The cellular organization of different regions of the crista epithelium from the frog posterior semicircular canal was studied by light, transmission and scanning microscopy. The sensory epithelium consists of hair cells surrounded by supporting cells and basal cells located close to the basement membrane. Three types of hair cells, namely club-like, cylindrical and pear-like cells differentially distributed along the crista could be recognized on the basis of their shape. Club-like cells are located only in the peripheral regions, cylindrical cells both in the central and in the peripheral regions, and pear-like cells appear segregated into the intermediate regions. Sensory cells of the central region are characterized by a ciliary apparatus consisting of stereocilia usually shorter—and in some cases less numerous—than those of cells of the other regions. The presence of large evaginations of the apical membrane of hair cells and of several vesicles of microexocytosis demonstrates that receptor cells have a considerable secretory activity. This secretory activity is also proven by the presence in the supranuclear region of hair cells of numerous Golgi complexes. Moreover, the presence of two kinds of Golgi complexes, one constituted by dilated cisternae containing a moderately electron-dense material and the other made up of flattened electron-transparent cisternae, suggests a diversified secretion of material by the hair cells. This heterogeneous material may provide substances important for cupula formation and the composition of the endolymph.

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By means of immunocytochemical methods, immunoreactivity for the brain isoform of nitric oxide synthase (NOS-I) was recognized in numerous Leydig cells of the human testis as well as in MA-10 tumor and TM3 non-tumor mouse Leydig cell lines. Within the Leydig cell cytoplasm, immunocytochemical results suggested the occurrence of factors known to activate NOS-I such as glutamate and aspartate, as well as molecules involved in the regulation of the NOS-I activity such as calmodulin and Ca²⁺/calmodulin-dependent protein kinase II. Leydig cells, Sertoli cells, some endothelial cells of the testis, MA-10- and TM3 mouse Leydig cell lines exhibited a relatively strong NADPH-diaphorase enzyme activity as well. Double sequential immunostainings provided evidence that NOS-like immunoreactivity of the testicular Leydig cells is colocalized with testosterone, calmodulin, aspartate, glutamate, and Ca²⁺/calmodulin-dependent protein kinase II. Sodium nitroprusside treatment did not result in increased cGMP formation by MA-10- or TM3 mouse Leydig cells, suggesting that NO produced by these cells acts primarily in a paracrine fashion.
The NO produced by NOS-I immunoreactive Leydig cells may act as a messenger: 1) between neighbouring NOS-I positive and/or negative Leydig cells as well as to mediate the action of numerous intracellular and extracellular neuroactive substances and growth factors; 2) between Leydig cells and the muscle cells or pericytes of blood vessels to regulate local blood flow and permeability; and 3) between Leydig cells and peritubular myofibroblasts to influence their contraction and the permeability of the lamina propria.

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The distribution of somatostatin immunoreactivity was determined throughout the hypothalamus of the sheep and comparisons were made with the known distribution of somatostatin immunoreactivity in the rat. Immunopositive perikarya were present in the sheep periventricular region from as far rostral as the supraoptic recess of the third ventricle to the posterior optic chiasm. In the basal hypothalamus, a thick shell of immunopositive neurons surrounded the ventromedial nucleus (VMH), and there were also neurons in the caudal arcuate nucleus. Somatostatin immunoreactive fibres were concentrated in the dorsal VMH and arcuate nucleus as well as in the median eminence. The distribution in sheep was similar to that in rats, butimmunoreactive neurons around sheep VMH were distinctive, a characteristic that might relate to differences in growth hormone physiology in this species.

