Visualization of three-dimensional stigmoid body in FFPE and ultrathin sections of mouse.

Huntingtin-associated protein 1 (HAP1) is abundantly expressed in the neurons of the central nervous system and forms unique intracytoplasmic inclusions of unknown function called 'stigmoid bodies' (STBs). Transmission electron microscopy has revealed that the STBs are aggregates of granules containing cavities with a diameter of 0.5-3 µm. Small STBs fuse to form larger STBs, the size of which is said to vary depending on the developmental growth stage and brain region. Light microscopy can only reveal that these STBs have similar circular shapes due to its limited resolution. Therefore, light microscopy is only fit for the study of the STB distribution and quantitative changes. We, herein, suggest the adoption of correlative light and electron microscopy, which combines confocal laser scanning microscopy and scanning electron microscopy as the method allowing us to identify the huntingtin-associated protein 1-positive STBs in formalin-fixed paraffin-embedded (FFPE) sections. This approach allows us to study the three-dimensional morphology of immunolabeled objects in histopathological specimens. The STBs in FFPE sections of murine hypothalami reflected the transmission electron microscopic images of Epon-embedded STBs, although we were not able to observe any organelle covering the STBs of the FFPE sections. Furthermore, we were able to reconstruct the three-dimensional structure of the STB, and we identified it to be of spherical form, covered with mitochondria and rough endoplasmic reticulum, and bearing a cluster of cavities in the centre. In the future, we might gain new insights by comparing the three-dimensional structure of the STB between different neurons and under a variety of conditions.

Effects of exercise and bryostatin-1 on serotonin dynamics after cerebral infarction.

Although it has been suggested that the combination of exercise and bryostatin-1 administration may induce greater functional recovery than exercise alone, the detailed molecular mechanisms are not well known. Here, we examined the relationship between this combination treatment and monoamine dynamics in the cerebral cortex peri-infarction area to promote our understanding of these molecular mechanisms. Experimental cerebral cortex infarctions were produced by photothrombosis in rats. Voluntary exercise was initiated 2 days after surgery. Motor performance was then measured using the rotarod test. Monoamine concentrations in the perilesional cortex were analyzed by high-performance liquid chromatography. In behavioral evaluations, performance in the rotarod test was significantly increased by exercise. Moreover, performance in the rotarod test after the combination of exercise and bryostatin-1 administration was significantly greater than that after exercise alone. In the analysis of monoamines, serotonin (5-HT) concentrations were significantly higher in the groups treated with exercise and bryostatin-1. In addition, 5-HT turnover was significantly lower in the groups treated with exercise and bryostatin-1. Furthermore, the mean latency in the rotarod test showed a significant positive correlation with 5-HT levels. In immunohistochemical analysis, 5-HT immunoreactivity in the dorsal raphe nucleus was shown to be higher in the groups treated with exercise. In the present study, we detected changes in the levels of monoamines associated with the combined treatment of exercise and bryostatin-1 administration in the perilesional cortex. It has been suggested that this combination of therapies may affect 5-HT turnover and serve to increase local 5-HT concentrations in the perilesional area.

Phenotypic changes of AADC-only-immunoreactive cells in the alimentary canal of the laboratory shrew, Suncus murinus, induced by systemic administration of monoamine precursors.

In order to elucidate the function of anti-aromatic acid decarboxylase (AADC)-only-positive cells in the alimentary canal, 5-hydroxy-L-tryptophan (5-HTP) or L-3,4-dihydroxyphenylalanine (L-DOPA) was intraperitoneally injected into the laboratory shrew, Suncus murinus, and immunohistochemical studies were conducted on continuous sections of the alimentary canal using specific antisera against tyrosine hydroxylase (TH), AADC, dopamine (DA), and serotonin (5-HT). AADC-only-positive cells localized to the epithelial layer of the alimentary canal from the stomach to the large intestine. These AADC-only-positive cells became DA- and AADC-positive cells after L-DOPA injection, and 5-HT- and AADC-positive cells after 5-HTP injection. These results strongly indicate that the AADC-only-positive cells in the alimentary canal of Suncus murinus are capable of synthesizing DA and 5-HT simultaneously upon administration of L-DOPA and 5-HTP.

Functional recovery and alterations in the expression and localization of protein kinase C following voluntary exercise in rat with cerebral infarction.

Recently, it has become widely known that rehabilitative training after stroke brings about some improvement of paralysis and disability; however, not much is known about the relationship between paralysis recovery and the participation of plasticity-related molecules. Hence, the localization and level of expression of several proteins in the cerebral cortex of rat groups with/without voluntary exercise using a running wheel after photo thrombotic infarction were examined in this study. In behavioral evaluation, the mean latency until falling from a rotating rod in the group with voluntary exercise at 6 days after infarction was significantly longer than that in the group without exercise. Immunohistochemical localization of c-Fos protein after behavioral test occurred in the area surrounding the infarction core in the exercise group. In protein expression analysis, protein kinase C (PKC), growth-associated protein 43 (GAP43) and phosphorylated at serine 41 GAP43 (p-GAP43) were significantly increased after voluntary exercise compared with those in rats without exercise. Expression of PKC immunoreactivity was observed in layer III of the perilesional cortex in rats with exercise, and the intracellular localization in the pyramidal neurons was mainly translocated to the plasma membrane. The expression and localization of these proteins may be related to the underlying mechanisms of exercise-induced paralysis recovery, that is, neuronal plasticity and remodeling of cortical connections through the phosphorylation of GAP43 by interaction with PKC. In the present study, the participation of at least some of the modulators associated with the improvement of motor deficit adjacent to the brain lesion might have been detected.

