Many intracellular calcium responsive proteins contain a common structural feature, “EF-hand motif” within their sequence. Among them, parvalbumin, calbindin and calretinin are localized in mostly separate subsets of neurons in the brain although they are co-expressed in some neurons. They are considered to participate in the regulation of the concentration of intracellular calcium ion. On the other hand, S100 β is primarily expressed in glial cells in the brain. This review introduces the molecular feature of these proteins and recent works on their distribution in the adult and developing brain with respect to the anatomical and functional view as well as questions to be clarified in the future.

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Migration inhibitory factor-related proteins-8 (MRP8) and MRP14 comprise a heterodimeric molecule. This complex molecule consisting of 8kDa and 14kDa subunits (MRP8/14 complex) contains two chracteristic Ca²⁺-binding regions named EF hands in each subunit. The MRP8/14 complex is expressed mainly in peripheral blood neutrophils and monocytes but not in resting tissue macrophages. It is also induced in human cultured leukemia cells including HL-60 promyelocytic leukemia cells by treatment with differentiation-inducing agents. The MRP8/14 complex also appears in human sera. Its serum level is high in patients with cystic fibrosis, rheumatoid arthritis, sarcoidosis, and other chronic diseases. To date, several groups of investigators have demonstrated this complex's modulation of cellular kinase reactions, its configurational changes in a Ca²⁺-dependent manner, and its transition from the cytosol to the membrane during neutrophil activation. Such observations may be an important clue to understanding the functional role of MRP8 and MRP14.

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Neurotransmitter release in the synapse is accomplished by the vesicular exocytosis, the membrane fusion between the synaptic vesicles and the presynaptic plasma membrane. At least three molecular systems are involved in the membrane fusion in the synapse: the Rab3A-RabGDI-Rabphilin-3A system, the NSF-SNAP-SNARE system, and the fusion pore formation system. In the first system, Rab3A, a small GTP-binding protein, functions in two interconvertible forms like a molecular switch: the GDP-bound inactive “off” form which is recognized by Rab GDP dissociation inhibitor (GDI) and the GTP-bound active “on” form which is recognized by Rabphilin-3A. RabGDI specifically binds to the GDP-bound “off” form of Rab3A and translocates it from the membrane to the cytoplasm. Rabphilin-3A localized on the membrane specifically binds to the GTP-bound “on” form. Whether Rab3A is in the form of the GTP-bound “on” or the GDP-bound “off” clearly determines its own localization on the membrane or in the cytoplasm. By immunoelectron microscopy, Rab3A is indeed localized at the synaptic vesicles, the presynaptic plasma membrane, and the cytoplasm near the active zones, where the membrane fusion takes place. Taken together, Rab3A-RabGDI-Rabphilin-3A system probably regulates the targeting and docking of the synaptic vesicles at the active zone. The NSF-SNAP-SNARE system further forms a vesicle fusion apparatus. Finally, the fusion pore formation system directly forms the channel between the synaptic vesicle and the presynaptic plasma membrane, through which neurotransmitter is released. In the nerve growth cone, a membrane fusion machinery similar to that in the synapse may be involved in the plasmalemmal expansion of the growth cone. For further histochemical approaches toward the molecular architecture of the membrane fusion machinery, the stoichiometric detection of the proteins by the immunoelectron microscopy with a modified pre-embedding 1-nm gold particle (nanogold)-silver enhancement method is very useful.

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To analyze dynamically the development of small follicles in the immature rat, a cohort of follicles was set up by labelling a certain group of oocyte's nuclei in the embryonal stage. The pregnant rats were transabdominally injected with bromodeoxyuridine (BrdU) in the 17th day of pregnancy. The litters were sacrificed at 1, 2, 3 and 4 weeks after birth to localize BrdU in the ovaries and the livers by immunohistochemistry with anti-BrdU antibody. The BrdU staining was evident mainly in the nuclei of oocytes in primordial follicles through 1 to 4 weeks, while live cells were not stained after the 2nd week, indicating that the fetal oocyte escaped the dilution of BrdU uptaken due to arrest of its mitosis. The primordial follicles occupied 100% of the BrdU-labelled follicles at the 1st week, 98.8% the 2nd week, 90.9% the 3rd week, and 88.5% the 4th week. However, the majority of antral follicles were unlabeled though only a few vesicular follicles showed BrdU incorporation even at the 4th week. This result suggests about 10% in a given cohort of primordial follicles were developed into the next growing follicles during this period.

