In the last decade, the molecular mechanisms of apoptosis, a major type of active cell death (type I cell death) have largely been clarified in mammalian cells. Particularly, the caspase family of proteinases has been shown to play crucial roles in the execution of apoptosis. Differing from apoptosis, type II cell death is known to be associated with autophagosomes/autolysosomes and appear in the developing nervous system (CLARKE, 1990). We have previously shown that delayed neuronal death occurring in the CA1 pyramidal layer of the gerbil hippocampus after brief forebrain ischemia is apoptotic in nature and autophagosomes/autolysosomes abundantly appear in the neurons before DNA fragmentation. To further understand the roles of autophagosomes/autolysosomes in active cell death, we examined the apoptosis of PC12 cells using morphological and biochemical techniques. PC12 cells are known to undergo apoptosis when cultured in the absence of serum. In such an environment, the mitochondrial pathway of apoptosis is activated; cytochrome c is released from mitochondria, and caspase-9/caspase-3 are activated. We have first examined morphological features of PC12 cells during the apoptotic process following serum deprivation, and found that autophagy is induced from the early stage of the process in the cells before typical nuclear changes. When autophagy is inhibited in the cells by 3-methyladenine, an autophagy inhibitor, they are largely protected from apoptosis. In relation to the induction of autophagy in PC12 cells following serum deprivation, immunoreactivity, protein amounts, and the proteolytic activity of lysosomal proteinases, particularly cathepsins B and D, are all greatly altered; those of cathepsin B drastically decrease in the cells from the early stage of serum-deprived cultures, whereas those of cathepsin D increase. Moreover, PC12 cells overex-pressing cathepsin D undergo apoptosis more rapidly in serum-deprived cultures than wild-type cells, whereas those overexpressing cathepsin B increase the viability. These lines of evidence suggest that autophagy is involved in PC12 cell death following serum deprivation, this type of cell death being regulated by lysosomal proteinases, cathepsins B and D, downstream autophagy.

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The formation of unique vacuoles in tenotomized rat soleus muscle fibers was examined by light and electron microscopy. After tenotomy at both proximal and distal tendons, virtually all muscle fibers underwent characteristic degenerative changes with a disorganization of myofibrils called the central core lesion, but eventually recovered. At 3 days after tenotomy, some muscle fibers showed small vacuoles in the sarcoplasm of the end segments, which were larger in diameter and paler in staining than those of the control fibers in light microscopy. At 5 days, more fibers formed larger vacuoles together with the extensive disorganization of myofibrils. Such vacuole formation was more conspicuous in the distal end than in the proximal end. At 1 week the myofibrillar disorganization was most extensive in the central areas, and vacuoles were considerably enlarged in some fibers to occupy most of the sarcoplasm near the fiber ends. Vacuoles decreased in number and size with time and could rarely be seen at 4 weeks postoperative. In thin-section electron microscopy, the early forms of vacuoles were often connected with the T-system tubules. The limiting membrane of such vacuoles possessed many caveolae, some of which appeared to be continuous with the T-system networks. The vacuole membrane was closely associated with the sarcoplasmic reticulum to form dyadic connections. In later stages, the vacuole membrane was lined in part with the basal lamina. From these findings, it can be concluded that the vacuoles are sarcolemmal in nature and derived from the T-system. The significances of the vacuole formation are discussed with special reference to the mechanism and fate of the vacuoles and their clinical implications.

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It has previously been known that the developing parotid glands in humans and rats contain mucous cells in their terminal clusters and acini, but these cells disappear within a short period of time. Using rat parotid glands, IKEDA and AIYAMA (1997, 1999) suggested that the mucous cells might change into serous cells in the early postnatal period, but it is uncertain whether mucous cells appear only in the developing parotid gland of a few species such as humans and rats, or whether the cell transformation actually occurs. To clarify these points, the present study investigated the developing mouse parotid glands.
  Light microscopy showed cells with secretory granules that stained extensively with PAS and alcian blue in the terminal clusters of a 1-day-old mouse parotid gland. Mucous cell numbers in the terminal clusters and the acini reached a peak on day 5 and decreased on day 7. By day 10, the mucous cells had disappeared altogether. Thus, the presence of mucous cells in the developing mouse parotid gland was confirmed.
  Electron microscopy showed granules of low electron density and bipartite granules in the mucous cells. Bipartite granules and highly electron-dense granules sometimes co-existed in a single cell. Immuno-electron microscopy revealed a positive reaction for amylase to the low-electron-density granules and the low-electron-density portions of the bipartite granules, in addition to the highly electron-dense granules and the electron-dense cores of the bipartite granules. No mucous cells with nuclei displaying characteristics of apoptosis were recognizable.
  Lectin histochemistry both at the light and electron microscopic levels showed that the secretory granules in the mouse parotid gland mucous cells had sugar residues similar to those of the mature serous granules.
  These findings demonstrate that mucous cells appear in the early postnatal mouse parotid gland, and that almost all of these cells may be converted into serous cells.

