Downregulation of functional Reelin receptors in projection neurons implies that primary Reelin action occurs at early/premigratory stages

Reelin signaling is essential for correct development of the mammalian brain. Reelin binds to apolipoprotein E receptor 2 and very low-density lipoprotein receptor and induces phosphorylation of Dab1. However, when and where these reactions occur is essentially unknown, and the primary function(s) of Reelin remain unclear. Here, we used alkaline phosphatase fusion of the receptor-binding region of Reelin to quantitatively investigate the localization of functional Reelin receptors (i.e., those on the plasma membrane as mature forms) in the developing brain. In the wild-type cerebral cortex, they are mainly present in the intermediate and subventricular zones, as well as in radial fibers, but much less in the cell bodies of the cortical plate. Functional Reelin receptors are much more abundant in the Reelin-deficient cortical plate, indicating that Reelin induces their downregulation and that it begins before the neurons migrate out of the intermediate zone. In the wild-type cerebellum, functional Reelin receptors are mainly present in the cerebellar ventricular zone but scarcely expressed by Purkinje cells that have migrated out of it. It is thus strongly suggested that Reelin exerts critical actions on migrating projection neurons at their early/premigratory stages en route to their final destinations, in the developing cerebral cortex and cerebellum. Copyright © 2009 Society for Neuroscience.This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture (M.H., A.B.), Ono Medical Research Foundation, and Kanae Foundation for the Promotion of Medical Science (M.H.). T.H. is a Research Fellow of Japan Society for the Promotion of Science. J.M.L. is a Ramón y Cajal Research Fellow funded by Grant SAF2004-07685 and Fundación Mutua Madrileña.Peer Reviewe

Altered Insulin Receptor Signalling and β-Cell Cycle Dynamics in Type 2 Diabetes Mellitus

Insulin resistance, reduced β-cell mass, and hyperglucagonemia are consistent features in type 2 diabetes mellitus (T2DM). We used pancreas and islets from humans with T2DM to examine the regulation of insulin signaling and cell-cycle control of islet cells. We observed reduced β-cell mass and increased α-cell mass in the Type 2 diabetic pancreas. Confocal microscopy, real-time PCR and western blotting analyses revealed increased expression of PCNA and down-regulation of p27-Kip1 and altered expression of insulin receptors, insulin receptor substrate-2 and phosphorylated BAD. To investigate the mechanisms underlying these findings, we examined a mouse model of insulin resistance in β-cells – which also exhibits reduced β-cell mass, the β-cell-specific insulin receptor knockout (βIRKO). Freshly isolated islets and β-cell lines derived from βIRKO mice exhibited poor cell-cycle progression, nuclear restriction of FoxO1 and reduced expression of cell-cycle proteins favoring growth arrest. Re-expression of insulin receptors in βIRKO β-cells reversed the defects and promoted cell cycle progression and proliferation implying a role for insulin-signaling in β-cell growth. These data provide evidence that human β- and α-cells can enter the cell-cycle, but proliferation of β-cells in T2DM fails due to G1-to-S phase arrest secondary to defective insulin signaling. Activation of insulin signaling, FoxO1 and proteins in β-cell-cycle progression are attractive therapeutic targets to enhance β-cell regeneration in the treatment of T2DM

A Rapid Optical Clearing Protocol Using 2,2'-Thiodiethanol for Microscopic Observation of Fixed Mouse Brain

Elucidation of neural circuit functions requires visualization of the fine structure of neurons in the inner regions of thick brain specimens. However, the tissue penetration depth of laser scanning microscopy is limited by light scattering and/or absorption by the tissue. Recently, several optical clearing reagents have been proposed for visualization in fixed specimens. However, they require complicated protocols or long treatment times. Here we report the effects of 2,2'-thiodiethanol (TDE) solutions as an optical clearing reagent for fixed mouse brains expressing a yellow fluorescent protein. Immersion of fixed brains in TDE solutions rapidly (within 30 min in the case of 400-mu m-thick fixed brain slices) increased their transparency and enhanced the penetration depth in both confocal and two-photon microscopy. In addition, we succeeded in visualizing dendritic spines along single dendrites at deep positions in fixed thick brain slices. These results suggest that our proposed protocol using TDE solution is a rapid and useful method for optical clearing of fixed specimens expressing fluorescent proteins

Three-Dimensional Analysis of Cell Division Orientation in Epidermal Basal Layer Using Intravital Two-Photon Microscopy

<div><p>Epidermal structures are different among body sites, and proliferative keratinocytes in the epidermis play an important role in the maintenance of the epidermal structures. In recent years, intravital skin imaging has been used in mammalian skin research for the investigation of cell behaviors, but most of these experiments were performed with rodent ears. Here, we established a non-invasive intravital imaging approach for dorsal, ear, hind paw, or tail skin using R26H2BEGFP hairless mice. Using four-dimensional (x, y, z, and time) imaging, we successfully visualized mitotic cell division in epidermal basal cells. A comparison of cell division orientation relative to the basement membrane in each body site revealed that most divisions in dorsal and ear epidermis occurred in parallel, whereas the cell divisions in hind paw and tail epidermis occurred both in parallel and oblique orientations. Based on the quantitative analysis of the four-dimensional images, we showed that the epidermal thickness correlated with the basal cell density and the rate of the oblique divisions.</p></div

A summarized schematic of the proliferation and migration of the epidermal basal cells, based on our results.

