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The bone mineral density (BMD) of calcanei dissected from 30 cadavers (14 males and 16 females; ages 61 to 100) was measured by dual-energy X-ray absorptiometry (DXA) and quantitative computed tomography (QCT), while the stiffness was measured by ultrasound bone densitometry (USD). There was high correlation between BMD by DXA and average bone density by QCT (R = 0.94), but poor correlation between BMD by DXA and stiffness by USD (R = 0.55). However, the correlation coefficient of stiffness to BMD was much higher in the female calcanei (R = 0.74) than in the male calcanei (R = 0.32). Analyzing the three elements, stiffness, speed of sound (SOS) and broad-band ultrasound attenuation (BUA), it was clear that SOS rather than stiffness correlated strongly to BMD by DXA and to bone density of trabeculated bones by QCT (R = 0.618; 0.699 respectively). The value of BUA shows more relationship to the width of the calcanei than the area of X-ray projection from the side (R = 0.561).

By means of immunohistochemistry using anti-γ-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.

The patterns of 892 synapses with participating GABA elements were examined in the rat anterior cingulate cortex, using a preembedding immunohistochemical method. Somas and dendrites of pleomorphic GABA neurons received both GABA and unstained terminals at a ratio of 2 to 5, forming axo-somatic and axo-dendritic synapses. Some GABA terminals displayed axo-axonic contacts with either GABA or unstained terminals at an almost equal ratio. Occasionally, two GABA elements forming a synapse in turn showed further contacts with common or separate postsynaptic elements to constitute large synaptic complexes. Triadic arrangement of GABA terminals was noted adjacent to a nonGABA terminal forming an axo-dendritic synapse. The other GABA terminals formed synapses with nonGABA pyramidal cells on their apical dendrites, somas and axon initial segments. The majority of these GABA terminals, including small round vesicles, displayed symmetric contacts. A quarter of the examined synapses were found to be composed of contacts between two GABA elements. The high incidence of synapses between two GABA elements suggested the presence of a particular local network of GABA system in a restricted cortical area.

To clarify the origin of the cells appearing in a stab wound of the parietal cortex, bone marrow cells from inbred Lewis rats were labeled in vitro with 3H-uridine and injected intravenously into 36 rats of the same strain Just before or after the brain had been stabbed. The animals were sacrificed from 1 to 15 days after the injection of labeled cells. The cells in the wound were examined and the percentage of the various types recorded. Radioautographs were prepared of the wound and the surrounding neuropil, as well as of the corresponding region on the intact side; and they were searched for labeled cells. One day after the stabbing, the wound consisted of a central region containing fluid and red blood cells, and a marginal region of disorganized nervous tissue including granulocytes, monocytes and macrophages. Around the wound, the neuropil appeared normal, except for the perivascular areas appearing more cellular and distended than normally. During the next few days, the granulocytes and monocytes gradually disappeared from the wound. Meanwhile, the macrophages, which initially often showed monocytic features, became increasingly numerous. However, after the seventh day, the macrophages decreased in number, while many small cells appeared in the wound. These displayed a scanty basophilic cytoplasm rich in phagosomes and a nucleus with dense chromatin clumps similar to that seen in microglia A search of the radioautographs revealed no labeled cell on the intact side, but 220 labeled cells on the injured side. Most of these cells were within the wound itself, and a few in the surrounding neuropil. Within the wound, labeled granulocytes and monocytes were common one day after injection, but disappeared after the first three days. Few labeled macrophages were observed at one day but many at three days; none were labeled after the seventh day, however, possibly due to turnover and loss of the 3H-uridine labeled RNA. In the neuropil surrounding the wound, a few labeled cells identified as microglia were observed during the first three days after injection. They were located in perivascular areas, in satellite position to neurons and within the nearby subependymal layer It is concluded that, under the influence of a stab wound, blood cells of bone marrow origin enter the wound and the surrounding neuropil. The presence of macrophages with monocytic features within the wound indicates that the monocytes transform into macrophages. The small cells displaying phagosomes and a microglia-like nucleus, which become numerous after the seventh day, are tentatively interpreted as intermediates in the transformation of the monocytederived macrophages into microglia. Finally, the early labeled cells identified as microglia in the surrounding neuropil are interpreted as a direct transformation of monocytes into microglia.

