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Astrocyte

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Baljit S. Khakh – 1st expert on this subject based on the ideXlab platform

  • visualizing Astrocyte morphology using lucifer yellow iontophoresis
    Journal of Visualized Experiments, 2019
    Co-Authors: Stefanie Moye, Blanca Diazcastro, Mohitkumar R Gangwani, Baljit S. Khakh

    Abstract:

    Astrocytes are essential components of neural circuits. They tile the entire central nervous system (CNS) and are involved in a variety of functions, which include neurotransmitter clearance, ion regulation, synaptic modulation, metabolic support to neurons, and blood flow regulation. Astrocytes are complex cells that have a soma, several major branches, and numerous fine processes that contact diverse cellular elements within the neuropil. In order to assess the morphology of Astrocytes, it is necessary to have a reliable and reproducible method to visualize their structure. We report a reliable protocol to perform intracellular iontophoresis of Astrocytes using fluorescent Lucifer yellow (LY) dye in lightly fixed brain tissue from adult mice. This method has several features that are useful to characterize Astrocyte morphology. It allows for three-dimensional reconstruction of individual Astrocytes, which is useful to perform morphological analyses on different aspects of their structure. Immunohistochemistry together with LY iontophoresis can also be utilized to understand the interaction of Astrocytes with different components of nervous system and to evaluate the expression of proteins within the labelled Astrocytes. This protocol can be implemented in a variety of mouse models of CNS disorders to rigorously examine Astrocyte morphology with light microscopy. LY iontophoresis provides an experimental approach to evaluate Astrocyte structure, especially in the context of injury or disease where these cells are proposed to undergo significant morphological changes.

  • reducing Astrocyte calcium signaling in vivo alters striatal microcircuits and causes repetitive behavior
    Neuron, 2018
    Co-Authors: Xinzhu Yu, Giovanni Coppola, Peyman Golshani, Anna M W Taylor, Jun Nagai, Christopher J Evans, Baljit S. Khakh

    Abstract:

    Summary Astrocytes tile the central nervous system, but their functions in neural microcircuits in vivo and their roles in mammalian behavior remain incompletely defined. We used two-photon laser scanning microscopy, electrophysiology, MINIscopes, RNA-seq, and a genetic approach to explore the effects of reduced striatal Astrocyte Ca2+ signaling in vivo. In wild-type mice, reducing striatal Astrocyte Ca2+-dependent signaling increased repetitive self-grooming behaviors by altering medium spiny neuron (MSN) activity. The mechanism involved Astrocyte-mediated neuromodulation facilitated by ambient GABA and was corrected by blocking Astrocyte GABA transporter 3 (GAT-3). Furthermore, in a mouse model of Huntington’s disease, dysregulation of GABA and Astrocyte Ca2+ signaling accompanied excessive self-grooming, which was relieved by blocking GAT-3. Assessments with RNA-seq revealed Astrocyte genes and pathways regulated by Ca2+ signaling in a cell-autonomous and non-cell-autonomous manner, including Rab11a, a regulator of GAT-3 functional expression. Thus, striatal Astrocytes contribute to neuromodulation controlling mouse obsessive-compulsive-like behavior.

  • new transgenic mouse lines for selectively targeting Astrocytes and studying calcium signals in Astrocyte processes in situ and in vivo
    Neuron, 2016
    Co-Authors: Rahul Srinivasan, Giovanni Coppola, Tsaiyi Lu, Hua Chai, Ji Xu, Ben S Huang, Peyman Golshani, Baljit S. Khakh

    Abstract:

    Summary Astrocytes exist throughout the nervous system and are proposed to affect neural circuits and behavior. However, studying Astrocytes has proven difficult because of the lack of tools permitting Astrocyte-selective genetic manipulations. Here, we report the generation of Aldh1l1 -Cre/ERT2 transgenic mice to selectively target Astrocytes in vivo. We characterized Aldh1l1 -Cre/ERT2 mice using imaging, immunohistochemistry, AAV-FLEX-GFP microinjections, and crosses to RiboTag, Ai95, and new Cre-dependent membrane-tethered Lck-GCaMP6f knockin mice that we also generated. Two to three weeks after tamoxifen induction, Aldh1l1 -Cre/ERT2 selectively targeted essentially all adult (P80) brain Astrocytes with no detectable neuronal contamination, resulting in expression of cytosolic and Lck-GCaMP6f, and permitting subcellular Astrocyte calcium imaging during startle responses in vivo. Crosses with RiboTag mice allowed sequencing of actively translated mRNAs and determination of the adult cortical Astrocyte transcriptome. Thus, we provide well-characterized, easy-to-use resources with which to selectively study Astrocytes in situ and in vivo in multiple experimental scenarios.

