Abstract
Mural cells (smooth muscle cells and pericytes) are integral components of brain blood vessels that play important roles in vascular formation, blood–brain barrier maintenance, and regulation of regional cerebral blood flow (rCBF). These cells are implicated in conditions ranging from developmental vascular disorders to age-related neurodegenerative diseases. Here we present complementary tools for cell labeling with transgenic mice and organic dyes that allow high-resolution intravital imaging of the different mural cell subtypes. We also provide detailed methodologies for imaging of spontaneous and neural activity-evoked calcium transients in mural cells. In addition, we describe strategies for single- and two-photon optogenetics that allow manipulation of the activity of individual and small clusters of mural cells. Together with measurements of diameter and flow in individual brain microvessels, calcium imaging and optogenetics allow the investigation of pericyte and smooth muscle cell physiology and their role in regulating rCBF. We also demonstrate the utility of these tools to investigate mural cells in the context of Alzheimer’s disease and cerebral ischemia mouse models. Thus, these methods can be used to reveal the functional and structural heterogeneity of mural cells in vivo, and allow detailed cellular studies of the normal function and pathophysiology of mural cells in a variety of disease models. The implementation of this protocol can take from several hours to days depending on the intended applications.
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Data availability
The mouse lines described in this protocol can be obtained from JAX and the rest upon request from the developer (see the ‘Animals’ section under ‘Materials’). All the images presented in the figures are derived from raw data with minor contrast modifications. For the calcium time-lapse imaging, the raw movies can be obtained from supplementary data in ref. 2. All other data are available upon request.
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Acknowledgements
This work was supported by the following grants from the National Institutes of Health: R01-HL106815, R01-NS089734, R21-NS087511, R21-NS088411, and R21-AG048181 to J.G., F32-NS090820 and R00‐NS099469 to R.A.H., and R01-NS111980-02 to A.B., and the American Heart Association: 17POST33670779 to K.M.
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All authors contributed to experimental work shown in this protocol. L.T. and J.G. wrote the protocol; all authors contributed to editing the manuscript. J.G. supervised the projects.
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Key references using this protocol:
Hill, R. A. et al. Neuron 87, 95−110 (2015): https://doi.org/10.1016/j.neuron.2015.06.001
Damisah, E. C. et al. Nat. Neurosci. 20, 1023−1032 (2017): https://doi.org/10.1038/nn.4564
Lacar, B. et al. J. Neurosci. 32, 16435−16448 (2012): https://doi.org/10.1523/JNEUROSCI.1457-12.2012
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Tong, L., Hill, R.A., Damisah, E.C. et al. Imaging and optogenetic modulation of vascular mural cells in the live brain. Nat Protoc 16, 472–496 (2021). https://doi.org/10.1038/s41596-020-00425-w
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DOI: https://doi.org/10.1038/s41596-020-00425-w
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