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The distribution of immunoreactive glutamate was compared in the pineal complex (pineal and frontal organs) and retina of frogs (Rana esculenta, R. arvalis, R. ridibunda, R. catesbeiana, Bufo viridis, Bombinator igneus) by postembedding immuno-electron microscopy. Similar to retinal photoreceptors (rods and cones), bipolars and ganglion cells, the rod- and conelike photoreceptors and the neurons of the pineal and frontal organs exhibited glutamate immunoreactivity. Synaptic terminals of photoreceptor cells on secondary neurons of the pineal complex and retina were strongly immunoreactive. The pineal tract and the fibers of the frontal nerve also displayed glutamate immunoreactivity. There was no essential difference in the immunoreactivity of the retinal and pineal elements among the species studied.
The similar histology of the pineal complex and retina of the frog and the high correlation of their binding sites of antiglutamate immunosera allow us to assume that glutamate performs a similar role in the pineal complex as is already known for the retina. The high immunoreactivity of the presynaptic region of pinealocytic processes and axons of secondary neurons suggests the role of a neurotransmitter for this excitatory amino acid in the efferent pathways of the pineal complex.

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In the canine proximal colon, tissue near the submucosal surface of the circular muscle layer produces spontaneous mechanical contractions, synchronized with electrical slow waves. Comparative physiological examination of tissue strips from various regions of the submucosa and circular muscle coat revealed that the characteristic smooth muscle tissue of the innermost sublayer of the circular muscle is required for this rhythmical phenomenon. Histological examination showed that tissues containing special smooth muscle cells form an inner sublayer of the circular muscle coat. These innermost muscle cells were distinguishable from the bulk circular muscle cells by the following features: 1) flattened and shorter shapes of the cell and nucleus, 2) numerous caveolae on the cell surface, 3) abundant mitochondria, and 4) frequent gap-junction formations. Neither slow waves nor spontaneous mechanical rhythmicities were recorded from the submucosal connective tissue or from the bulk circular muscle tissue without the inner sublayer. The thicker smooth muscle cells found in the submucosal border specimens were identical in histological features to the bulk circular muscles which produced no slow waves and no spontaneous contractions. Cellular elements in the interstitium, such as fibroblasts, mast cells and macrophages, were found in all tissue strips that were physiologically examined. Nerve elements were found in all the specimens; however, there was a unique nerve network probably corresponding to the plexus entericus (submucosus) extremus described by STACH (1972). It was concluded, therefore, that the inner sublayer characterized by special smooth muscle cells with a delicate nerve plexus is essential for producing spontaneous activities of the circular muscle coat in the canine proximal colon.

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Fibroblast-like type B cells are known to produce hyaluronic acid (HA), but the process of its degradation remains unknown. In order to examine the possible route for the degradation of HA in normal human Synovium, histochemical and immunohistochemical techniques were applied to the synovial tissue, using biotinylated HA binding region (HABR) and antibodies against CD44 and cathepsin B. Reaction products for HA and CD44 were detected on the cell surface of all synovial lining cells, while half of these lining cells contained intracellular stainings of HA and CD44. Electron microscopically, the lining cells containing intracellularly stained HA and CD44 extended cytoplasmic processes (type A cells), while the other lining cells possessed a smooth cell surface (type B cells). By light microscopic double staining, the intracellular stainings of HA and CD44 appeared co-localized in the cells immunopositive for cystatin β, an endogenous cysteine proteinase inhibitor which has been shown to be localized in alveolar macrophages and osteoclasts. Moreover, these intracellular stainings of HA and CD44 were co-localized with immunodeposits for cathepsin B, a representative cysteine proteinase in lysosomes. In the extracellular staining of HA, dot-like reaction products appeared on fibrous structures with a periodicity of 41.7nm. These results suggest that Type A cells in the normal human synovium participate in the degradation of HA by its CD44 mediated intake. Furthermore, HA may be closely associated with fibrous structures, probably type III collagen molecules.