Michael V. Sofroniew – 2nd expert on this subject based on the ideXlab platform

  • Astrocyte scar formation aids central nervous system axon regeneration
    Nature, 2016
    Co-Authors: Joshua E. Burda, Yan Ao, Riki Kawaguchi, Giovanni Coppola, Baljit S. Khakh, Timothy J Deming, Mark Anderson, Timothy M Oshea, Michael V. Sofroniew

    Abstract:

    Transected axons fail to regrow in the mature central nervous system. Astrocytic scars are widely regarded as causal in this failure. Here, using three genetically targeted loss-of-function manipulations in adult mice, we show that preventing Astrocyte scar formation, attenuating scar-forming Astrocytes, or ablating chronic astrocytic scars all failed to result in spontaneous regrowth of transected corticospinal, sensory or serotonergic axons through severe spinal cord injury (SCI) lesions. By contrast, sustained local delivery via hydrogel depots of required axon-specific growth factors not present in SCI lesions, plus growth-activating priming injuries, stimulated robust, laminin-dependent sensory axon regrowth past scar-forming Astrocytes and inhibitory molecules in SCI lesions. Preventing astrocytic scar formation significantly reduced this stimulated axon regrowth. RNA sequencing revealed that Astrocytes and non-Astrocyte cells in SCI lesions express multiple axon-growth-supporting molecules. Our findings show that contrary to the prevailing dogma, Astrocyte scar formation aids rather than prevents central nervous system axon regeneration.

  • Astrocyte scar formation AIDS central nervous system axon regeneration
    Nature, 2016
    Co-Authors: Mark A. Anderson, Joshua E. Burda, Yilong Ren, Yan Ao, Timothy M Shea, Riki Kawaguchi, Giovanni Coppola, Baljit S. Khakh, Timothy J Deming, Michael V. Sofroniew

    Abstract:

    ARTICLE doi:10.1038/nature17623 Astrocyte scar formation aids central nervous system axon regeneration Mark A. Anderson1*†, Joshua E. Burda1*, Yilong Ren1†, Yan Ao1, Timothy M. O’Shea1, Riki Kawaguchi2, Giovanni Coppola2, Baljit S. Khakh3, Timothy J. Deming4 & Michael V. Sofroniew1 Transected axons fail to regrow in the mature central nervous system. Astrocytic scars are widely regarded as causal in this failure. Here, using three genetically targeted loss-of-function manipulations in adult mice, we show that preventing Astrocyte scar formation, attenuating scar-forming Astrocytes, or ablating chronic astrocytic scars all failed to result in spontaneous regrowth of transected corticospinal, sensory or serotonergic axons through severe spinal cord injury (SCI) lesions. By contrast, sustained local delivery via hydrogel depots of required axon-specific growth factors not present in SCI lesions, plus growth-activating priming injuries, stimulated robust, laminin-dependent sensory axon regrowth past scar-forming Astrocytes and inhibitory molecules in SCI lesions. Preventing astrocytic scar formation significantly reduced this stimulated axon regrowth. RNA sequencing revealed that Astrocytes and non-Astrocyte cells in SCI lesions express multiple axon-growth-supporting molecules. Our findings show that contrary to the prevailing dogma, Astrocyte scar formation aids rather than prevents central nervous system axon regeneration.

