Takao Ichimura

Fine-structural study of the pineal body of the monkey (Macaca fuscata) with special reference to synaptic formations.

SummaryVarious types of synaptic formations on pinealocytes and pineal neurons were found in the pineal body of Macaca fuscata. Axo-somatic synapses of the Gray type-II category were detected on the pinealocyte cell body. Gap junctions and ribbon synapses were observed between adjacent pinealocytes. About 70 nerve-cell bodies were detected in one half of the whole pineal body bisected midsagittally. They were localized exclusively deep in the central part. When examined electron-microscopically, they were found to receive ribbon-synapse-like contacts from pinealocytic processes. They also received synaptic contacts of the Gray type-I category on their dendrites, and those of the Gray type-II category on their cell bodies from nerve terminals of unknown origin. All these synapse-forming axon terminals contained small clear vesicles. Thus, the pineal neurons of the monkey, at least in part, are suggested to be derived from the pineal ganglion cells in the lower vertebrates and not from the postganglionic parasympathetic neurons. The functional significance of these observations is discussed in relation to the innervation of the pineal body of the monkey.

Structural components in the synaptic cleft captured by freeze-substitution and deep etching of directly frozen cerebellar cortex

SummaryStructural components in the synaptic cleft were examined in cerebellar excitatory synapses by conventional electron microscopy and by rapid freezing followed by freeze-substitution or deep etching. Two transverse components and one parallel element were identified in the clefts of rapidly frozen and freeze-substituted synapses: (i) bridging fibrils, 4–6 nm in diameter, that span the cleft; (ii) columnar pegs, 4–6 nm wide and 8–15 nm high, projecting from the postsynaptic surface; and (iii) intervening fine fibrils running parallel to the apposed synaptic membranes. These were more clearly visible in deep-etched synapses, although the postsynaptic pegs were difficult to distinguish from intramembrane particles in the cross-fractured postsynaptic membranes. Deep etching also revealed other fibrils on the cytoplasmic surface of the postsynaptic membrane. These appear to contact the membrane surface or the intramembrane particles. Freeze-substituted materials also displayed the fibrillar components in the postsynaptic dense fuzz, but failed to display the presynaptic dense projections typically observed in thin sections or deep-etched replicas of the conventionally fixed materials. The bridging fibrils are likely to play a mechanical role in holding the synapse together, while the short pegs may be integral parts of the receptor molecules.

Apical tubular network in the rat kidney proximal tubule cells studied by thick-section and scanning electron microscopy.

Abstract.The apical cytoplasm of several absorbing epithelia contains well-developed apical tubules (AT) which contribute to membrane recycling from endocytic vacuoles to the apical cell membrane. In this study, we examined three-dimensional structures of the AT in rat kidney proximal tubule cells by transmission and scanning electron microscopy. In thin sections, the AT appeared as straight tubules with a rather constant diameter (70–90 nm), but others were curved and, occasionally, branching. No AT were labeled with the marker for the external cell surface (ruthenium red) or exhibited histochemical enzyme activity for lysosomal hydrolase (acid phosphatase). After intravenous injection of horseradish peroxidase, it was absorbed in the kidney proximal tubule cells and the AT were labeled with HRP reaction products. Stereo-viewing of the labeled AT in thick sections revealed that they formed an interconnected tubular network. Scanning electron microscopy allowed a three-dimensional view of the AT, in which a network of branching and anastomosing tubules was revealed. These observations indicate that the AT are intracellular endosomal compartments which form an extensive tubular network in the apical cytoplasm. The possibility that this apical tubular network serves as a large membrane store for membrane recycling is discussed.

Three-dimensional fine structure of elastic fibers in the perivascular space of some circumventricular organs as revealed by high-voltage electron microscopy.

Detailed fine structure of elastic fibers and their distribution in the brains of rats, guinea pigs, and cats were examined by conventional and high-voltage (HVEM) electron microscopes. Small elastic fibers were observed in the perivascular space of fenestrated capillaries in circumventricular organs such as the area postrema, the subfornical organ, and the pineal body. The amorphous component of elastic fibers was identified as elastin by its affinity for orcein and specific susceptibility to elastase. Stereoscopic observation by HVEM revealed the right-turned double-stranded helical structure of microfibrils of elastic fibers. Some physiological roles of these elastic fibers are discussed as related to a possible cerebrospinal fluid absorptive system in the central nervous system.

Fine structure of basement membranes of the capillary endothelium and perivascular astrocyte in some circumventricular organs by three-dimensional SEM.