<p>(<b>A</b>) Schematic of parallel and oblique division. Parallel division generates two basal cells. Because the basal cell density is maintained, the number of parallel divisions is similar to the number of cells migrating to the suprabasal layer. Thus, after a parallel division occurs, a basal cell might gradually migrate into the suprabasal layer over at least 4 hours. On the other hand, oblique division generates one basal cell and another cell that is translocated into a suprabasal layer, without slow migration. Thus, oblique divisions might play an important role in maintaining the rapid upward stream of keratinocytes in thick epidermis. (<b>B</b>) A simple model of the maintenance of the thin and thick epidermis. The epidermis can be roughly considered as having three layers: a proliferative basal layer (layer a), a differentiated cell layer (layer b), and a cornified layer (layer c). Assuming that the increase and the decrease in cell numbers in each layer is approximately equal to the number of cell divisions (as described in the text), then the basal cell density, the number of cell divisions, and oblique division rate are increased in thick epidermis compared with thin epidermis. Thus, the upward stream of the keratinocytes in thick epidermis is more rapid than in thin epidermis.</p

Multi-point Scanning Two-photon Excitation Microscopy by Utilizing a High-peak-power 1042-nm Laser

The temporal resolution of a two-photon excitation laser scanning microscopy (TPLSM) system is limited by the excitation laser beam's scanning speed. To improve the temporal resolution, the TPLSM system is equipped with a spinning-disk confocal scanning unit. However, the insufficient energy of a conventional Ti:sapphire laser source restricts the field of view (FOV) for TPLSM images to a narrow region. Therefore, we introduced a high-peak-power Yb-based laser in order to enlarge the FOV. This system provided three-dimensional imaging of a sufficiently deep and wide region of fixed mouse brain slices, clear four-dimensional imaging of actin dynamics in live mammalian cells and microtubule dynamics during mitosis and cytokinesis in live plant cells

Correcting spherical aberrations in a biospecimen using a transmissive liquid crystal device in two-photon excitation laser scanning microscopy

Two-photon excitation laser scanning microscopy has enabled the visualization of deep regions in a biospecimen. However, refractive-index mismatches in the optical path cause spherical aberrations that degrade spatial resolution and the fluorescence signal, especially during observation at deeper regions. Recently, we developed transmissive liquid-crystal devices for correcting spherical aberration without changing the basic design of the optical path in a conventional laser scanning microscope. In this study, the device was inserted in front of the objective lens and supplied with the appropriate voltage according to the observation depth. First, we evaluated the device by observing fluorescent beads in single-and two-photon excitation laser scanning microscopes. Using a 25x water-immersion objective lens with a numerical aperture of 1.1 and a sample with a refractive index of 1.38, the device recovered the spatial resolution and the fluorescence signal degraded within a depth of +/- 0.6 mm. Finally, we implemented the device for observation of a mouse brain slice in a two-photon excitation laser scanning microscope. An optical clearing reagent with a refractive index of 1.42 rendered the fixed mouse brain transparent. The device improved the spatial resolution and the yellow fluorescent protein signal within a depth of 0-0.54 mm

Comparison of the epidermal structures.

<p>(<b>A-H</b>) Orthogonal images and reconstructed three-dimensional images of the following skin areas: dorsum (<b>A</b>, <b>B</b>), ear (<b>C</b>, <b>D</b>), hind paw (<b>E</b>, <b>F</b>), and tail (<b>G</b>, <b>H</b>). Scale bar = 100 μm. The arrow and arrowhead show the interscale and scale, respectively. (<b>I</b>, <b>J</b>) The average epidermal thickness without the cornified layer (<b>I</b>) and the thickness of the cornified layer (<b>J</b>). These data were obtained from 21 (dorsum), 15 (ear), 15 (hind paw), 14 (interscale), and 14 (scale) points across 5–7 mice per group and compared using the Steel-Dwass test. The error bars represent the standard deviations **<i>P</i> < 0.01; n.s., not significant. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163199#pone.0163199.s013" target="_blank">S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163199#pone.0163199.s014" target="_blank">S4</a> Tables. (<b>K</b>) The relationship between the basal cell density and epidermal thickness (<b>I</b>). The correlation coefficient was 0.837. The dashed line is the regression line. The error bars represent the standard deviations. The statistical significance of the differences in the basal cell densities between the body regions was shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163199#pone.0163199.s015" target="_blank">S5 Table</a>.</p