AP192R Sigma-AldrichDonkey Anti-Mouse IgG Antibody, Rhodamine conjugate, Species Adsorbed

It has been established that a postsynaptic scaffolding protein, gephyrin, is essential for anchoring two main groups of inhibitory receptors, GABA(A) receptors (GABA(A) Rs) and glycine receptors (GlyRs), to the postsynaptic sites of neurons. The present study was primarily attempted to examine if expression patterns of gephyrin might be different between jaw-closing (JC) and jaw-opening (JO) motoneurons. The JC- and JO-motoneurons in the rat trigeminal motor nucleus (Vm) were located in the dorsolateral (Vm.dl) and ventromedial (Vm.vm) divisions, respectively (Mizuno et al.,1975). Thus, immunoreactivity (IR) for gephyrin was investigated in the Vm: immunofluorescence histochemistry for gephyrin was combined with retrograde tract-tracing of fluorogold (FG), which was injected into nerves innervating JC-muscles or nerves innervating JO-muscles; neuronal cells were counterstained with propidium iodide (PI). The Vm.dl was discriminated from the Vm.vm by the presence of vesicular glutamate transporter 1 (VGLUT1)-immunopositive axon terminals, which were distributed in the Vm.dl but not in the Vm.vm (Pang et al., J Comp Neurol 2009;512:595-612). Gephyrin-IR showed a punctate pattern of fluorescence, and motoneuronal profiles were coated with small clusters of gephyrin-immunopositive puncta throughout the Vm. The distribution density of such clusters was apparently higher in the Vm.dl than in the Vm.vm; this was confirmed quantitatively by a method similar to that described by Lorenzo et al. (Eur J Neurosci 2006;23:3161-3170). On the basis of the present results, possible correlation between the distribution density of gephyrin clusters in the submembrane region of Vm motoneurons and that of axon terminals making inhibitory synapses on Vm motoneurons was discussed.

Conjugation in Tetrahymena thermophila involves a developmental program consisting of three prezygotic nuclear divisions, pronuclear exchange and fusion, and postzygotic and exconjugant stages. The conjugation junction structure appears during the initiation of conjugation development, and disappears during the exconjugant stage. Many structural and functional proteins are involved in the establishment and maintenance of the junction structure in T. thermophila. In the present study, a zinc finger protein-encoding gene ZFR1 was found to be expressed specifically during conjugation and to localize specifically to the conjugation junction region. Truncated Zfr1p localized at the plasma membrane in ordered arrays and decorated Golgi apparatus located adjacent to basal body. The N-terminal zinc finger and C-terminal hydrophobic domains of Zfr1p were found to be required for its specific conjugation junction localization. Conjugation development of ZFR1 somatic knockout cells was aborted at the pronuclear exchange and fusion conjugation stages. Furthermore, Zfr1p was found to be important for conjugation junction stability during the prezygotic nuclear division stage. Taken together, our data reveal that Zfr1p is required for the stability and integrity of the conjugation junction structure and essential for the sexual life cycle of the Tetrahymena cell.

We report the first demonstration of the embryonal patch patterns of endocrine organs and the polyclonality of hormone-producing cell populations using chimeric mice produced by aggregation of C57BL/6-Tg(CAG-EGFP)C14-Y01-FM131Osb transgenic mice and BALB/C mice. Confocal laser scanning microscopy (CLSM) analysis for enhanced green fluorescent protein (EGFP) and immunohistochemistry with anti-EGFP antibody revealed that all endocrine organs of chimeric mice had a mosaic appearance of EGFP-positive patches and EGFP-negative patches. The patches composed of EGFP-positive cells were distinctive in their size and shape. The pituitary patches were large and irregular, representing a geographical pattern. In contrast, parathyroid, pancreatic islet, and adrenal medulla patches were small and demarcated, representing an island-like pattern. Thyroid follicles and adrenal cortex cords showed a mixture of monophenotypia and polyphenotypia, indicating polyclonal embryonic origin. Furthermore, we studied the tissue clonality of hormone-producing cell populations in the pituitary, thyroid, and pancreatic islets using a combination method of CLSM for EGFP and immunohistochemistry for hormones. All the pituitary cell populations of GH, prolactin, TSH, FSH, LH, and ACTH, the calcitonin-producing cell population in the thyroid, and the insulin- and glucagon-producing cell populations in pancreatic islets had mosaic patterns in EGFP expression in the chimeric mice, suggesting polyclonal embryonic origin. In conclusion, the different patch patterns of the endocrine organs could contribute to the understanding of embryonic development and organization of endocrine organs. Furthermore, we clearly demonstrate that all hormone-producing cell populations are of polyclonal embryonic origin, derived from more than two progenitor cells.

Immunohistochemical techniques were employed to examine orexin-like immunoreactivities in the pituitary of the Nile tilapia (Oreochromis niloticus). Rabbit anti-orexin-A serum and mouse anti-orexin-B monoclonal antibodies were used as primary antibodies. Orexin-B immunoreactive cells corresponded to luteinizing hormone (LH)- or thyroid-stimulating hormone (TSH)-containing cells, and all LH- and TSH-containing cells were immunoreactive for orexin-B. However, we found no orexin-A immunoreactive cells in the pituitary. In the Nile tilapia, an orexin-B-like substance may be secreted from LH- or TSH-containing cells and may regulate pituitary function, rather than the orexin-A-like substance in the pituitaries of Japanese seaperch and medaka.

