BT reduction and we could obtain the much better method of demonstrating aldolase than that of Allen and Bourne.The optimal incubating mixture was composed of 1) 10 ml 0.02 M sodium fructose-1,6-diphosphate, 2) 5 mg DPN, 3) 10 mg nitro-BT, 4) 10 ml of 0.05 M arsenate-HCl buffer (pH 7.6). Fresh frozen section, which were fixed briefly in 80% cold ethanol, gave a better staining results. The distribution of aldolase of some organs of rat and the validity and limitation of the method were described.

Demonstration by degeneration silver method of the ascending projection from the locus ceruleus.

SummaryIn order to demonstrate the ascending projection from the locus ceruleus by degeneration silver method, the nucleus was destroyed unilaterally in rats and the brains were stained by a modified Fink-Heimer method following various survival times. Additional experiments included the bilateral destruction of the locus ceruleus and the administration of 6-OH-DOPA.For the demonstration of degenerated terminals in the cerebral cortex it is essential to choose an optimal survival time of 4 or 5 days, keep the time of fixation below 1 week, and perform treatments with permanganate and with silver at 37°C.The trajectory and distribution of the cerulo-cortical tract as revealed by the silver degeneration methods is in agreement with the results of the histofluorescence method, with some minor differences. The ascending fibers run through Forels tegmental fascicle, turn rostro-ventrally to the subthalamus, enter and cross the internal capsule, pass through the ventral part of the caudate-putamen and distribute to the entire cerebral cortex. The tract is mainly ipsilateral, though some fibers are crossing.

Descending projection of the nucleus tegmentalis latero-dorsalis to the spinal cord; studied by the horseradish peroxidase method following 6-hydroxy-DOPA administration.

Descending projection of the nucleus tegmentalis laterodorsalis (TLD) to the spinal cord was investigated using the horseradish peroxidase (HRP) method following the systemic administration of 6-hydroxy-DOPA (6-OH-DOPA). 6-OH-DOPA pretreatment resulted in an intense labeling of the cell group at the laterocaudal portion of TLD after the injection of HRP to the lower segments of the spinal cord. Whereas in untreated rats, cells at the laterocaudal portion of TLD were faintly labeled. Such indirect effect correlated with a reduction in the number of fluorescent catecholamine nerve terminals in the spinal cord after 6-OH-DOPA injection, suggesting that the descending projection from laterocaudal portion of TLD to the spinal cord might be influenced by the catecholamine neurons in the central nervous system.

Fine structure of the area postrema of the rabbit brain

SummaryThe area postrema of the rabbit, which was perfused with glutaraldehyde and postfixed in osmium tetroxide, was observed under the electron microscope. This area showed neuronal and neuroglial structures similar to those of ordinary cerebral tissue, except for rich blood capillaries, which were surrounded by conspicuous perivascular spaces. Parenchymal cells included a moderate number of small neurons and large numbers of specific astrocyte-like cells. The neuropil consisted of a small number of thin myelinated and many non-myelinated nerve fibers of varying calibers, axo-dendritic synapses, and neuroglial cell processes, leaving no spaces between them. The axons and synaptic terminals contained moderate amounts of granular vesicles, which were similar in size to those found in the hypothalamus and were supposed to contain catecholamine. Glycogen paticles were demonstrated mainly in the cytoplasm of the astrocyte-like cells.

The Fine Structural Organization of the Locus Coeruleus in the Rat with Reference to Noradrenaline Contents

SummaryCombination of glyoxylic acid perfusion and postfixation in permanganate was used in an electron microscopic study of the locus coeruleus (LC) of the rat to give good preservation of fine structure and a reproducible demonstration of noradrenaline (NA) storage granules. Medium-sized LC cells (18 × 30 μm) contained a moderate number of small granular vesicles (SGV) and a few large granular vesicles (LGV), mainly near the Golgi apparatus. Dendritic branches were identified by their SGV content up to the tip. Dendrites were occasionally in close contact with each other or with the soma of LC cells, forming dendro-dendritic or dendro-somatic contacts. Numerous axon terminals containing many SGV and some LGV were observed in the neuropil, and they tended to contact dendrites and somata of LC cells or dendrites of unknown origin. These neuronal contacts were devoid of synaptic specializations except for an array of dense perpendicular lines between the juxtaposed membranes. Small oval cells (10 × 15 μm) devoid of SGV occurred frequently in the peripheral part of the nucleus, and they were occasionally in direct contact with LC cells.

Fine structure of rat cerebellar noradrenaline terminals as visualized by potassium permanganate 'in situ perfusion' fixation method.

Abstract The fine structure of noradrenaline-containing nerve terminals in the rat cerebellum was investigated by means of modified potassium permanganate fixation, which revealed a number of small cored vesicles in the terminals. In the molecular layer, noradrenaline-containing terminals mainly made a synaptic like contact with tertiary or secondary Purkinje cell dendrites, while in the Purkinje cell layer no direct contact between noradrenaline-containing terminals and Purkinje cell soma was demonstrated in this study. In the granular layer, noradrenaline-containing nerve terminals were found in its superficial part, particularly around the glomeruli. Noradrenaline-containing nerve terminals in the granular layer mostly make a close contact with the dendrites of the granule cells. As well as confirming the noradrenergic input to Purkinje cells, the results show that noradrenaline might directly influence the granule cells.