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A novel method for culturing stellate astrocytes reveals spatially distinct Ca2+ signaling and vesicle recycling in astrocytic processes
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jgp_201611607.pdf | Published version | 4.53 MB | Adobe PDF | View/Open |
Title: | A novel method for culturing stellate astrocytes reveals spatially distinct Ca2+ signaling and vesicle recycling in astrocytic processes |
Authors: | Wolfes, AC Ahmed, S Awasthi, A Stahlberg, MA Rajput, A Magruder, DS Bonn, S Dean, C |
Item Type: | Journal Article |
Abstract: | Interactions between astrocytes and neurons rely on the release and uptake of glial and neuronal molecules. But whether astrocytic vesicles exist and exocytose in a regulated or constitutive fashion is under debate. The majority of studies have relied on indirect methods or on astrocyte cultures that do not resemble stellate astrocytes found in vivo. Here, to investigate vesicle-associated proteins and exocytosis in stellate astrocytes specifically, we developed a simple, fast, and economical method for growing stellate astrocyte monocultures. This method is superior to other monocultures in terms of astrocyte morphology, mRNA expression profile, protein expression of cell maturity markers, and Ca2+ fluctuations: In astrocytes transduced with GFAP promoter–driven Lck-GCaMP3, spontaneous Ca2+ events in distinct domains (somata, branchlets, and microdomains) are similar to those in astrocytes co-cultured with other glia and neurons but unlike Ca2+ events in astrocytes prepared using the McCarthy and de Vellis (MD) method and immunopanned (IP) astrocytes. We identify two distinct populations of constitutively recycling vesicles (harboring either VAMP2 or SYT7) specifically in branchlets of cultured stellate astrocytes. SYT7 is developmentally regulated in these astrocytes, and we observe significantly fewer synapses in wild-type mouse neurons grown on Syt7−/− astrocytes. SYT7 may thus be involved in trafficking or releasing synaptogenic factors. In summary, our novel method yields stellate astrocyte monocultures that can be used to study Ca2+ signaling and vesicle recycling and dynamics in astrocytic processes. |
Issue Date: | 1-Jan-2017 |
Date of Acceptance: | 11-Nov-2016 |
URI: | http://hdl.handle.net/10044/1/78673 |
DOI: | 10.1085/jgp.201611607 |
ISSN: | 0022-1295 |
Publisher: | Rockefeller University Press |
Start Page: | 149 |
End Page: | 170 |
Journal / Book Title: | Journal of General Physiology |
Volume: | 149 |
Issue: | 1 |
Copyright Statement: | © 2017 Wolfes et al. This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). |
Keywords: | Physiology 0606 Physiology 1116 Medical Physiology |
Publication Status: | Published |
Online Publication Date: | 2016-12-01 |
Appears in Collections: | Department of Brain Sciences |