The use of fluorescent chemical indicator dyes enables the dynamic and quantitative imaging of intracellular sodium concentrations and activity-related sodium transients in astrocytes.
Here we describe different approaches for the loading of cellular networks or single astrocytes with sodium-sensitive indicators in brain tissue. Fluorescence signals can then be detected and analyzed with conventional camera-based, wide-field imaging or by employing high-resolution multi-photon microscopy. We furthermore explain strategies for the induction of local and global sodium transients in astrocytes. Finally, we illustrate how fluorescence signals derived from such imaging experiments can be converted into absolute changes of sodium concentration in astrocytes based on an in situ calibration procedure.
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
The authors wish to thank Simone Durry and Claudia Rodrigo for expert technical support and Joel Nelson for comments on the manuscript. The studies in the authors laboratory this manuscript were supported by grants from the German Research Foundation (DFG) to C.R.R (Special Priority Programme “Glial Heterogeneity” (SPP 1757), Ro 2327/8-1, 2).
Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450Google Scholar
Cornell-Bell AH, Finkbeiner SM, Cooper MS et al (1990) Glutamate induces calcium waves in cultured astrocytes: long-range glial signaling. Science 247:470–473CrossRefGoogle Scholar
Minta A, Tsien RY (1989) Fluorescent indicators for cytosolic sodium. J Biol Chem 264:19449–19457PubMedGoogle Scholar
Lamy CM, Chatton JY (2011) Optical probing of sodium dynamics in neurons and astrocytes. NeuroImage 58:572–578CrossRefGoogle Scholar
Bindocci E, Savtchouk I, Liaudet N et al (2017) Three-dimensional Ca2+ imaging advances understanding of astrocyte biology. Science 356(6339)CrossRefGoogle Scholar
Mondragao MA, Schmidt H, Kleinhans C et al (2016) Extrusion versus diffusion: mechanisms for recovery from sodium loads in mouse CA1 pyramidal neurons. J Physiol 594:5507–5527CrossRefGoogle Scholar
Kafitz KW, Meier SD, Stephan J et al (2008) Developmental profile and properties of sulforhodamine 101-labeled glial cells in acute brain slices of rat hippocampus. J Neurosci Methods 169:84–92CrossRefGoogle Scholar
Stosiek C, Garaschuk O, Holthoff K et al (2003) In vivo two-photon calcium imaging of neuronal networks. Proc Natl Acad Sci U S A 100:7319–7324CrossRefGoogle Scholar
Langer J, Gerkau NJ, Derouiche A et al (2017) Rapid sodium signaling couples glutamate uptake to breakdown of ATP in perivascular astrocyte endfeet. Glia 65:293–308CrossRefGoogle Scholar
Langer J, Rose CR (2009) Synaptically-induced sodium signals in hippocampal astrocytes in situ. J Physiol 587:5859–5877CrossRefGoogle Scholar
Langer J, Stephan J, Theis M et al (2012) Gap junctions mediate intercellular spread of sodium between hippocampal astrocytes in situ. Glia 60:239–252CrossRefGoogle Scholar
Gerkau NJ, Rakers C, Durry S et al (2017) Reverse NCX attenuates cellular sodium loading in metabolically compromised cortex. Cereb Cortex 9:1–7Google Scholar
Kleinhans C, Kafitz KW, Rose CR (2014) Multi-photon intracellular sodium imaging combined with UV-mediated focal uncaging of glutamate in CA1 pyramidal neurons. J Vis Exp. https://doi.org/10.3791/52038
Karus C, Gerkau NJ, Rose CR (2017) Differential contribution of GLAST and GLT-1 to network sodium signaling in the early postnatal hippocampus. Opera Medica et Physiologica 3:71–83Google Scholar
Rose CR, Ransom BR (1996) Intracellular sodium homeostasis in rat hippocampal astrocytes. J Physiol 491:291–305CrossRefGoogle Scholar
Meier SD, Kovalchuk Y, Rose CR (2006) Properties of the new fluorescent Na+ indicator CoroNa green: comparison with SBFI and confocal Na+ imaging. J Neurosci Methods 155:251–259CrossRefGoogle Scholar
Karus C, Mondragao MA, Ziemens D et al (2015) Astrocytes restrict discharge duration and neuronal sodium loads during recurrent network activity. Glia 63:936–957CrossRefGoogle Scholar