Biology subjects

Osanai, Y.

Publications and source records attributed to Osanai, Y..

6 recordsLinked to original sources

bioRxiv2025

Visualization and Morphological Analysis of Individual Oligodendrocytes in the Mouse White Matter

Myelin formation by oligodendrocytes is essential for the regulation of the conduction velocity and proper brain function. To ensure accurate information processing in response to experiences such as sensory stimuli and learning, oligodendrocytes adjust their number and morphology. In addition, oligodendrocyte morphology changes with senescence and in the presence of neurodegenerative diseases. Thus, visualizing oligodendrocytes and analyzing their morphology is crucial for understanding how our brains change under such conditions. Herein, we describe the methods for labeling and analyzing the morphologies of individual oligodendrocytes in mouse white matter at the light microscopic level. PDGFRa-CreERT2:Tau-mGFP and PLP-CreERT2:Tau-mGFP mice enable us to visualize and analyze later-born or early-born oligodendrocyte morphology. In addition, sparse oligodendrocyte labeling with attenuated rabies virus expressing GFP enables the visualization and morphological analysis of individual oligodendrocytes in various brain white matter regions without the need for transgenic animals. Furthermore, the combination with immunostaining in thick tissues enables the identification of labeled oligodendrocytes and myelin sheaths, as well as their interactions with neuronal axons. These methods are suitable for revealing how oligodendrocytes adapt their morphologies depending on environmental stimuli or pathological conditions.

neuroscience↗

bioRxiv2025

GABA mediates experience-dependent regulation of myelination in the mouse visual pathway.

Myelination in the visual pathway is critical for transmitting visual information from retina to the brain. Reducing visual experience shortens myelin sheath length and slows the conduction velocity of the optic nerve. However, the mechanism underlying such experience-dependent myelination is unclear. Here, we found that closing both eyes, binocular deprivation (BD), during the juvenile period less affects the optic nerve myelination than monocular deprivation (MD) via GABA signaling. RNA-seq analysis of optic nerves from MD and BD mice revealed that GABAergic signaling is downregulated on the deprived side of MD compared to the intact side and BD. Inhibition of GABAergic signaling during the juvenile period resulted in myelin sheath shortening and excessive oligodendrocyte generation in normal mice, similar to the changes observed in MD mice. Enhancing GABAergic signaling rescued the myelin sheath shortening and excessive oligodendrocyte generation in the optic nerve of MD mice. Furthermore, we identified novel GABAergic neurons located within the optic nerve, whose neurites form belt-like presynaptic structures with the oligodendrocyte lineage cells, suggesting a potential source of the GABAergic inputs into oligodendrocytes. Our results indicate that the myelination of visual pathway is maintained by binocular visual inputs via intra-nerve GABA signaling.

neuroscience↗

bioRxiv2023

Axonal selectivity of myelination by single oligodendrocytes established during development in mouse cerebellar white matter

Myelin formation by oligodendrocytes regulates conduction velocity and functional integrity of neuronal axons. While individual oligodendrocytes form myelin sheaths around multiple axons and control the functions of neural circuits in the brains, it remains unclear if oligodendrocytes selectively form myelin sheaths around the specific types of axons. In this study, we developed a method for observing a single oligodendrocyte and its myelin sheaths around different types of axons in the mouse cerebellar white matter. This was achieved by combining sparse fluorescent labeling of oligodendrocytes by attenuated rabies virus and subsequent immunostaining for axonal markers along with tissue clearing. We revealed that approximately half of the oligodendrocytes showed a preferential myelination of axons originating from Purkinje cells in the adult mice. The preference for Purkinje cell axons was more pronounced during development, when the process of myelination within cerebellar white matter was initiated; over 90% of oligodendrocytes preferentially myelinated the Purkinje cell axons. The transgenic mice that label early born oligodendrocytes showed that their myelin sheaths were predominantly formed around Purkinje cell axons in the adult cerebellar white matter. These results suggest that a significant proportion of oligodendrocytes preferentially myelinate functionally distinct axons in the cerebellar white matter, and the axonal preference of myelination is established during development.

neuroscience↗

bioRxiv2022

5' transgenes drive leaky expression of 3' transgenes in inducible bicistronic vectors