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We examined the effects of the developing cerebral cortex (CC) and septo-preoptic medial basal hypothalamus (S-MBH) on the development of LHRH neurons in vitro. The serum-free basal culture medium (BCM) was supplemented with CC or S-MBH extracts prepared from 18.5-day-old embryos or from 2-day-old newborns, and the olfactory placode (NAP) of 12-day-old embryos was cultured. The migration of LHRH neurons was found on Day 3 in the cultures supplemented with the embryonic S-MBH extract (Group 3), where the cell development proceeded showing a numerical increase of the cells and the elongation of neurites. In cultures supplemented with the newborn S-MBH extract (Group 5), the cell development was less intensive in comparison with that of Group 3, while in cultures which had no brain extracts (Group 1), the neurons failed to survive a long term culture. The effects of the CC were less than of S-MBH extracts. Analysis of the protein composition of the extracts by electrophoretic and immunoblotting examinations demonstrated a protein spot of 70-kD in the embryonic S-MBH extract. Because the protein spot was identified to be alphafetoprotein (AFP), we further examined the effects of AFP. When the anti-AFP immunoglobulin was added to the Group 3 culture, the stimulative effects of the embryonal extract were inhibited, and the addition of AFP to Group 1 cultures did not show stimulative effects. We conclude that the developing S-MBH, the migrating target of LHRH neurons, contains some essential factors for the development of LHRH neurons, but further analysis is needed to determine the chemical natures of these factors.

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We applied conventional and freeze-fracture electron microscopy to study intercellular contacts between the processes of fibroblasts in the myotendon of the rat exterior digitorium longus. The results showed well defined gap junctions between the cell processes, while other cell junctional structures such as tight junctions and desmosomes were not recognizable. The present study suggests that the gap junctions represent a structure to coordinate the activities of fibroblasts distributed in the myotendon of the muscle.

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Proliferating cell nuclear antigen (PCNA) in a useful marker for monitoring cell proliferation in most species. The immunostaining of PCNA on tissue cryosections, however, has been hampered by a loss of PCNA immunoreactivity during the staining process. The need for both identifying the actual phenotypes of proliferating cells and differentiating them from other tissue components prompted us to establish reliable techniques for PCNA immunostaining on cryosections and to apply these to double immunostainings with other markers. We tested various fixing conditions for rat tissue cryosections and the effect derived from unmasking with pepsin digestion to restore PCNA immunoreactivity after fixation. For single immunostainings, the unmasking was effective in most fixing conditions tested. Particularly, 4% paraformaldehyde/0.05% glutaraldehyde fixation followed by 0.001% pepsin digestion resulted in the strongest immunoreactivity for PCNA and the best morphology, and was the first choice for double immunostainings with relatively stable second antigens. Alternatively, periodate-lysine-paraformaldehyde fixation was also applicable to other second antigens which are labile to the former treatment. These techniques can serve in the collection of important information from frozen tissues regarding the relationship between PCNA and other markers of interest which are usually susceptible to routine formalin and/or paraffin embedding.

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The regional distribution of calcitonin generelated peptide (CGRP)-immunoreactive nerve fibers in the epithelium throughout the nasal cavity was determined using semiserial cryostat sections and whole mount preparations.
Both respiratory and olfactory epithelia displayed, in their basal portion, an extensive nerve plexus projecting beaded nerve fibers toward the luminal surface. However, the density of the intraepithelial CGRP nerve fibers conspicuously varied according to regions. They were abundant on the septal mucosa, the ventromedial side of the nasoturbinates and the dorsal surface of the maxilloturbinates, while less numerous on the lateral side of the naso- and maxilloturbinates and on the lateral nasal wall. Another difference in the regional distribution of nerves was recognized between the anterior and posterior portions of the respiratory area: the anterior portion received a denser supply of intraepithelial CGRP fibers than the posterior portion.
Characteristically, the transepithelial CGRP-immunoreactive nerve fibers were densest on the anteroventral aspect of the nasoturbinates and on the anterodorsal surface of the maxilloturbinates. Some of them appeared to penetrate through the epithelium to come into contact with the lumen of the nasal cavity.
These results suggest that the CGRP fibers in the epithelium display a region-specific distribution, apparently disposed more densely over the areas which are more directly exposed to inhaled air, possibly that containing irritants and toxicants.

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