Giovanni Coppola – 3rd expert on this subject based on the ideXlab platform

  • reducing Astrocyte calcium signaling in vivo alters striatal microcircuits and causes repetitive behavior
    Neuron, 2018
    Co-Authors: Xinzhu Yu, Giovanni Coppola, Peyman Golshani, Anna M W Taylor, Jun Nagai, Christopher J Evans, Baljit S. Khakh

    Abstract:

    Summary Astrocytes tile the central nervous system, but their functions in neural microcircuits in vivo and their roles in mammalian behavior remain incompletely defined. We used two-photon laser scanning microscopy, electrophysiology, MINIscopes, RNA-seq, and a genetic approach to explore the effects of reduced striatal Astrocyte Ca2+ signaling in vivo. In wild-type mice, reducing striatal Astrocyte Ca2+-dependent signaling increased repetitive self-grooming behaviors by altering medium spiny neuron (MSN) activity. The mechanism involved Astrocyte-mediated neuromodulation facilitated by ambient GABA and was corrected by blocking Astrocyte GABA transporter 3 (GAT-3). Furthermore, in a mouse model of Huntington’s disease, dysregulation of GABA and Astrocyte Ca2+ signaling accompanied excessive self-grooming, which was relieved by blocking GAT-3. Assessments with RNA-seq revealed Astrocyte genes and pathways regulated by Ca2+ signaling in a cell-autonomous and non-cell-autonomous manner, including Rab11a, a regulator of GAT-3 functional expression. Thus, striatal Astrocytes contribute to neuromodulation controlling mouse obsessive-compulsive-like behavior.

  • new transgenic mouse lines for selectively targeting Astrocytes and studying calcium signals in Astrocyte processes in situ and in vivo
    Neuron, 2016
    Co-Authors: Rahul Srinivasan, Giovanni Coppola, Tsaiyi Lu, Hua Chai, Ji Xu, Ben S Huang, Peyman Golshani, Baljit S. Khakh

    Abstract:

    Summary Astrocytes exist throughout the nervous system and are proposed to affect neural circuits and behavior. However, studying Astrocytes has proven difficult because of the lack of tools permitting Astrocyte-selective genetic manipulations. Here, we report the generation of Aldh1l1 -Cre/ERT2 transgenic mice to selectively target Astrocytes in vivo. We characterized Aldh1l1 -Cre/ERT2 mice using imaging, immunohistochemistry, AAV-FLEX-GFP microinjections, and crosses to RiboTag, Ai95, and new Cre-dependent membrane-tethered Lck-GCaMP6f knockin mice that we also generated. Two to three weeks after tamoxifen induction, Aldh1l1 -Cre/ERT2 selectively targeted essentially all adult (P80) brain Astrocytes with no detectable neuronal contamination, resulting in expression of cytosolic and Lck-GCaMP6f, and permitting subcellular Astrocyte calcium imaging during startle responses in vivo. Crosses with RiboTag mice allowed sequencing of actively translated mRNAs and determination of the adult cortical Astrocyte transcriptome. Thus, we provide well-characterized, easy-to-use resources with which to selectively study Astrocytes in situ and in vivo in multiple experimental scenarios.

  • Astrocyte scar formation aids central nervous system axon regeneration
    Nature, 2016
    Co-Authors: Joshua E. Burda, Yan Ao, Riki Kawaguchi, Giovanni Coppola, Baljit S. Khakh, Timothy J Deming, Mark Anderson, Timothy M Oshea, Michael V. Sofroniew

    Abstract:

    Transected axons fail to regrow in the mature central nervous system. Astrocytic scars are widely regarded as causal in this failure. Here, using three genetically targeted loss-of-function manipulations in adult mice, we show that preventing Astrocyte scar formation, attenuating scar-forming Astrocytes, or ablating chronic astrocytic scars all failed to result in spontaneous regrowth of transected corticospinal, sensory or serotonergic axons through severe spinal cord injury (SCI) lesions. By contrast, sustained local delivery via hydrogel depots of required axon-specific growth factors not present in SCI lesions, plus growth-activating priming injuries, stimulated robust, laminin-dependent sensory axon regrowth past scar-forming Astrocytes and inhibitory molecules in SCI lesions. Preventing astrocytic scar formation significantly reduced this stimulated axon regrowth. RNA sequencing revealed that Astrocytes and non-Astrocyte cells in SCI lesions express multiple axon-growth-supporting molecules. Our findings show that contrary to the prevailing dogma, Astrocyte scar formation aids rather than prevents central nervous system axon regeneration.