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To elucidate the role of proliferating cell nuclear antigen (PCNA) in vivo, PCNA immunohistochemistry combined with ³H-thymidine autoradiography was performed on 2 types of regenerating rat livers. One group of rats underwent 2/3 partial hepatectomy (PH), and the other group 1/3 PH. After ³H-thymidine labeling, the livers were fixed with paraformaldehyde (PFA) and methanol, and paraffin-embedded. In methanol-fixed tissues, the serial changes of the PCNA-positive index (PCNA-PI) corresponded well with those of the ³H-thymidine-labeling index (³H-thymidine-LI) after each PH. In PFA-fixed tissues, however, the PCNA-PI was 26% before PH, when the ³H-thymidine-LI was below 1%. Furthermore, the 1/3 PH group, despite a continued low ³H-thymidine-LI, showed a rise in PCNA-PI to a level comparable to that in the 2/3 PH group. From these results, we propose 3 phases of PCNA expression. In PFA-fixed tissues, PCNA that was detectable before PH was considered pooled in G₀-phase nucleoplasm (Phase I), and some or many of the PCNA-positive cells failed to enter the S-phase in the 1/3 PH group, reflecting the increased PCNA not bound to DNA replicons (Phase II). In methanol-fixed tissues, PCNA was detected only in the S-phase, indicating its direct involvement in DNA synthesis (Phase III). Therefore, determining PCNA-PI in methanol-fixed tissues is useful to evaluate proliferative activity, whereas that in PFA-fixed tissues must be assessed with great caution.

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Tenascin (TN) is transiently expressed in some organs and tumors at various developmental stages. It has also been reported that tenascin was a major extracellular matrix component in a developing nervous system, and played an important role in neurite outgrowth and pattern formation of the cerebral and cerebellar cortices.
In the present study, we examined, under light and electron microscopes, the localization of TN in the cervical region of rat spinal cord during the late prenatal and early postnatal stages by means of conventional and immunohistochemical methods. In the specimens stained with hematoxylin-eosin and thionin, the following results were obtained: the structure of spinal cord was developing at the postnatal stages and attained a definite form around the 5th day after birth. Nissl bodies in neurons were maturing during this period and the processes of neurons elongated toward white matter. Concomitantly, neuroglia were also increased in the white matter.
In accordance with it, luxol fast blue (LFB) and sudan black B stainable substances appeared in the white matter, especially ventral and lateral funiculi around the 5th day of age.
The results of immuno-light microscopical observation showed that TN-labeling was observed only in the gray matter of the spinal cord at the 18th day of gestation, but both in gray and white matters from the 20th day of gestation to the 5th day after birth. The most intense TN-labeling was detected in the boundary of gray and white matter in the ventral and lateral funicili at the 2nd to 5th day after birth. As the development proceeded, up to the 2nd week after birth, TN-labeling was mainly distributed in the white matter except for the ventral region of dorsal funiculus and almost completely disappeared from the gray matter. Here, it was also noticed that the ventral region of dorsal funiculus became stainable with LFB or sudan black B between the 2nd and 3rd week after birth and at the same stage of development, TN-labeling became detectable in this region.
At the electron microscopical level, TN-labeling was detected in the intercellular spaces of the gray matter during the 1st to 10th day of age. That is, TN was expressed associating with the cytoplasmic membrane and processes of neurons and neuroglia. TN-labeling was also observed along the cytoplasmic membrane of myelinating oligodendrocytes and in the cytoplasm of neuroglia, especially oligodendrocytes in the white matter at the 10th day to the 2nd week after birth. Clear reaction products for TN could not be detected in the synaptic region.
From this evidence, it is concluded that TN mediates the interaction of neuron and neuroglia during late prenatal and early postanatal stages and plays an important role in elongation of neuronal processes and migration of neuroglia as well as the myelination in the developing rat spinal cord.