Peculiar tonsil-like structure near vagina of the laboratory shrew, Suncus murinus.

After finding tonsil-like structures near the entrance of vagina of a laboratory shrew (Suncus murinus), which we subsequently designated as vaginal tonsils, we performed detailed immunohistochemical and developmental studies. The location of T and B cells in the vaginal tonsils differed from that in the palatine tonsils or that in the lymphoid nodes of other animals. The boundary between the germinal center region and the region encompassing follicular interfollicular tissue was not clear. B cells were widely distributed and very dense in the parenchyma, but they were scattered in the epithelial area (B cells were present in around 90% of the vaginal tonsil tissue). In contrast, T cells were scattered in the parenchyma and in the epithelial area (T cells were present in around 10% of the vaginal tonsil tissue). B cells were more prominent than T cells throughout the development of these structures and the epithelium was invaded by many immigrating cells. The size of the vaginal tonsils changed during postnatal development. Vaginal tonsils are structurally similar to other tonsils, and they may function to protect the vagina from infection.

Light and electron microscopic observation of intracytoplasmic inclusion bodies in the locus coeruleus of the hamster.

The authors previously demonstrated that intracytoplasmic inclusion bodies (1-3 microm) in the mouse locus coeruleus under light and electron microscopy are characteristically stained using the Holmes modified method. We reported that one inclusion body existed in almost all neurons of the locus coeruleus. The present study examined whether similar inclusion bodies are present in the Syrian hamster (weight, about 60 g). Paraffin sections stained with the modified Holmes' method dis played numerous small inclusion bodies in the cytoplasm of cells in the locus coeruleus. Epon sections (1 microm thick) stained using toluidine blue were observed under light microscopy, and numerous small inclusion bodies were again observed. Under electron microscopy observation, inclusion bodies (<1 microm in diameter) predominantly comprised small granular materials, similar to those described by previous investigators. Although inclusion bodies were devoid of a limiting membrane, the relation ship to cytoplasmic organelles was unclear. However, free and polyribosomes were occasionally noted in close proximity to inclusion bodies. Inclusion bodies may thus be formed from ribosomes. Intracytoplasmic inclusion bodies in the hamster locus coeruleus differed in appearance compared with inclusion bodies in the mouse locus coeruleus.

Glucose concentration-dependent potentiation of insulin secretion by a new chemical entity, KCP256.

The insulinotropic activity of KCP256 [(R)-8-benzyl-2-cyclopentyl-7, 8-dihydro-4-propyl-1H-imidazo[2,1-i]purin-5(4H)-one hydrochloride] was examined using MIN6 cells (a pancreatic beta-cell line) and pancreatic islets isolated from rats. Unlike sulfonylurea anti-diabetic drugs, KCP256 dose-dependently (0.1-10 microM) enhanced insulin secretion from MIN6 cells and its insulinotropic effect was exerted only at high concentrations of glucose (8.3-22 mM) but not at low concentrations of glucose (3.3-5.5 mM). Furthermore, the action mechanism of KCP256 was different because, unlike sulfonylurea drugs, KCP256 did not displace the binding of [3H]glibenclamide, and did not inhibit the 86Rb+ efflux nor K(ATP) channel activity. In isolated islets, KCP256 also enhanced insulin secretion in a dose- and a glucose-concentration-dependent manner. Plasma levels of insulin after glucose challenge in KCP256-administrated rats were higher than those in vehicle-administrated animals, indicating that KCP256 can enhance insulin secretion in vivo. Since the insulinotropic activity of KCP256 only occurs at high concentrations of glucose, this novel drug may exhibit a decreased risk of drug-induced hypoglycemia compared with sulfonylurea drugs when treating patients with diabetes.

Cerebellar fastigial neurons send bifurcating axons to both the left and right superior colliculus in cats.

Anesthetized cats were injected with 2% Fast Blue and 0.5% Nuclear Yellow into the intermediate and deep layers of the left and right superior colliculus, respectively. In the right caudal part of the cerebellar fastigial nucleus (cFN), double-labeling was found in 38.5% of the neurons labeled with Fast Blue, and in 11.5% of the neurons labeled with Nuclear Yellow. In the left cFN, 52.2% of the neurons labeled with Fast Blue and 11.0% of the neurons labeled with Nuclear Yellow were double-labeled. The results suggest a role of bifurcating fastigial fibers in cerebellar visual control.