Scanning electron microscopy revealed numerous macrophagic ameboid cells on the ependymal surface of all brain ventricles in neonatal rats. Macrophagic ameboid cells aggregated in the sulcus medianus of the fossa rhomboidea, the recessus of the cerebral aqueduct and the recessus infundibuli, i. e. the ventromedial floor of the ventricular cavity covered mainly with non-ciliated ependyma. Macrophagic ameboid cells were numerous in the first few days after birth, often intermingling with extravasated erythrocytes. Subsequently, these cells decreased in number until 10 days after birth. Thus, it was rather difficult to find such ameboid cells in the brain ventricles of 21-day-old rats. Intravenous injection of primuline, a fluorescence dye used as a cytoplasmic marker in the previous study, enhanced the appearance of the ameboid cells and caused them to remain longer on the ventricular surface.

Following repeated subcutaneous administrations of 6-OHDA into neonatal rats (a dose; 100μ/g bw), the effect on the brain development was examined. The striatal dopamine fluorescence never disappeared completely even immediately after multiple administrations of 6-OHDA, however a slight reduction of the fluorescence lasted for a long time. Semithin sections stained with toluidine blue showed numerous dark necrotic neurons appearing at a 15-times higher ratio than that in controls. Such necrotic neurons always accompanied a few granular deposits around the wavy surface. In a certain area of the striatum (0.898mm2), there were no clear changes in the cell population in neurons and glia, in the ratio of glia to neurons, and in the ratio of each cell type of glia to the total glia, but some neuronal cell loss was speculated to occur slowly.The ultrastructural alteration observed at 28, 35 and 42 postnatal days in 6-OHDA treated rats was characterized by an appearance of myelin-like membranous lamellar bodies in the boutons synapsing with neural soma. In some cases intact small synaptic vesicles were seen around the lamellar body which probably rose from cell membranes in the synaptic area. Most of the necrotic neurons corresponded to the post-synaptic side for the boutons including lamellar bodies. Necrotic neurons displayed a dense cytoplasmic matrix, an increase of polysomes and cisternal swelling in rough ER and in the Golgi apparatus. Membranous lamellar bodes were occasionally observed in the periphery of satellite glial cells, probably oligodendrocytes.In conclusion, a long lasting reduction of striatal dopamine caused by 6-OHDA might disturb the function around the synaptic area and the metabolic regulation in the postsynaptic striatal neuron, thus resulted in the appearance of rather chronic morphological alterations in those areas.

Following unilateral destruction of the substantia nigra of the rat, terminal degenerations in the neostriatum were examined under an electron microscope. The early changes that appeared at 15hrs after destruction were characterized by the increased electron density of the terminals and an accumulation of glycogen particles in the astrocytal processes surrounding such terminals and in the perivascular areas. The postsynaptic sides of the early degenerating terminals were often presumed to be dendritic spines with a fluffy material and the spine apparatus. Some were small dendrites with neurotubules.The degenerating terminals gradually increased the electron density, and irregularly shaped dense bodies appeared in these terminals. At 2 and 3 days after destruction, degenerating terminals became shrunken into dense bodies of 0.5-0.7μ in diameter. They were completely enclosed by the astrocytal swollen processes with glycogen particles, and then digested by these glial processes. At 7 days after destruction, there was no dense degenerating terminal, whereas the degeneration of myelinated axons remained till 2 weeks, showing dense axoplasm and disintegrated lamellar structures of the myelin sheaths. These results indicate that the terminal degeneration of the dopamine neuron in the neostriatum appears within a few days and disappears after 7 days, corresponding well to those results obtained by the Fink-Heimer method.

Microglial cells are absent from the corpus callosum of newborn rats. In the hope of finding out when and how microglial cells appear with age, 3H-thymidine was given intraperitoneally as single or three shortly spaced injections to 5-day-old rats weighing about 15 g; and these animals were sacrificed at various time intervals from 2 hours to 35 days later. Pieces of corpus callosum were taken near the superior lateral angle of the lateral ventricles; and semithin sections were radioautographed and stained with toluidine blue. The corpus callosum of 5-day-old rats is composed of loosely arranged unmyelinated fibers and scattered cells. Among these cells, microglia are rare; there are a few astrocytes, many immature glial cells, rare pericytes, and 6-7% of phagocytic “ameboid cells” consisting of a few monocytes and many macrophages. In the animals sacrificed two hours after 3H-thymidine administration, label is present only in immature cells and “ameboid cells.” As time elapses and the fibers of corpus callosum become myelinated, oligodendrocytes and, later, microglial cells appear. At the age of 12 days, microglial cells are present in substantial number; and by 19 days, the number doubles to reach a plateau. Many of the new microglial cells are labeled, e.g., 78.1% in 12-day-old animals (7 days after 3H-thymidine administration). The labeled microglial cells must have come from the transformation of cells that acquired label early, that is, from the immature cells or the “ameboid cells.” The height of the peaks of labeling – 59.8% at nine days for immature cells and 77.8% at 12 days for “ameboid cells” – points to the latter as precursors of the highly labeled microglial cells. Furthermore, the “ameboid cells” disappear as microglial cells appear and there are transitional elements between these two cell types. Cell counts suggest that about a third of the “ameboid cells” transform into microglial cells, while the others degenerate and die. Thus, the microglial cells which appear in the corpus callosum during the first three weeks of life result from transformation of the “ameboid cells” – a group of macrophages showing various stages of transition from monocytes. As for the occasional microglial cell appearing after the third week or in the adult, they presumably come directly from monocytes. In either case, monocytes would be the intial precursors.

