Immunohistochemical methods identified serotonin, and the peptides bombesin and caerulein, in the skin of the teleosts Lepadogaster candollei and Mastacembelus erytrotaenia. In both species, the secretory content of epidermal sacciform cells reacted positively for all three substances. These results are compared with studies on the skin glands of amphibians, which also contain multiple active compounds, and on various neuroendocrine cells of fish. The precise functions of the secretions are not known.

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Scanning electron microscopy of vascular casts of the pancreas from monkies, cattle, pigs, dogs, cats, rabbits, guinea pigs and mice showed certain species differences in the occurrence of intralobular and interlobular islets and in the microcirculatory pattern of these islets.
Interlobularly located islets were frequently found in the mouse and guinea pig, as has been previously established in the rat (MURAKAMI and FUJITA, 1992); they emitted insulo-venous efferent vessels directly draining into veins. In contrast, the intralobular islets in the guinea pig usually issued insulo-acinar portal vessels continuous with the lobular capillary network. In the mouse, they usually emitted both the insulo-acinar portal and insulo-venous efferent vessels. The insulo-venous efferent vessels, including those of the interlobular islets, could partly be portal in nature since they occasionally issued portal branches directed to the lobular capillary network. In rabbits, cats, dogs, pigs, cattle and monkies, as in men (MURAKAMI et al., 1992), essentially all islets in the pancreas were intralobular in location and usually emitted the portal vessels only.
In the mouse and rabbit, as in the rat (MURAKAMI and FUJITA, 1992), the islet received afferent vessels in its superficial aspect and issued efferent vessels from its deep aspect. In the Formosan monkey, as previously reported in the rhesus monkey (FUJITA and MURAKAMI, 1973), the afferent vessels ususlly ran deep into the islet which emitted vessels from its superficial aspect. In other animals examined in this study, as in humans (MURAKAMI et al., 1992), no consistent rule concerning the microcirculatory pattern within the islet could be determined.

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The structure and distribution of the initial lymphatics in whole mount preparations of the mesentery and intestinal walls of the Japanese monkey (Macaca fuscata) were studied using an enzyme-histochemical method (KATO et al., 1991, 1993). The lymphatic walls, colored dark brown by their positive 5′-nucleotidase (5′-Nase) activity, were clearly distinguished from the blood vessels (especially capillaries and arterioles) which were colored blue due to their positive alkaline phosphatase activity. The specificity and localization of both enzyme reactions were confirmed by comparative histochemical studies of the same specimen under a light microscope and scanning or transmission electron microscopes. Application of this staining method takes advantages of the overview preparation of flat membranous organs.
The mesenterial area was generally lobulated in distribution with collecting lymphatics and blood vessels. At about the center of each lobule enclosed by the vessels, 5′-Nase-positive initial lymphatics assumed tubulosacclar shapes, branching antler-like figure to form dense networks in the main lymph vascular pathway. Their apical parts revealed marked knob-like blind endings demarcated by a thin endothelial wall. No direct interconnection was recognizable between the lymphatic space and the tissue interstitium as a prelymphatic fluid pathway. In such preparations, 5′-Nase-positive lymphatic islands could be found isolated from the lymphatic network.

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Immunocytochemical study was performed in the Bufo marinus retina to reveal the localization of γ-aminobutyric acid (GABA)-immunoreactivity in neuropeptide Y (NPY)-, substance P (SP)-, serotonin (5HT)- and tyrosin hydroxylase (TH)-immunoreactive amacrine cells. GABA-immunoreactivity was present in all NPY-, in some of the SP-and 5HT-containing amacrine cells, but not in TH-immunoreactive amacrine cells. Among the 5HT-immunoreactive amacrine cells, a population of 5HT-synthesizing and most of the 5HT-accumulating cells were GABA-immunoreactive. These results indicate that neuropeptide- and GABA-immunoreactivity are colocalized in amacrine cells of the anuran retina. We propose a possible co-transmission for two classical neurotransmitters (GABA and 5HT) in some of the 5HT-containing amacrine cells.