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Cyanobacteria are photosynthetic prokaryotes. They have thylakoidal membrane that carry out oxygenic photosynthesis. They grow autotrophically in a general way. Nostoc muscorum M-14, a species of cyanobacteria, show exceptionally heterotrophic growth. DAB-histochemistry proved occurrence of cytochrome oxidase activity in thylakoidal membrane. Activity of cytochrome oxidase was quantitatively estimated by stereological morphometry based on modified Weibel's method. Activity of cytochrome oxidase increased by 3.5 times in heterotrophic cells compared with autotrophic cells. The enhancement was coincident with the increment of oxygen consumption measured with oxygen electrode. Quantitative DAB-histochemistry elucidated that heterotrophic condition modulates respiratory activity in the thylakoidal membrane of cyanobacteria. Presence of respiratory activity in photosynthetic membrane of cyanobacteria suggests evolutionary ancient nature of the membrane.

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The suprachiasmatic nucleus (SCN) obtained from the newborn rat was successfully cultured in vitro for more than 4 weeks by the roller tube method. The slices which were 400μm thick and 1×1mm wide at the time of extraction, became thinner and wider week by week, and at the end of 4 weeks, the slices had spread (2×2mm wide) and flattened out to a monolayer or two-cell layers. The culture was composed of three parts: the ependymal zone consisting of piled up ependymal cells in the central part, the neuronal zone consisting of two tightly-packed neuronal cell masses located adjacent to the ependymal zone, and the surrounding glial cell-dispersed zone mainly composed of astrocytes expressing glial fibrillary acidic protein (GFAP). GFAP-immunoreactive astrocyte processes were also found in the neuronal zone, and the distributional density was higher in the peripheral (ventral and lateral) part than the medial part. Cells in the neuronal zone at 4 weeks in vitro increased in size to about twice that at the day of excsion. An electron microscopic study revealed that neurons having little cytoplasm with well developed rough endoplasmic reticulum formed many axodendritic and asymmetrical synapses in the neuronal zone. The present findings suggest that survived SCN neurons extended processes to form a complex intranuclear neuronal network in the neuronal zone in vitro similar to that in vivo. The SCN slice culture system may be useful for analyzing the neuronal network generating circadian rhythms in vitro.

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The expression of peptidergic traits was analyzed in the slice culture of the suprachiasmatic nucleus (SCN) by the roller tube technique. Since the slice thickness was one to three cell layers after three weeks in vitro, the in situ hybridization and immunocytochemistry could be applied directly to the slices without further cutting. We found the expression of peptides and their messenger RNAs in neurons of the neuronal zone occurring in a topographic distinct pattern; vasopressin (AVP) containing neurons in the dorsomedial part near the ependymal zone and vasoactive intestinal peptide (VIP) containing neurons in the ventrolateral part of the neuronal zone. VIP-neurons aligned on the border of the neuronal zone and dispersed cell zone, projected fibers in the fixed directions; the thick process to the center of the neuronal zone, and the thin process to the opposite direction. For somatostatin (SOM) expressing neurons, two types of SOM mRNA containing cells were detected; the larger cells expressing a stronger signal located in and near the ependymal layer, and smaller cells expressing a weaker signal in the dorsomedial part of the neuronal zone. The larger SOM neurons in vitro correspond to the periventricular SOM neurons in vivo, the smaller SOM neurons in vitro correspond to the SOM neurons in the SCN in vivo. Rhythmic AVP release occuring in vivo were also recorded in the medium of the slice culture system by enzyme immunoassay. The similarity of the distribution pattern of VIP, AVP and SOM neurons in vitro and in vivo, and persistent rhythmic release of AVP in vitro, suggest that the peptidergic neurons develop with the original neuronal network being preserved and secrete peptides as they do in vivo.

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