Molecular cloning techniques enabling contemporaneous expression of two or more protein-coding sequences in a cell type of interest provide an invaluable tool for understanding the molecular regulation of cellular functions. DNA recombination employing the Cre-lox system is commonly used as a molecular switch for inducing the expression of recombinant proteins encoded within a bicistronic cassette. In such an approach, the two protein-coding sequences are separated by a 2A peptide or internal ribosome entry site (IRES), and expression is designed to be strictly Cre-dependent by using a lox-STOP-lox cassette or flip-excision (FLEX) switch. However, low-level or leaky expression of recombinant proteins is often observed in the absence of Cre activity, potentially compromising the utility of this approach. To investigate the mechanism of leaky gene expression, we generated pCAG-lox-GFP-STOP-lox-Transgene A-2A-Transgene B vectors, which are designed to express nuclear-targeted GFP in the absence of Cre, and express both transgenes A and B after Cre-mediated recombination. We found that cells transfected with these bicistronic vectors exhibited low-level Cre-independent expression specifically of the transgene positioned 3' of the 2A peptide. We observed similar results in vivo by viral transduction of the adult mouse cerebral cortex with AAV-mutagenesis of putative transcription factor binding sites that the 5' transgene confers promoter-like activity that drives expression of the 3' transgene. Finally, we demonstrate that inclusion of an additional lox-STOP-lox cassette between the 2A sequence and 3' transgene dramatically reduces the extent of Cre-independent leaky gene expression. Our findings highlight that caution should be applied to the use of Cre-dependent bicistronic constructs when tight regulation of transgene expression is desired and provide a guide to preventing leaky gene expression when the expression of more than one protein is required.

molecular biology↗

bioRxiv2021

A Novel Pharmacogenetic Model for Highly Efficient Ablation of Oligodendrocyte Progenitor Cells in the Adult Mouse CNS

Approaches to investigate adult oligodendrocyte progenitor cells (OPCs) by targeted cell ablation in the rodent central nervous system have been limited by methodological challenges resulting in only partial and transient OPC depletion. We have developed a novel pharmacogenetic model of conditional OPC ablation, eliminating 98.6% of all OPCs throughout the brain. By combining recombinase-based transgenic and viral strategies for targeting OPCs and ventricular-subventricular zone (V-SVZ)-derived neural precursor cells (NPCs), we found new PDGFRA-expressing cells born in the V-SVZ repopulated the OPC-deficient brain starting 12 days after OPC ablation. Our data reveal that OPC depletion induces V-SVZ-derived NPCs to generate vast numbers of PDGFRA+ NG2+ cells with the capacity to migrate and proliferate extensively throughout the dorsal anterior forebrain. Further application of this novel approach to ablate OPCs will advance knowledge of the function of both OPCs and oligodendrogenic NPCs in health and disease.

neuroscience↗

bioRxiv2020

Selective and controlled myelin formation by individual interfascicular oligodendrocytes in the mouse corpus callosum

Single oligodendrocytes produce myelin sheath around multiple axons in the central nervous system. Interfascicular oligodendrocytes (IOs) in the white matter are aligned in rows and facilitate nerve conduction, but their detailed morphologies remain largely unknown. In the present study, we three-dimensionally reconstructed seven IOs in a row in the murine corpus callosum using serial block face-scanning electron microscopy (SBF-SEM). These morphologically polarized IOs extended a thick process with numerous branches from the cytoplasm-rich part of the cell and formed myelin sheaths preferentially around distant axons. The multiple branched processes of each IO myelinated multiple axons having similar diameters with restricted myelin thicknesses, indicating that individual IOs have their own myelination profiles even on distinct target neurons. Consistent with the finding, the IOs transduced and visualized with the rabies viral vector expressing GFP showed statistically significant variations in the myelination patterns. We further reconstructed the sheath immediately adjacent to those derived from the seven IOs; the thicknesses of both sheaths were significantly correlated despite emanating from different IOs. These results proposed a rule that myelination by individual IOs, regulated by interaction with ensheathed axons, is selective in specific axons and at the same time orchestrated at the whole cell level. Main pointsCallosal oligodendrocytes form myelin sheaths preferentially around distant axons. Each oligodendrocyte myelinate multiple axons having similar diameters with restricted myelin thicknesses. The thicknesses of adjacent myelin sheaths are similar.

neuroscience↗