Color Imaging with Multimodal Three-Photon Microscopy

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Potential of Multiscale Astrocyte Imaging for Revealing Mechanisms Underlying Neurodevelopmental Disorders

Astrocytes provide trophic and metabolic support to neurons and modulate circuit formation during development. In addition, astrocytes help maintain neuronal homeostasis through neurovascular coupling, blood–brain barrier maintenance, clearance of metabolites and nonfunctional proteins via the glymphatic system, extracellular potassium buffering, and regulation of synaptic activity. Thus, astrocyte dysfunction may contribute to a myriad of neurological disorders. Indeed, astrocyte dysfunction during development has been implicated in Rett disease, Alexander’s disease, epilepsy, and autism, among other disorders. Numerous disease model mice have been established to investigate these diseases, but important preclinical findings on etiology and pathophysiology have not translated into clinical interventions. A multidisciplinary approach is required to elucidate the mechanism of these diseases because astrocyte dysfunction can result in altered neuronal connectivity, morphology, and activity. Recent progress in neuroimaging techniques has enabled noninvasive investigations of brain structure and function at multiple spatiotemporal scales, and these technologies are expected to facilitate the translation of preclinical findings to clinical studies and ultimately to clinical trials. Here, we review recent progress on astrocyte contributions to neurodevelopmental and neuropsychiatric disorders revealed using novel imaging techniques, from microscopy scale to mesoscopic scale

Micro-magnetic resonance imaging of ex vivo mouse embryos with potato starch suspension

Summary: Potato starch suspension (PSS) holds promise as a solution to issues, such as air bubbles and specimen motion, associated with micro-magnetic resonance imaging (micro-MRI) of ex vivo embryos. Here, we present a protocol for using PSS when scanning specimens with micro-MRI. We describe steps for preparing samples and potato starch with phosphate-buffered saline. We then detail steps for specimen immersion and micro-MRI scanning. This protocol will enable micro-MRI of not only embryos but also other specimens, such as insects.For complete details on the use and execution of this protocol, please refer to Tsurugizawa et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Foxg1 Coordinates the Switch from Nonradially to Radially Migrating Glutamatergic Subtypes in the Neocortex through Spatiotemporal Repression

The specification of neuronal subtypes in the cerebral cortex proceeds in a temporal manner; however, the regulation of the transitions between the sequentially generated subtypes is poorly understood. Here, we report that the forkhead box transcription factor Foxg1 coordinates the production of neocortical projection neurons through the global repression of a default gene program. The delayed activation of Foxg1 was necessary and sufficient to induce deep-layer neurogenesis, followed by a sequential wave of upper-layer neurogenesis. A genome-wide analysis revealed that Foxg1 binds to mammalian-specific noncoding sequences to repress over 12 transcription factors expressed in early progenitors, including Ebf2/3, Dmrt3, Dmrta1, and Eya2. These findings reveal an unexpected prolonged competence of progenitors to initiate corticogenesis at a progressed stage during development and identify Foxg1 as a critical initiator of neocorticogenesis through spatiotemporal repression, a system that balances the production of nonradially and radially migrating glutamatergic subtypes during mammalian cortical expansion

Dual-color Three-photon Microscopy for Deep Imaging of Neural Tissue (Poster)

International audienceWe demonstrate dual-color three-photon imaging of red and green fluorescent proteins in neural tissue using a novel ultrafast laser design providing simultaneous excitation at 1300 and 1700 nm

Dual-color deep-tissue three-photon microscopy with a multiband infrared laser source

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Direct Readout of Neural Stem Cell Transgenesis with an Integration-Coupled Gene Expression Switch

International audienceStable genomic integration of exogenous transgenes is essential in neurodevelopmental and stem cell studies. Despite tools driving increasingly efficient genomic insertion with DNA vectors, transgenesis remains fundamentally hindered by the impossibility of distinguishing integrated from episomal transgenes. Here, we introduce an integration-coupled On genetic switch, iOn, which triggers gene expression upon incorporation into the host genome through transposition, thus enabling rapid and accurate identification of integration events following transfection with naked plasmids. In vitro, iOn permits rapid drug-free stable transgenesis of mouse and human pluripotent stem cells with multiple vectors. In vivo, we demonstrate faithful cell lineage tracing, assessment of regulatory elements, and mosaic analysis of gene function in somatic transgenesis experiments that reveal neural progenitor potentialities and interaction. These results establish iOn as a universally applicable strategy to accelerate and simplify genetic engineering in cultured systems and model organisms by conditioning transgene activation to genomic integration