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-µm-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.

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In the postnatal mammalian brain, neural stem cells of the ventricular-subventricular zone continue to generate doublecortin (Dcx)-expressing immature neurons. Throughout life, these immature neurons migrate to the olfactory bulb through the rostral migratory stream (RMS). In this study, we investigated the distribution of these putative immature neurons using enhanced green fluorescent protein (EGFP) expression in the area surrounding the RMS of the juvenile Dcx-EGFP mice. Through the combined use of an optical clearing reagent (a 2,2'-thiodiethanol solution) and two-photon microscopy, we visualized three-dimensionally the EGFP-positive cells in the entire RMS and its surroundings. The resulting wide-field and high-definition images along with computational image processing methods developed in this study were used to comprehensively determine the position of the EGFP-positive cells. Our findings revealed that the EGFP-positive cells were heterogeneously distributed in the area surrounding the RMS. In addition, the orientation patterns of the leading process of these cells, which displayed the morphology of migrating immature neurons, differed depending on their location. These novel results provide highly precise morphological information for immature neurons and suggest that a portion of immature neurons may be detached from the RMS and migrate in various directions.

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