Abstract
Astrocytes are glial cells carrying out complex homeostatic functions in the healthy and diseased central nervous system (CNS). It has so far been impossible to reliably culture adult astrocytes and the results of studies on astrocytes outside of their normal environment are challenging to interpret. Consequently, most culture studies use astrocytes isolated from postnatal rodents. Yet cultured astrocytes do not display their complex three-dimensional in vivo morphology, and transcriptomes of cultured astrocytes vary significantly from those of acutely isolated astrocytes (Cahoy et al., J Neurosci 28:264–278, 2008). Astrocyte isolation for culture experiments, and the cutting of acute brain slices, induces astrocyte reactivity similar to a severe acute injury. In response to CNS injury, such as moderate or severe focal traumatic brain injury (TBI), astrocytes can change in cell number, physiological state, gene and protein expression, secretome, and morphology, in a process termed reactive astrogliosis. This makes the use of methods that inherently induce astrogliosis (e.g., dissociation of brain tissue for culture or sectioning of brains for acute brain slices) challenging, especially when conditions are studied that present with changes in astrocyte function that are milder and/or of a different nature.
In this methods chapter, we will describe a technical approach that allows one to study astrocytes in the intact brain using two-photon in vivo imaging. We will use mild TBI as an example of how to use this approach to compare astrocyte function in the same animal before and after an injury.
Here we describe the use of a noninvasive label-free method (Choi et al., J Biomed Opt 16:075003, 2011) to increase astrocyte Ca2+ using optical femtosecond pulsed laser activation. We will provide systematic instruction of the surgical technique, which when done properly, allows in vivo astrocyte imaging in the same experimental animal before the injury as well as over the course of days, weeks, and even months after injury. We will also elaborate on challenges in astrocytic Ca2+ imaging and how different image acquisition settings can affect the readout of astrocyte Ca2+ oscillations.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Cahoy JD, Emery B, Kaushal A, Foo LC, Zamanian JL, Christopherson KS, Xing Y, Lubischer JL, Krieg PA, Krupenko SA, Thompson WJ, Barres BA (2008) A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J Neurosci 28(1):264–278
Choi M, Ku T, Choi K, Choi C, Yoon J (2011) Label-free optical activation of astrocyte in vivo. J Biomed Opt 16(7):075003
MacVicar BA, Newman EA (2015) Astrocyte regulation of blood flow in the brain. Cold Spring Harb Perspect Biol 7(5):a020388
Bazargani N, Attwell D (2016) Astrocyte calcium signaling: the third wave. Nat Neurosci 19(2):182–189
Takano T, Han X, Deane R, Zlokovic B, Nedergaard M (2007) Two-photon imaging of astrocytic Ca2+ signaling and the microvasculature in experimental mice models of Alzheimer's disease. Ann N Y Acad Sci 1097:40–50
Smith N, Kress B, Lu Y, Chandler-Militello D, Benraiss A, Nedergaard M (2018) Fluorescent Ca2+ indicators directly inhibit the Na,K-ATPase and disrupt cellular functions. Sci Signal 11(515):eaal2039
Gee JM, Smith NA, Fernandez FR, Economo MN, Brunert D, Rothermel M, Morris SC, Talbot A, Palumbos S, Ichida JM, Shepherd JD, West PJ, Wachowiak M, Capecchi MR, Wilcox KS, White JA, Tvrdik P (2014) Imaging activity in neurons and glia with a Polr2a-based and Cre-dependent GCaMP5G-IRES-tdTomato reporter mouse. Neuron 83(5):1058–1072
Shih AY, Mateo C, Drew PJ, Tsai PS, Kleinfeld D (2012) A polished and reinforced thinned-skull window for long-term imaging of the mouse brain. J Vis Exp 61:3742
Yardeni T, Eckhaus M, Morris HD, Huizing M, Hoogstraten-Miller S (2011) Retro-orbital injections in mice. Lab Anim 40(5):155–160
Kimbrough IF, Robel S, Roberson ED, Sontheimer H (2015) Vascular amyloidosis impairs the gliovascular unit in a mouse model of Alzheimer’s disease. Brain 138(12):3716–3733
Watkins S, Robel S, Kimbrough IF, Robert SM, Ellis-Davies G, Sontheimer H (2014) Disruption of astrocyte–vascular coupling and the blood–brain barrier by invading glioma cells. Nat Commun 5:4196 https://www.nature.com/articles/ncomms5196#supplementary-information
Shimizu S (2004) CHAPTER 32 - routes of administration. In: Hedrich HJ, Bullock G (eds) The laboratory mouse. Academic Press, London, pp 527–542
Science Education Database JoVE (2018) Lab Animal Research Compound Administration IV JoVE
Shen Z, Lu Z, Chhatbar PY, O'Herron P, Kara P (2012) An artery-specific fluorescent dye for studying neurovascular coupling. Nat Methods 9(3):273–276
Fiacco TA, Agulhon C, McCarthy KD (2009) Sorting out astrocyte physiology from pharmacology. Annu Rev Pharmacol Toxicol 49:151–174
Stobart JL, Ferrari KD, Barrett MJP, Gluck C, Stobart MJ, Zuend M, Weber B (2018) Cortical circuit activity evokes rapid astrocyte calcium signals on a similar timescale to neurons. Neuron 98(4):726–735.e724
Mulligan SJ, MacVicar BA (2004) Calcium transients in astrocyte endfeet cause cerebrovascular constrictions. Nature 431(7005):195–199
Acknowledgments
This work was supported by the National Institute of Neurological Disorders and Stroke at the National Institutes of Health (grant number R01NS105807).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
1 Electronic Supplementary Materials
3D reconstruction of astrocytes and TRITC-Dextran labeled vasculature before TBI. Imaging laser wavelength 870 nm. Zoom factor 1.5 (MP4 4684 kb)
3D reconstruction of astrocytes and TRITC-Dextran labeled vasculature 1 day after TBI. Imaging laser wavelength 870 nm. Zoom factor 1.5 (MP4 4583 kb)
3D reconstruction of astrocytes and TRITC-Dextran labeled vasculature 3 days after TBI. Imaging laser wavelength 870 nm. Zoom factor 1.5 (MP4 4642 kb)
Astrocyte endfeet laser stimulation cause Ca2+ wave generation and constriction of the arteriole. Imaging wavelength 870 nm 10% laser power. Stimulation laser power 30%, duration 100 ms. Zoom factor 1.3. Red line indicates the laser stimulation ROI (MP4 6903 kb)
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Shandra, O., Robel, S. (2019). Imaging and Manipulating Astrocyte Function In Vivo in the Context of CNS Injury. In: Di Benedetto, B. (eds) Astrocytes. Methods in Molecular Biology, vol 1938. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9068-9_16
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9068-9_16
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-9067-2
Online ISBN: 978-1-4939-9068-9
eBook Packages: Springer Protocols