The three components of the basement membrane, the lamina lucida, lamina basalis, and lamina reticularis, were examined stereoscopically with a high-resolution scanning electron microscope. Brains of 12 rats and 3 guinea pigs were used. In the lamina reticularis, microfibrils connect to collagen fibrils with one end and anchor to the external surface of the lamina basalis with the other. Their helical substructure, the pitch of helices 70-80 nm and maximum width 15-20 nm on scanning electron micrographs, was reconfirmed. In the lamina lucida, short cross-bridge filaments spanned the basal lamina and the applied plasma membrane. They measured 10-15 nm in diameter, 30-90 nm in length, and were distributed with a regular spacing fo 40-60 nm. It is discussed that the basement membrane serves as the anchorage of the microfibrils and of the cross-bridge filaments and mediates the interconnection between the plasma membrane and extracellular connective tissue elements.

Three-dimensional architecture of the tubular endocytic apparatus and paramembranous networks of the endoplasmic reticulum in the rat visceral yolk-sac endoderm

The three-dimensional architecture of the tubular endocytic apparatus and the endoplasmic reticulum in the rat yolk-sac endoderm was investigated after loading with horseradish peroxidase-conjugated concanavalin A by intrauterine administration. After 30 min, small vesicles (50–150 nm in diameter), small tubules (80–100 nm in diameter) and large vacuoles (0.2–1.0 μm in diameter) in the apical cytoplasm were labeled with the tracer, but lysosomes (1.0–3.5 μm in diameter) in the supranuclear cytoplasm were not labeled until 60 min after loading. Stereo-viewing of the labeled small tubules in thick sections revealed that they were not isolated structures but formed three-dimensional anastomosing networks, which were also confirmed by scanning electron microscopy after maceration with diluted osmium tetroxide. Their earlier labeling with the endocytic tracer, localization in the apical cytoplasm and three-dimensional network formation indicated that the labeled small tubules represented tubular endosomes (tubular endocytic apparatus). These well-developed membranous networks provided by the tubular endosomes are suggested to facilitate the receptor-mediated endocytosis and transcytosis of the maternal immunoglobulin in the rat yolk-sac endoderm. Scanning electron microscopy further revealed lace-like networks of the smooth endoplasmic reticulum near the lateral plasma membrane. Their possible involvement in transport of small molecules or electrolytes is discussed.

Occurrence of unusual tubular invaginations of the plasma membrane in smooth muscle cells of the lamprey, Lampetra japonica

Numerous tubular structures were observed in the surface region of smooth muscle cells making up the vascular walls in the lamprey, Lampetra japonica; they were designated as surface tubules. The limiting membrane of the surface tubules was connected to the plasma membrane, allowing communication of the lumen of the tubule with the extracellular space. Tannic acid reacted with osmium, serving as an extracellular marker, penetrated into the tubules but not into the intracellular organelles, such as the endoplasmic reticulum and the Golgi complex. The surface tubules were grouped in longitudinal parallel rows, separated from each other by tubule-free areas where dense plaques were present. Each tubule was fairly cylindrical (approximately 60 nm in diameter) and often ramified into two or three branches with a blind end. Occasionally, these tubules were encircled by the sarcoplasmic reticulum which was located immediately beneath the plasma membrane. Similar tubules were also observed in the surface region of vascular endothelial cells and fibroblasts in the adventitial connective tissue. The possibility that the surface tubules in the present observations are analogous to the smooth muscle caveolae or the striated muscle T-tubule is discussed.

IgG transcytosis is accelerated but its pathway is not disrupted by Brefeldin-A

To see the action of Brefeldin-A (Bref-A) to transcytosis of the immunoglobulin G (IgG), we have examined the early stage of endocytosis and intracellular transport of IgG and transferrin (Tf) in epithelial cells of the rat yolk sac in culture with and without Bref-A. In the absence of Bref-A, the endocytosed IgG and Tf appeared in large apical endosomes during the first 5-15 min. At 15 min Tf-recycling endosomes and IgG-transferring vesicles also appeared in the apical or basolateral cytoplasm, respectively. At 15 min in the presence of Bref-A, clouds of Tf-recycling endosomes accumulated in the apical cytoplasm, while distinct IgG-transferring vesicles were distributed further to the basolateral cytoplasm. This differential action of Bref-A to IgG-transferring vesicles and Tf-recycling vesicles suggested an unexplored mechanism of sorting and basolateral transport of IgG.