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The major pelvic ganglion in both the rat and guinea pig has been extensively studied because of its anatomical simplicity. To clarify the target specific neural pathway in the diffusely distributed pelvic ganglia of larger animals, the pelvic plexus of the female dog was investigated by retrograde tracing and immunohistochemistry. The whole mount staining of the pelvic plexus with acetylcholinesterase histochemistry revealed 70-100 ganglia of varying sizes. Neurons retrogradely labeled from the rectum were mainly found in ganglia located in the dorso-caudal part of the plexus. The majority of these were non-catecholaminergic, immunoreactive for either calbindin (Calb) or neuropeptide Y (NPY), and characteristically associated with baskets of enkephalin (ENK)-immunoreactive varicose fibers. Neurons projecting to the utero-vaginal walls were distributed in ganglia located in the ventro-caudal part of the plexus. These mainly consisted of two major neuron groups: catecholaminergic Calb-immunoreactive neurons, and non-catecholaminergic neurons containing nitric oxide synthase (NOS) and/or vasoactive intestinal peptide (VIP), which were preferentially associated with a network of ENK-immunoreactive varicose fibers. Neurons retrogradely labeled from the urinary bladder mainly occurred in ganglia located around the junction between the ureter and the bladder. These consisted of catecholaminergic Calb neurons and non-catecholaminergic neurons containing Calb or NOS. Only a few ENK-immunoreactive fibers were found within the clusters of catecholaminergic neurons. These results indicate that organ specific neurons are located in separate ganglia and have both a distinctive composition of neuron types as well as different innervation by preganglionic fibers.

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The locations of cell bodies of sympathetic neurons projecting to the stomach, the duodenum, the ileum, the colon, the spleen and the pancreas have been studied using retrograde tracing. Projections arose from both pre- and paravertebral ganglia. In the rat, the prevertebral ganglia are the paired coeliac ganglia lying caudo-lateral to the root of the coeliac artery, paired splanchnic ganglia in the abdominal segments of the greater splanchnic nerves, unpaired superior mesenteric and inter-renal ganglia and the inferior mesenteric ganglia. The projections from the prevertebral sympathetic ganglia to the different parts of the gut were organised somatotopically. The most rostral ganglia (splanchnic, coeliac, and superior mesenteric ganglia) contained neurons innervating all regions of the gastrointestinal tract, the pancreas and the spleen. The inter-renal and inferior mesenteric ganglia, located more caudally, contained neurons innervating the distal part of the gut (distal ileum and colon). The innervation of the spleen and the pancreas came from the closest ganglia (sympathetic chains, splanchnic and coeliac ganglia). This organotopic organisation was not found in the sympathetic chain ganglia; the innervation of all organs came predominantly from the lower part of the thoracic chains.
  A large proportion of the retrogradely labelled nerve cells in the splanchnic ganglia received nitric oxide synthase immunoreactive innervation probably from the spinal cord. In the other prevertebral ganglia, most of the neurons received nitric oxide synthase immunoreactive innervation and/or bombesin immunoreactive innervation. This leads to the conclusion that, in these ganglia, many neurons receive projections from the gastrointestinal tract in addition to the spinal cord.

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Amylin (AMY) is a 37 amino acid peptide of pancreatic origin that has been localized in peripheral and central nervous structures. Both peripheral and central injection of the peptide causes various effects, including anorectic behavior in rats. Prompted by previous reports showing that the anorectic effect of AMY is mediated by histamine release, we immunohistochemically investigated possible relationships between these two systems at the light microscopical level. Monkey (Macaca fuscata japonica) hypothalamus specimens were submitted to immunohistochemical double staining procedures using AMY and histidine decarboxylase (HDC) antisera. AMY-immunoreactive neurons were found widely distributed in several nuclei of the monkey hypothalamus including the supraoptic, paraventricular, perifornical, periventricular, ventro-medial, arcuate, and tuberomammillary nuclei. We detected AMY-immunoreactive nerve fibers throughout the hypothalamus, the median eminence and hypothalamus-neurohypophysial tract. Although AMY- and HDC-immunoreactive neuronal cell bodies occupied distinct hypothalamic zones, many HDC-immunoreactive cell bodies and dendrites, particularly those in the periventricular, arcuate, and rostral tuberomammillary regions, were surrounded by numerous AMY-immunoreactive nerve fiber varicosities. These findings demonstrate for the first time the presence of a discrete number of AMY-immunoreactive neurons in the monkey hypothalamus and add morphological support to the experimental data demonstrating that AMY probably exerts its influence on food intake via the histaminergic system.

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This study examined the occurrence and morphological features of serous-type cells in human sublingual gland, using immunocytochemistry for lysozyme. Lysozyme-positive cells usually formed demilunes and occasionally their own acini. They were also found among cells of an intercalated duct and in its immature acinus consisting of a small number of secretory cells. All these serous cells could be classified as seromucous cells because they simultaneously revealed reactivity for mucus, i.e., a periodic acid-Schiff (PAS) and a periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) reaction under the light- and electron-microscope, respectively. Immunogold labeling of lysozyme in the seromucous cells was distributed on variously sized secretory granules. These usually possessed a single electron-dense spherule in an electron-lucent matrix, while granules of a homogenous structure were also present. Lysozyme-positive cells filled with large, lucent secretory granules could hardly be morphologically distinguished from the lysozyme-negative mucous cells; they corresponded to “intermediate” cells designated under the light microscope. All “immature” secretory cells with only a few secretory granules were also lysozyme-positive seromucous cells. The present study demonstrated that the seromucous cells in the human sublingual glands conform closely with those in the human labial glands (MIYAZAKI et. al., 1998).