The corpus callosum of young rats was examined to clarify the behavior of the three subtypes of oligodendrocytes (the large organelle-rich “light oligodendrocytes,” the smaller and more densely stained cells referred to as “medium oligodendrocytes,” and the even smaller and denser “dark oligodendrocytes”). It was hoped to find out whether cells of the three subtypes undergo division and how they are related to one another. 3H-thymidine was given intraperitoneally as single or three shortly spaced injections to a first group of 19- to 20-day old rats weighing about 40 g, and to a second group of 25-day old rats weighing about 80 g. The animals were sacrificed at various time intervals from 2 hours to 35 days after 3H-thymidine administration. Pieces of corpus callosum were taken near the superior lateral angle of the lateral ventricles; and semithin sections were radioautographed and stained with toluidine blue. Two hours after 3H-thymidine injection, label is virtually absent from light, medium and dark oligodendrocytes, from microglia, and probably from astrocytes, but is present in about 10% of the immature glial cells, which include the poorly differentiated glioblasts and the partially differentiated oligodendroblasts and astroblasts. Hence, the cells undergoing DNA synthesis and mitosis in the corpus callosum are these three types of immature cells. During the week that follows the administration of 3H-thymidine, label appears in oligodendrocytes and astrocytes, which presumably have arisen from the initially labeled immature cells. The oligodendrocytes acquire label in a sequential manner: the light cells show label first and their labeling index reaches a peak at the seven-day interval; the medium oligodendrocytes become labeled next with a labeling peak toward the 14- and 21-day intervals and, finally, the dark oligodendrocytes with a peak around the 28-day interval. Analysis by the method of Zilversmit et al. (1942-1943) provides precise details on the sequence: immature cells presumed to be oligodendroblasts give rise to light oligodendrocytes which, after four to seven days, transform into medium oligodendrocytes which, after another 11 to 18 days, transform into dark oligodendrocytes. The dark cells may persist indefinitely or turn over at a very slow rate. It is concluded that oligodendrocytes arise from the last division of oligodendroblasts and develop in three main periods; a light stage lasting less than a week, a medium stage lasting about two week, and a very long lasting dark stage.

Divergent projections of catecholamine (CA) neurons of the locus coeruleus have been studied by fluorescent retrograde double labeling in conjunction with monoamine histofluorescence technique. The present results indicate that the coerulo-cortical CA system is composed of two types of neurons. A predominant type possesses few divergent axons innervating a restricted region, while the other type projects widely to various areas of the cerebral cortex. The existence of divergent axonal projections of single CA neurons in the locus coeruleus to the cerebellum and the spinal cord, to the frontal cortex and the cerebellum, is also demonstrated.

The mesencephalic nucleus of the trigeminal nerve of the male adult rat was studied under electron microscopes and the following results were obtained.1. The neuronal cytoplasm is characterized by a network of Nissl substance composed of rosette-like polysomes and free ribosomes rather than granular reticula. Numerous Golgi complexes are situated apart from the cell nucleus, accompanying several lysosomal dense bodies. Lipofuscin-like dense bodies also often appear at a distance from the Golgi complexes.2. Some neurons of the mesencephalic nucleus exhibit soma-somatic junctions with an intercellular space of about 110-300A. These junctions are not considered temporary or vestigial structures, because of their frequent occurrence and the linear array of mitochondria. It might be questioned whether this type of junction acts as an electrical synapse in the central nervous system of the rat.3. Excepting the soma-somatic junctions, the neuronal surface shows irregular projections (or crests) and depressions. In an extreme case, a dendrite is deeply infolded within the cleft of the cytoplasm formed by soma spines (crests), but the significance of this structure is unknown.4. The afferent terminals form axo-somatic synapses on the irregular surface of the perikarya. We classified these synaptic terminals into S-type and F-type (UCHIZONO, BODIAN) according to the forms of their synaptic vesicles and discussed their possible origins.

