The distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) was examined in the upper thoracic segment of the spinal cord in rat. Under the light microscope, NADPH-d positive cell bodies and fibers were readily detected in the following areas: 1) the dorsal horn; 2) the dorsolateral funiculus and lateral spinal neurons; 3) spinal autonomic region, consisting of the nucl. intermediolateralis pars funicularis, nucl. intermediolateralis pars principalis, nucl. intercalatus spinalis and nucl. intercalatus pars paraependymalis; and 4) in the white matter lateral to the nucl. intermediolateralis pars funicularis. In the nucl. intermediolateralis pars principalis, the positive dendrites, running in bundles, were directed medially in the gray matter towards the central canal as well as laterally in the white matter towards the pia mater. The medially-directed positive dendrites fomed a subependymal plexus around the central canal. A dense bundle of NADPH-d positive fibers were also observed running longitudinally.
Combined retrograde tracing with fluorogold and NADPH-d histochemistry study revealed that some of the NADPH-d positive neurons, due to their fluorescence labelling, were sympathetic preganglionic neurons that innervated the superior cervical ganglion. Under the electron microscope, the reaction products in the neurons of the nucl. intermediolateralis pars principalis were deposited in their nuclear envelope, rough endoplasmic reticulum, mitochondria and Golgi apparatus.
In the neuropil, three types of synaptic configurations were oberved: between NADPH-d negative axon terminals and NADPH-d positive dendrites, between NADPH-d positive axon terminals and NADPH-d negative dendrites, and between NADPH-d positive axon terminals and NADPH-d positive dendrites. These synaptic configurations suggest that the neurons are regulated by nitric oxide released from both pre- and post-synapticelements. The sources of the NADPH-d positive axon terminals associated with the neurons remain unclear although the possibility of their being derived from supraspinal origins has to be considered.
The ultrastructural demonstration of NADPH-d reaction product in the three major types of glial cells suggests that nitric oxide might be produced by these cells, but its functional significance awaits further investigation.

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The presence or absence of type-VI collagen in mechanoreceptors in the lips and palate of the mouse and gerbil was assessed histochemically. When fresh palatine mucosae and the lips of mice were incubated in 20mM ATP solution, numerous fibrous long spacing fibers with a periodicity of 100nm appeared in the interlamellar spaces of Meissner's corpuscles, simple lamellated corpuscles, and the peripheral connective tissue of Ruffini's nerve endings, irrespective of age. In addition, when the palatine mucosae of mice and gerbils were stained immunohistochemically with anti-type-VI collagen, an intensely positive reaction was constantly observed in Meissner's corpuscles. Furthemore, a less intense immunoreaction to anti-type-VI collagen was recognized in the inner core of simple lamellated corpuscles and Ruffini-like formations in the labial mucosae. From these results, we conclude that type-VI collagen is a major constituent of the interstitial connective tissue of cutaneous mechanoreceptors.

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The mucosal surface of the human lacrimal sac represents an area exposed to exogenous agents including potentially harmful microorganisms. The human lacrimal sac was examined histochemically to identify glycoproteins, and immunohistochemically to identify secretory IgA. Neutral and acid glycoconjugates were detected mainly in the cytoplasm of the surface cells of the columnar stratified epithelial lining. The same reactions were recognized in occasional clusters of secretory cells forming intraepithelial glands in the lining of the lacrimal sac. The presence of secretory IgA in the cytoplasm of the apical epithelial cells was demonstrated. The results indicate that the lacrimal sac mucosa possesses certain active defense mechanisms against ascending infections.