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Light microscopic observations of healthy adult rat brain sections stained with anionic iron colloid indicated that 5-10% of neurons in the hippocampal subiculum and all neurons in the medial cerebellar nucleus possessed an intensely positively charged perineuronal net. This net was demonstrated to react to oxine, and therefore suggested to consist of guanidino compounds. It was further shown that the intensely positively charged perineuronal net, in accordance with the intensely negatively charged perineuronal net of proteoglycans, was digested by chondroitinase ABC, hyaluronidase, and collagenase, but not by endo-alpha-N-acetylgalactosaminidase. This finding suggested that the former positively charged net might be linked to the latter negatively charged one.

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Kidney-derived aspartic protease-like protein (KAP), initially identified in the mouse kidney, is a novel aspartic protease exclusively expressed in the lung and spleen as well as the kidney. Its orthologues have been identified in the human and rat, and termed napsin. We performed in situ hybridization analysis to determine the cellular expression of napsin mRNA in the kidney, Iung, and lymphatic organs of adult mice and to demonstrate, for the first time, its expression patterns in ontogeny. In the adult mouse kidney, extremely intense signals for napsin mRNA were observed in the proximal straight and convoluted tubules, in agreement with a previous study. The first signals for napsin mRNA during nephrogenesis occurred selectively in mesonephric tubules at embryonic day 13, and in metanephric tubules from embryonic day 14. In the lung, a distribution restricted to type II alveolar cells or their precursors was found from embryonic day 15, at the onset of type II cell differentiation, to the adult stage. In the spleen, the mRNA was expressed in lymph nodules of the white pulp and the marginal zone - namely. B-lymphocyte-rich regions - from postnatal day 0 to adult. The lymph node and Peyer’s patch displayed similar expression patterns, but T cell-dependent areas in these organs and the thymus lacked such signals. These findings suggest that mouse napsin possesses crucial functional roles not only in the kidney but also in the lung and lymphatic tissues, even during fetal stages.

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The expression and localization of AQP6 were examined in rat kidneys. In the kidney compartments, the expression was more intense in the outer medulla than in the cortex or inner medulla, and was negative in the glomerulus. During development, the AQP6 mRNA expression in the kidney was not detected in the fetus, but was recognized at birth, increased gradually by 4 weeks of age, and was unchanged thereafter. In situ hybridization demonstrated significant signals for AQP6 mRNA along the outer and inner medullary collecting ducts. Since the localization of the AQP6 mRNA-expressing cells was comparable to that of immunoreactive H⁺ ATPase-bearing cells in the collecting duct, they were identified as intercalated cells. No AQP6 mRNA signals were recognizable in other cells in the kidneys, including glomerular cells. No glomerular expression of AQP6 mRNA was confirmed by RT-PCR using total RNA extracted from the glomeruli. Immunohistochemistry using an antibody raised against recombinant rat AQP6 protein could localize the immunoreactivity in a population of collecting duct cells. Serial section observations indicated that the AQP6-immunoreactive cells corresponded to H⁺ATPase bearing intercalated cells.

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The three-dimensional ultrastructure of principal cells in the rat renal collecting duct was studied by scanning electron microscopy (SEM) using the NaOH digestion technique and the aldehyde prefix-osmium-DMSO-osmium method, as well as by transmission electron microscopy (TEM). Special reference was given to the basal labyrinth of the cells and its numerous ruffles and infoldings of the basal plasma membrane. Observations showed that, as the collecting duct descends from the cortical collecting duct (CCD) to the terminal portion of the inner medullary collecting duct (t-IMCD), the pattern of the labyrinth gradually simplified and the ruffles grew thinner. In the CCD, the labyrinth was conspicuously complicated in structure, being formed of tightly arranged ruffles of uniform shape and thickness (70 nm). From the outer medullary collecting duct (OMCD) to the initial portion of inner medullary collecting duct (i-IMCD), the labyrinth became less complicated due to the mingling of wide flattened ruffles. Also, the basal infoldings were reduced in depth (from 700 nm in CCD to 500-600 nm in i-IMCD). In the t-IMCD, the labyrinth was rudimental, and instead presented small grooves (300 nm in depth) which corresponded to indentions of the basal plasma membrane. The regional simplification of the labyrinth was accompanied by morphological changes in mitochondria suggesting their functional decline: the electron density and number of cristae were reduced, these being changed in shape from plate-like to vesicular. These morphological data readily account for the potential for active transport by the collecting duct, which is highest in the CCD and is decreased towards the t-IMCD, and which may function merely as an excretory duct of urine from the papilla. The present study three-dimensionally demonstrates fine-structural heterogeneity in different segments of the collecting duct of the rat kidney.

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