The fine structural changes during retrograde degeneration were studied in the trigeminal motor nucleus from 17hrs to 95 days following neurotomy of the 3rd division of the trigeminal nerve in the rat.The typical Nissl bodies composed of a parallel array of long granular endoplasmic reticulum presented derangement and fragmentation. Concomitantly an accumulation of polysomes became eminent in the perinuclear area adjacent to the nuclear indentation and the peripheral portion of the perikaryon. In the neuron with advanced degeneration, dilatation of cisterns of the granular endoplasmic reticulum and gradual change of polysomes to single free ribosomes were demonstrated. However, the motor neurons concerned gave few necrotic images, suggesting the recovery from the affection. Concernig the highly dark and shrunken neurons frequently observed in the affected side of the motor nucleus some possibilities were discussed.The lamellar cisterns of the Golgi apparatus were replaced by well-developed Golgi vesicles, coated vesicles and dense bodies. Neurofilaments and neurotubules moderately increased in number and irregularly dispersed without forming unique bundles. The mitochodria often showed indistinct cristae and a decrease in electron density of the matrix.In the neuropil some axon terminals supposed to be axon collaterals from the trigeminal mesencephalic neurons showed moderate abnormalities characterized by the high density of the axoplasm, glycogen accumulation and the occasional appearance of neurofilaments. Directly after neurotomy the microglial cells markedly proliferated as satellites, which migrated around the neurons separating synaptic endings from the synaptic area with finger-like processes. The necrotic neuron was completely enclosed and digested within a hypertrophied microglial cytoplasm. Astrocytal processes around the nerve cells and vascular walls accumulated glycogen particles shortly after operation. An astrocytal perikaryon showed hypertrophy with gliofilamentous proliferation but an increase in the number of astrocytes was obscure.

The trigeminal mesencephalic nucleus was studied with an electron microscope after neurotomy of the third division of the trigeminal nerve in rats. Neuronal ultrastructures were characterized by the changes of the cell organelles 17hrs after neurotomy. Especially numerous polysomes appeared in the perinuclear area and then migrated into the peripheral area. Concomitantly, the central area of the cytoplasm was filled with bundles of synthesized neurofilaments. Ten days after neurotomy, the Nissl pattern shifted to dispersing free ribosomes and granular endoplasmic reticulum. Necrotic neurons and degenerating processes of the affected neurons revealed dense matrix and often lamellated dense bodies. These neurons packed with fibrous or tubular substances showed loss of the Nissl substances after 40 and 95 days. In the neuropil, astrocyte end feet accumulated more glycogen particles than usual. The vascular walls showed remarkable changes after 17hrs. Pericytes were activated as phagocytes, which have dense cytoplasmic matrix and large masses of lysosomal dense bodies and phagocytotic vesicles. Slight changes were observed in the vascular walls and perikarya of the neurons of the contralateral mesencephalic nucleus as well.

Immunohistochemistry using antisera to glial fibrillary acidic protein (GFAP) was performed to observe astrocytal changes in the white matter of myelin-deficient Jimpy mice. In wild-type controls, astrocytes stained with the GFAP immunoreaction appeared in the cerebellar medulla by postnatal day 3 and in the corpus callosum by postnatal day 6. The immunoreactivity gradually intensified with age to reach a peak of the GFAP level around postnatal day 12. With the progress of myelinogenesis, GFAP positive astrocytes decreased in number and became sparse in the white matter. On the other hand, the white matter of Jimpy mutant mice revealed intensified GFAP immunoreactivity, corresponding to a numerical increase in astrocytes and an enlargement of their cytoplasm. This condition continued from postnatal day 12 up to the end of life. Such astrocytes made up a dense framework with thickened processes in the non-myelinated white matter, where oligodendrocytes were few. Under the electron microscope, an accumulation of gliofilaments and glycogen particles was apparent in the enriched astrocytal cytoplasm. Immunostaining was observed not only on gliofilament bundles but also rough endoplasmic reticulum and cytoplasmic matrix. The present results demonstrate that astrocytal hypertrophy and hyperplasia occur in the entire white matter of Jimpy mice.