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The chemical coding and projections of neurons containing neuropeptide Y (NPY) have been investigated in the myenteric plexus of the guinea-pig small intestine. Chemical coding was determined by investigating the colocalization of NPY immunoreactivity with the immunoreactivities for bombesin (BN), 5-HT, nitric oxide synthase (NOS), and somatostatin. Projections were determined by studying the consequences of nerve lesions created by myectomy and myotomy operations. NPY immunoreactivity was localized in four classes of myenteric neuron, anally projecting interneurons, neurons that projected anally and to the circular muscle, neurons projecting to the longitudinal muscle and in a small population of secretomotor neurons that projected to the mucosa. The interneurons and muscle motor neurons both had Dogiel type I morphology, whereas the secretomotor neurons had fine branching processes. Of the NPY-immunoreactive Dogiel type I neurons, 98% were also immunoreactive for NOS; conversely, 82% of NOS-immunoreactive neurons were immunoreactive for NPY. BN was also colocalized with NPY and NOS; 30% of the NPY-immunoreactive neurons were BN/NOS/NPY-immunoreactive. No nerve cells had BN and NPY immunoreactivity without NOS immunoreactivity.
The presence of NPY immunoreactivity was investigated in three classes of descending interneurons that are distinguished by their reactivities for somatostatin, 5-HT and NOS. NPY immunoreactivity was never colocalized with 5-HT or somatostatin, but most NPY-immunoreactive descending interneurons whose terminals formed pericellular baskets were also reactive for BN and NOS. The average projection lengths of the NPY interneurons was 2-3mm, in the anal direction.
Evaluation of immunoreactivity for BN, NOS and NPY revealed three major populations of anally directed circular muscle motor neurons, with BN/NOS/NPY, BN/NOS and NOS/NPY immunoreactivities. Examination of simultaneous labeling after nerve lesions showed that NOS/NPY neurons had short anal projections, averaging about 2-3mm, and neurons with BN immunoreactivity were longer, having average projections of about 5-8mm.

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Amylin (or islet amyloid polypeptide) is a 37 amino acid peptide originally isolated from amyloid deposits in the pancreas of non-insulin dependent diabetic patients. It has already been immunohistochemically localised within the B and D cells of pancreatic islets and in endocrine cells of the rat and human stomach and duodenum. In this phylogenetic study, a polyclonal antiserum raised against the carboxy-terminal tridecapeptide amide of human amylin was used to demonstrate and examine the distribution of amylin-immunoreactivity in the stomach and duodenum of various vertebrate species. Except for fish, gastrointestinal tracts of all the species studied contained amylin-immunoreactive endocrine cells. They were located chiefly in the lower half portion of the distal gastric body and pyloric glands, and in the lining epithelium of the duodenal villi and crypts. Many cells were elongated, triangular or oval, and had a cytoplasmic process that extended from the cell base along the basement membrane. Others had a bipolar feature that gave them a so-called “open” appearance. Double and triple staining procedures on the same tissue section showed that almost all the amylin-immunoreactive cells present in the gastroduodenal region also co-stored serotonin and chromogranin A, and displayed argyrophilia in Grimelius impregnation. On the other hand, almost all the serotonin-immunoreactive cells of this region co-stored amylin, whereas those in more distal gut regions did not. This finding suggests that those amylin-containing cells correspond to a subtype of gastroduodenal serotonin cells.

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The aim of this study was to establish whether or not mononuclear cells which appear in both the vitelline vessels and embryonic coelom in mice prior to liver hemopoiesis are specialized scavengers. Before the initiation of liver hemopoiesis, the majority of the embryonic blood cells were primitive erythroblasts derived from yolk sac hemopoietic foci. In addition, the peripheral blood contained a few free phagocytes as early as 10 days of gestation. The phagocytes devoured various cell elements such as degenerating erythroblasts and cell fragments. Ultrastructurally, they had long filamentous cytoplasmic projections on their cell surface, clear subsurface vacuoles or vesicles, lipid droplets, a few lysosomal granules, large heterogeneous phagolysosomes and residual bodies. Mononuclear phagocytes with ultrastructural features similar to those of the intravascular phagocytes also could be observed in the intraembryonic peritoneal cavities at 10 days of gestation; they sometimes engulfed possible mesothelial cells undergoing degeneration. Based on fine structural criteria, these intravascular and coelomic phagocytes were considered to be specialized scavenger macrophages with the function of clearing the blood and tissue fluid of whatever has been ingested. In so doing, they serve as the most primitive discriminating filter set in embryonic circulation.

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Neurons of intracerebellar nuclei in the mouse brain were demonstrated to possess a marked surface coat, formed 3-4 weeks after birth, which was stainable with cationic iron colloid or aldehyde fuchsin. Neurons with a similar surface coat were noted as relay or local interneurons in rather restricted areas such as the occipital cortex, retrosplenial cortex, zona incerta, hippocampal subiculum and spinal posterior horn. Dark neurons with condensed cytoplasm were also shown to be covered with the surface coat.
The surface coat was stained doubly with cationic iron colloid and aldehyde fuchsin. Digestion with hyaluronidase eliminated the stainability of the surface coat to both agents. Combined digestion with chondroitinase ABC, heparitinase and keratanase eliminated the cationic iron colloid staining of the surface coat, but did not interfere with the aldehyde fuchsin staining of the surface coat.
Electron microscopy of ultrathin sections revealed that the iron particles indicating sulfated proteoglycans were preferentially deposited in the perineuronal tissue spaces. Many neurons in the hippocampal subiculum possessed cell surface glycoproteins which were labeled with lectin Vicia villosa or soybean agglutinin and formed 1-2 weeks after birth. Double staining revealed that these lectin-labeled neurons were identical in part with the neurons reactive to the cationic iron colloid.
Dark neurons began to appear 3-4 weeks after birth. The formation of perineuronal sulfated proteoglycans and the appearance of dark neurons, both occurring during the weaning period, may reflect the morphological and physiological completion of the brain. Dark neurons are suggested to be exhausted cells that are restored to light or normal neurons after sleep.

