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Intracellular Ca(2+) transients are considered a primary signal by which astrocytes interact with neurons and blood vessels. With existing commonly used methods, Ca(2+) has been studied only within astrocyte somata and thick branches, leaving the distal fine branchlets and endfeet that are most proximate to neuronal synapses and blood vessels largely unexplored. Here, using cytosolic and membrane-tethered forms of genetically encoded Ca(2+) indicators (GECIs; cyto-GCaMP3 and Lck-GCaMP3), we report well-characterized approaches that overcome these limitations. We used in vivo microinjections of adeno-associated viruses to express GECIs in astrocytes and studied Ca(2+) signals in acute hippocampal slices in vitro from adult mice (aged ∼P80) two weeks after infection. Our data reveal a sparkling panorama of unexpectedly numerous, frequent, equivalently scaled, and highly localized Ca(2+) microdomains within entire astrocyte territories in situ within acute hippocampal slices, consistent with the distribution of perisynaptic branchlets described using electron microscopy. Signals from endfeet were revealed with particular clarity. The tools and experimental approaches we describe in detail allow for the systematic study of Ca(2+) signals within entire astrocytes, including within fine perisynaptic branchlets and vessel-associated endfeet, permitting rigorous evaluation of how astrocytes contribute to brain function.
Pubmed ID: 23589582 RIS Download
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Non-profit plasmid repository dedicated to helping scientists around the world share high-quality plasmids. Facilitates archiving and distributing DNA-based research reagents and associated data to scientists worldwide. Repository contains over 65,000 plasmids, including special collections on CRISPR, fluorescent proteins, and ready-to-use viral preparations. There is no cost for scientists to deposit plasmids, which saves time and money associated with shipping plasmids themselves. All plasmids are fully sequenced for validation and sequencing data is openly available. We handle the appropriate Material Transfer Agreements (MTA) with institutions, facilitating open exchange and offering intellectual property and liability protection for depositing scientists. Furthermore, we curate free educational resources for the scientific community including a blog, eBooks, video protocols, and detailed molecular biology resources.
View all literature mentionsBiomedical technology research center that develops computer-aided, advanced microscopy for the acquisition of structural and functional data in the dimensional range of 1 nm to 100 um, a range encompassing macromolecules, subcellular structures and cells. Novel specimen-staining methods, imaging instrumentsincluding intermediate high-voltage transmission electron microscopes (IVEMs) and high-speed, large-format laser-scanning light microscopesand computational capabilities are available for addressing mesoscale biological microscopy of proteins and macromolecular complexes in their cellular and tissue environments. These technologies are developed to bridge understanding of biological systems between the gross anatomical and molecular scales and to make these technologies broadly available to biomedical researchers. NCMIR provides expertise, infrastructure, technological development, and an environment in which new information about the 3D ultrastructure of tissues, cells, and macromolecular complexes may be accurately and easily obtained and analyzed. NCMIR fulfills its mission through technology development, collaboration, service, training, and dissemination. It aims to develop preparative methods and analytical approaches to 3D microscopy applicable to neurobiology and cell biology, incorporating equipment and implementing software that expand the analysis of 3D structure. The core research activities in the areas of specimen development, instrument development, and software infrastructures maximize the advantages of higher voltage electron microscopy and correlated light microscopies to make ambitious imaging studies across scales routine, and to facilitate the use of resources by biomedical researchers. NCMIR actively recruits outside users who will not only make use of these resources, but who also will drive technology development and receive training.
View all literature mentionsMus musculus with name C57BL/6J from IMSR.
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