Following an intravenous injection of a fluorescent dye, primuline, into neonatal rats, the distribution of ameboid cells and their fate in the central nervous system were examined under incident fluorescence, light and electron microscopes. In the ameboid cells labeled with primuline, the ingested fluorogen spread throughout the cytoplasm, in which it appeared as a whitish yellow fluorescence. Such labeled cells displayed increased acid phosphatase (ACPase) activity. Combining primuline labeling with the ACPase reaction allowed for the identification of ameboid cells even after the transformation mentioned below. Ameboid cells were distributed throughout the white matter in neonatal rats, but were few in the gray matter. Particularly, they appeared as cell clusters in the corona radiata of the corpus callosum, the subependymal layer surrounding the ventricles, the fornical commissure, the internal and external capsules, the cerebellar peduncles and the medulla. Around 8 to 14 days after birth, the ameboid cells which had so far appeared spherical became gradually elongated with a few branched cytoplasmic processes, and scattered in the white matter. Thereafter, most primuline labeled cells transformed into cells with a dark nucleus and a thin cytoplasm including small phagosomes, i.e., into cells with features of microglia. However, a small number of ameboid cells underwent degeneration. The data indicate that the ameboid cells observed in the white matter during the postnatal period transform into microglia. Since other investigations suggest that the ameboid cells are derived from blood monocytes, microglia could initially come from monocytes.

Various cells from the monocyte-macrophage line, a few neutrophils and some lymphocytes were found in the non-myelinated white matter of Jimpy mice, though there was no vascular injury to the brain parenchyma. Some of the lymphocytes appearing in the white matter were in close contact with macrophages, and occasionally with young oligodendrocytes. Such lymphocytes were mainly of the small type with few cell organelles in a scanty cytoplasm, though a few were of the medium type with a moderate amount of cell organelles in an enriched cytoplasm. Macrophages in the non-myelinated white matter displayed a highly heterogeneous cytoplasm. In particular, "cytoplasmic compartments" in macrophages displayed specific structures differing from the inclusion bodies generally seen in phagocytic cells. The contents of such "cytoplasmic compartments" were rather similar to parts of the macrophage's own cytoplasm, though they were isolated from the cytoplasm by a membrane and gaps. Our findings would seem to support the previous presumption that inherited myelin deficiency in Jimpy mice is induced by a T-cell mediated autoimmune response.

Following unilateral section of the trigeminal third division, the ipsilateral mesencephalic neurons showed severe shrinkage with central chromatolysis and nuclear eccentricity while the motor neurons showed mild swelling. Histochemical reactions in the mesencephalic neurons were mostly characterized by the decreased activity of phosphorylase after 16hrs and concomitant diminution of PAS-positive substances. The increased activities of G6PDH and ACP were recognized in almost all neurons of both nuclei and in adjacent neuroglial cells. The neuropil in the motor nucleus showed remarkable decrease of SDH, ALD and LDH activities after 10 days. The results obtained were discussed in relation to chromatolysis, protein synthesis and carbohydrate metabolism in the affected neurons and surrounding neuropil.

The locus coeruleus of the rat was studied with the Golgi method and electron microscope in an attempt to demonstrate morphological substrates for the functional significance of these monoamine neurons.The locus coeruleus is mainly composed of densely packed medium-sized, spindle-shaped nerve cells with frequent soma spines, while in the rostral part of the nucleus the medium-sized cells are less densely located and mingled with small oval cells. Most of the axons originating from the medium-sized nerve cells take a rostro-ventral course usually giving off an initial collateral. It is interesting that some of the axons demonstrate a curious recurrent way or run caudalwards to the medulla oblongata after a short rostro-ventral course. Afferent fibers are composed of thick and fine fibers, which make complicated neuropil around the nurons. The thick afferents enter the nucleus from a ventral to dorsal direction and take an undulating tortuous course among the neurons, revealing axonal swelling and giving off many terminal boutons.The cytoplasm of the medium-sized nerve cells demonstrates marked aggregates of rough surfaced ER and prominent development of Golgi complexes, and contains a large number of lysosomal dense bodies. Large granular vesicles as seen in the terminal bags are only small in number in the cytoplasm. Cytoplasmic protrusions are characteristic for the medium-sized nerve cells, being moderate in number, varying in shape and length (0.4-1.7μ) and constituting mostly the postsynaptic sites. Axo-somatic synapses are frequent which make contact with axon terminals similar to axo-soma spine synapses. The nuropil contains different kinds of axo-dendritic and axo-dendritic spine synapses. Occasionally one can observe thickened preterminal axons or axon terminals contacting several dendrites or dendritic spines. Astroglial cells fill up the interneuronal spaces and demonstrate large oval mitochondria with homogeneous content and occasional lipofuscin granules.