We examined the effects of single and multiple maternal glucocorticoid courses on apoptosis in the cerebral cortices of ovine fetuses (CC). Ewes received single dexamethasone or placebo courses at 104-106 or 133-135 days or multiple courses between 76-78 and 104-106 days gestation. In the single-course groups, ewes received four 6 mg dexamethasone or placebo injections every 12 hr for 48 hr. Multiple-course groups received the same treatment once per week for 5 weeks. Neuronal and nonneuronal apoptotic cell numbers per square millimeter were determined with TUNEL and NeuN staining and with caspase-3 enzyme activity on CC tissues harvested at 106-108 (70%) or 135-137 (90%) days of gestation. Apoptotic cell numbers and caspase-3 activity were 50% lower (P less than 0.02) after single placebo courses at 90% than 70% gestation; 90% of apoptotic cells were (P less than 0.01) nonneuronal at both ages. Nonneuronal apoptotic cells and caspase-3 activity were 40% and 20% lower (P less than 0.02) after single dexamethasone than placebo courses at 70%, but not 90%, gestation. Caspase-3 activity was 20% lower (P less than 0.01) after multiple dexamethasone than placebo courses, but apoptotic cell number did not differ. We conclude that nonneuronal apoptosis represents the major form of apoptosis in the CC at both 70% and 90% of gestation. Apoptosis in nonneuronal cells decreases with maturity and after a single course of dexamethasone at 70%, but not at 90%, gestation and not after multiple courses at 70% gestation. We speculate that a single course of glucocorticoids exerts maturational changes on the rate of apoptosis in the cerebral cortex of preterm ovine fetuses.

Intermediate filaments are frequently used in studies of developmental biology as markers of cell differentiation. To assess whether they can be useful to identify differentiating pancreatic endocrine cells, we examined the pattern of expression of nestin, cytokeratin 20, and vimentin on acetone-fixed cryosections of rat adult and developing pancreas. We also studied vimentin expression in mouse embryonic pancreas at E19. Cytokeratin 20 was found in all pancreatic epithelial cell lineages during the entire development of the rat gland and in the adult animals. Under our experimental conditions, therefore, cytokeratin 20 is not an exclusive marker of rat duct cells. Nestin was detected exclusively in stromal cells either in the adult or developing rat pancreas. Vimentin was observed within cells located in the primitive ducts of rat pancreas starting from E12.5. Their number rapidly increased, reaching its highest level in newborn animals. Vimentin was also spotted in alpha cells starting from E12.5 but disappeared soon after birth, likely identifying immature or recently differentiated alpha cells. In addition, vimentin was observed in duct and alpha cells of mouse developing pancreas showing that its expression in such cells is not an event restricted to the rat. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Abnormal brain development, induced by genetic influences or resulting from a perinatal trauma, has been recognized as a cause of seizure disorders. To understand how and when these structural abnormalities form, and how they are involved in epileptogenesis, it is important to generate and investigate animal models. We have studied one such model, a mouse in which deletion of the p35 gene (p35-/-) gives rise to both structural disorganization and seizure-like function. We now report that aberrant dentate development can be recognized in the organotypic hippocampal slice culture preparation generated from p35-/- mouse pups. In these p35-/- cultures, an abnormally high proportion of dentate granule cells migrates into the hilus and molecular layer, and develops aberrant dendritic and axonal morphology. In addition, astrocyte formation in the dentate gyrus is disturbed, as is the distribution of GABAergic interneurons. Although the p35-/- brain shows widespread abnormalities, the disorganization of the hippocampal dentate region is particularly intriguing since a similar pathology is often found in hippocampi of temporal lobe epilepsy patients. The abnormal granule cell features occur early in development, and are independent of seizure activity. Further, these aberrant patterns and histopathological features of p35-/- culture preparations closely resemble those observed in p35 knockout mice in vivo. This culture preparation thus provides an experimentally accessible window for studying abnormal developmental factors that can result in seizure propensity.

Immunohistochemical techniques were employed to investigate orexin-A-like and orexin receptor 1 (OX1R)-like immunoreactivities in the Xenopus pituitary gland. Orexin-A-immunoreactive cells were mainly scattered in the posterior half of the pars distalis. They corresponded to thyroid-stimulating hormone (TSH)-containing cells and so far have not corresponded to other types of pituitary adenocytes. On the other hand, OX1R-immunoreactive cells were mainly distributed in the anterior half of the pars distalis and corresponded to prolactin (PRL)-containing cells; however, we found that OX1R-immunoreactive cells did not correspond to other types of adenocytes in the Xenopus pituitary. These results suggest that an orexin-A-like substance secretes with and/or without TSH from TSH-containing cells and that the peptide modulates the functions of PRL-containing cells via OX1R in a paracrine fashion.

We demonstrated the occurrence of marked regeneration of the corticospinal tract (CST) after a single transection and failure of regeneration after a repeated transection in young rats. To provide convincing evidence for the complete transection and regeneration we used retrograde neuronal double labeling. Double-labeled neurons that took up the first tracer from the transection site and the second tracer from the injection site caudal to the transection site were observed in the sensorimotor cortex. The anterograde tracing method revealed various patterns of regeneration. In the most successful cases the vast majority of regenerated fibers descended in the normal tract and terminated normally whereas a trace amount of fibers coursed aberrantly. In the less successful cases fibers descended partly normally and partly aberrantly or totally aberrantly. To clarify the role of astrocytes in determining the success or failure of regeneration we compared expression of glial fibrillary acidic protein (GFAP), vimentin and neurofilament (NF) immunoreactivity (IR) in the lesion between single and repeated transections. In either transection, astrocytes disappeared from the CST near the lesion site as early as 3 h after lesioning. However, by 24 h after a single transection, immature astrocytes coexpressing GFAP- and vimentin-IR appeared in the former astrocyte-free area and NF-positive axons crossed the lesion. By contrast, after a repeated transection the astrocyte-free area spread and NF-positive axons never crossed the lesion. It appears likely that the major sign, and possibly cause of failure of regeneration is the prolonged disappearance of astrocytes in the lesioned tract area.