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For over a century it has been held that the Grandry corpuscle and the Merkel corpuscle are unique sensory organs in aquatic and nonaquatic birds, respectively. In other words, the Grandry and the Merkel cells are two varieties of the same receptor cell and never coexist together. Contrary to what has been believed, we found unmistakable Merkel cells in addition to Grandry cells in the lingual mucosa of the duck, Anas platyrhynchos var. domestica, an unprecedented observation in an aquatic bird. This study concerns the fine structure of these cells in the tongue of the duck. Although some ultrastructural features are shared by the Grandry and the Merkel cells, i. e., the presence of numerous dense-cored granules and microvillous projections at the cell surface, the morphology of these two types of cells clearly differ, especially in their size. Cells suggestive of stages intermediate between these two cells have never been recognizable. The present study indicates that the Grandry and the Merkel cells are not two varieties of the same cell in birds. It is suggested that the Merkel cell may exist in every vertebrate species, while the Grandry cell is unique to aquatic birds.

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Rapidly frozen and freeze-substituted submandibular glands of young female rats were embedded in Epon and processed for histochemical demonstration of calcium with the glyoxal bis (2-hydroxyanil) (GBHA) staining method. GBHA staining of thick Epon sections revealed discrete calcium reactions of moderate intensity in practically every secretory granule but not in other compartments of the acinar cells. The saliva in the excretory duct was also reactive with GBHA and showed a drastic decrease in staining intensity toward the distal segments of excretory ducts with larger diameters. In addition, the duct saliva contained numerous tiny particles that were highly GBHA reactive. Stromal cells and cells lining the excretory duct were totally free of reactions. In the acinar cells, X-ray analysis detected distinct peaks for calcium in secretory granules and smaller ones in the Golgi apparatus, while they were undetectable in the rough surfaced endoplasmic reticulum (RER), implicating post-RER calcium loading in the secretory pathway. Electron-dense deposits in the duct saliva showed distinct peaks both for calcium and phosphorus, though these appeared in the acinar secretory granules and other cytoplasmic regions lacked phosphorus.
Our observations thus demonstrated physiologcal calcium in the intra- as well as extracellular compartments of the submandibular gland, and further confirmed drastic changes in chemical composition along the synthetic and secretory pathways of the saliva, by both histochemical and X-ray microanalytical methods. GBHA staining of calcium combined with X-ray microanalysis is useful for an evaluation of the physiology and histo-pathological changes of the salivary glands associated with initial phases of microliths as well as sialoliths formation.

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The distribution of nitric oxide synthase (NOS) was determined in the gastrointestinal tract of the rainbow trout, Salmo gairdneri. NOS immunoreactivity and the NADPH diaphorase histochemical reaction were co-localized in nerve cells in the myenteric ganglia. However, only about 60% of NADPH diaphorase-stained nerve cells in the vagus nerve trunks were immunoreactive for NOS. Reactive myenteric nerve cells were found throughout the gastrointestinal tract, comprising about 10-15% of all nerve cells. Reactive nerve cells and fibres appeared in the myenteric ganglia and nerve trunks. The circular muscle was innervated by reactive nerve fibres throughout the gastrointestinal tract. Some NOS-containing cell bodies were also in this layer. The submucous plexus contained reactive nerve fibres in each region of the gut; in the large intestine a few reactive nerve cell bodies were also seen in this plexus. The muscle in the mucosal folds of the large intestine was densely innervated. The observations suggest that nitric oxide is an enteric transmitter in teleost fish, as it is in mammals.

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The formation of intrafusal muscle fibers of developing muscle spindles in the tenuissimus muscle of the Chinese hamster was examined by electron microscopy from birth to 7 days postnatal.
At birth, at least two nuclear bag myotubes with a small aggregation of nuclei were already recognizable within a spindle capsule, establishing side-to-side contact with each other without the intercalation of a basal lamina. By the third postnatal day, most muscle spindles contained a full adult set of four intrafusal myotubes effected by the formation of nuclear chain myotubes due to the fusion of myoblasts. The intrafusal myotubes were usually enclosed in a common basal lamina and closely apposed without an interposing basal lamina, thus forming a muscle bundle. Sensory endings were thereby confined to the outer surface of the muscle bundle. After the third day, closely packed intrafusal myotubes generally separated into independent muscle fibers which were innervated by sensory endings and ensheathed in attenuated inner capsule cells. Pairings of fibers and sensory cross terminals between fibers were frequently found in the equatorial region. These phenomena suggest that the incomplete separation of the bundled myotubes at an early stage of postnatal development may persist into adulthood.