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Microvascular changes during the development of follicles in bovine ovaries were studied by scanning electron microscopy of corrosion casts. A clear vascular plexus of ovarian follicles appeared at the stage when secondary follicles were 200-400 μm in diameter. The plexus consisted initially of a thin, roughly structured and single-layered capillary network.
During follicular development, the microvascular architecture of antral follicles was arranged as two independent vascular plexuses. The inner plexus, which received a spiral arteriole, consisted of a dense sinusoidal capillary network with an arterial and a venous layer; it functioned as an independent microcirculatory unit. The inner plexus developed from the capillary plexus of the theca interna of the secondary follicles. The outer plexus, which anastomosed with several stromal capillaries, consisted of a thin, coarse and basket-like capillary plexus. The outer plexus was formed from the stromal capillary plexus as a consequence of the rapid enlargement of developing antral follicles.

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The testis, the epididymis and the prostate are immunologically suppressed organs in which allogeneic tissue grafts can survive for a long time. In the present study, morphological features of these three organs after systemic administration of immunopotentiators was investigated in mice to determine whether or not this treatment can affect their immunosuppressive circumstances. The animals were intravenously injected with adjuvants, Bordetella pertussigens, then killed 7-10 days later for histological examination. The results showed that the testicular interstitium was completely free from leukocyte infiltration but that the accessory glands (the prostate, the coagulating gland and the seminal vesicle), the vas deferens, the epididymis and the ductuli efferentes received extravasation of leukocytes into their interstitial tissues. This indicates that the testis is resistant to leukocyte infiltration compared with the epididymis and the prostate.

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The effect of synthetic glucocorticoid, dexamethasone (DEX), on the fine structure of the fetal growth hormone (GH) cell was examined in rats on Days 17-19 of gestation by the protein A-gold method of immuno-electron microscopy. GH cells appeared on Day 19 in the control fetuses. The DEX-induced GH cells of Day 17 fetuses displayed more immature features than those of Day 18, which closely resembled GH cells found in the control fetus on Day 19. Cytological changes which indicate increased secretory activity such as the development of the rough endoplasmic reticulum or enlargement of the Golgi complex, and an accumulation of secretory granules were evident only in the DEX-treated fetuses on Day 19. It is concluded that DEX not only accelerates the development of GH cells, as observed on Day 17 or 18, but also brings them to a functionally stimulated state by Day 19.

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Mouse fetal livers of 12 days gestation were transplanted beneath the kidney capsule of syngeneic castrated male hosts. Recipients received a single intraperitoneal injection of 5mg estriol (E₃) immediately after transplantation and were sacrificed 4, 7 and 14 days later. Control mice received injections of solvent only. The fetal liver grafts of the E₃ groups showed remarkable growth compared with the control grafts at each corresponding time. In the grafted fetal livers of the E₃ groups, many more basophilic hepatocytes appeared for much longer periods, and the early formation of wide sinusoids was noted. Although hemopoietic activity had almost ceased a short time after transplantation in the control grafts, many prominent extrasinusoidal erythroid, granulocytic and megakaryocytic hemopoietic foci developed and persisted to the end of the experiments in the grafted fetal livers of the E₃ groups. In the next experiment, the recipients received three injections of 0.5-5mg E₃ at 5-day intervals, and were sacrificed 21 days after transplantation. Control animals received solvent only in the same manner. The fetal liver grafts of the E₃ groups also showed remarkable, dose dependent growth. The growth was particularly striking at doses of 3-5mg E₃. In the grafts of these groups, basophilic hepatocytes were predominant, and hepatic cords and sinusoids were well formed. Moreover, sinusoidal erythropoiesis was intense. These results provide evidence for the possible role of E₃, secreted from the placenta into the fetal circulation, in the development of the fetal liver.

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