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The present study was performed to understand the regeneration of the intralobular duct and acinus of partially injured salivary glands. Sections of rat submandibular glands irradiated with YAG laser were investigated histologically, and the proliferative activity of regenerating tissue was also investigated immunohistochemically with anti 5-bromo-2′-deoxyuridine (BrdU) monoclonal antibody.
After YAG laser irradiation, duct-like structures continuous to the remaining ducts were regenerated at the periphery of the injured lobule. At the distal end of the duct-like structures, squamous cells were shown to form epithelial clusters. Additionally, immature acinar cells appeared budding off from the duct-like structures. During regeneration, BrdU-positive cells were observed in these duct-like structures, epithelial clusters, and immature acini.
This study showed that duct-like structures, epithelial clusters and immature acini appear during the regeneration process of partially injured lobules, and that each structure proliferated in order to contribute to the rapid regeneration of the salivary gland.

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An antiserum against basic fibroblast growth factor (bFGF), characterized by immunoblot, was firstly used to immunolocalize bFGF-like materials in dorsal root ganglion (DRG) neurons, spinal motoneurons and their processes innervating the gastrocnemius muscle, and then secondly to determine the direction of bFGF transport in the processes by nerve ligation or nerve section experiments. Light and electron-microscopic immunohistochemistry revealed that significant numbers of DRG neurons (mainly of the large type), spinal motoneurons and their processes supplying the muscle are immunoreactive for bFGF, and that the bFGF neurons have diffuse immunoreaction deposits over the entire cytoplasmic areas except for the Golgi complex, mitochondria and microfilaments. Nerve terminals and glial processes with bFGF were rarely seen in contact with or in close apposition to the immunopositive neurons. At sixteen hours, 3 days and 7 days after unilateral ligation or transection of the sciatic nerve, bFGF fibers accumulated in a time-dependent way in the proximal but not in the distal stump; nerve transection caused a more intense accumulation of bFGF-immunoreactive fibers than did nerve ligation. These findings suggest that bFGF in large DRG neurons and motoneurons is transported anterogradely into the peripheral tissue, possibly without being incorporated into secretory granules or synaptic vesicles. They raise the possibility that bFGF within the transected nerve fibers is more actively conveyed during regeneration than in the ligated nerve fibers.

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Acetylcholinesterase (AChE) activities in the prenatal rat lower limb were investigated by both cytochemistry and immunocytochemistry.
Results indicate that the epidermal cells show immunoreactions of AChE at a limited stage at prenatal day 15, and mesenchymal cells which are occasionally in contact with the basal lamina or with the adjacent myotubes begin to show AChE activities at prenatal day 17. Such AChE-positive mesenchymal cells, involved in the formation of the muscular tissues, have almost disappeared in the subepidermis by prenatal day 19.
This suggests that AChE independent of the neuromuscular system may be involved in the mesenchymal cell differentiation especially in the inductive process during myogenesis.

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To clarify whether ATP synthase and side chain cleavage enzymes (SCC) can coexist in a mitochondrion of the adrenal cortical cell, and whether the functional heterogenity of mitochondria can occur in an adrenal cortical cell, the immunostainability of these two enzymes in mitochondria was compared by electron microscope using pre- and post-embedding methods.
In the pre-embedding method, the inner membrane of most mitochondria in the adrenal cortical cell was positively stained for SCC, 11β-hydroxylase, and ATP synthase, but among these immunopositive mitochondria we often observed negatively immunoreacted ones in the same adrenal cortical cell. In the positively stained mitochondria, immunoreaction products were evenly localized along the inner mitochondrial membrane. We therefore think that these differences in the immunostainability are caused by technical artifacts, namely the inadequent penetration of the antibody.
In the post-embedding immunocytochemistry, gold particles showing the presence of the enzymes were observed on all mitochondria. Two different antibodies, anti-ATP synthase antibody and anti-cytochrome P-450scc antibody, which were labelled with gold particles of varying diameter (5nm, 10nm each) could be observed on all the mitochondria of the bovine adrenal cortical cells. No heterogeneities as to the stainability for both ATP synthase and SCC were detected in mitochondria of the rat and bovine adrenal cortical cells.
These results indicate that in the rat and bovine adrenal cortical cells, both ATP synthase and SCC are evenly and simultaneously present on the inner membrane of all mitochondria. Hence, mitochondria in the adrenal cortical cell seem to be homogeneous in their steroid and ATP synthesizing abilities.

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