Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Imaging and optogenetic modulation of vascular mural cells in the live brain

Nature Protocols volume 16, pages 472–496 (2021)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1
Fig. 2: Generation of triple transgenic mice for mural cell labeling and identification.
Fig. 3: Distinct morphology of mural cells revealed by sparse labeling.
Fig. 4: NeuroTrace 500/525 and TO-PRO-3 exclusively label capillary pericytes in live mouse brain.
Fig. 5: Imaging the cortical microvasculature during transient cerebral ischemia and reperfusion.
Fig. 6: In vivo calcium imaging of mural cells.
Fig. 7: In vivo single-photon optogenetic stimulation of ChR2 or ArchT-expressing mural cells.
Fig. 8: In vivo two-photon optogenetic stimulation of ChR2-expressing mural cells.

Similar content being viewed by others

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.

References

  1. Armulik, A., Genove, G. & Betsholtz, C. Pericytes: developmental, physiological, and pathological perspectives, problems, and promises. Dev. Cell 21, 193–215 (2011).

    Article  CAS  PubMed  Google Scholar 

  2. Hill, R. A. et al. Regional blood flow in the normal and ischemic brain is controlled by arteriolar smooth muscle cell contractility and not by capillary pericytes. Neuron 87, 95–110 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Gerhardt, H. & Betsholtz, C. Endothelial–pericyte interactions in angiogenesis. Cell Tissue Res. 314, 15–23 (2003).

    Article  PubMed  Google Scholar 

  4. Armulik, A., Abramsson, A. & Betsholtz, C. Endothelial/pericyte interactions. Circ. Res. 97, 512–523 (2005).

    Article  CAS  PubMed  Google Scholar 

  5. Gaengel, K., Genove, G., Armulik, A. & Betsholtz, C. Endothelial–mural cell signaling in vascular development and angiogenesis. Arterioscler. Thromb. Vasc. Biol. 29, 630–638 (2009).

    Article  CAS  PubMed  Google Scholar 

  6. Armulik, A. et al. Pericytes regulate the blood–brain barrier. Nature 468, 557–561 (2010).

    Article  CAS  PubMed  Google Scholar 

  7. Winkler, E. A., Bell, R. D. & Zlokovic, B. V. Central nervous system pericytes in health and disease. Nat. Neurosci. 14, 1398–1405 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Farkas, E. & Luiten, P. G. Cerebral microvascular pathology in aging and Alzheimer’s disease. Prog. Neurobiol. 64, 575–611 (2001).

    Article  CAS  PubMed  Google Scholar 

  9. Bell, R. D. et al. Pericytes control key neurovascular functions and neuronal phenotype in the adult brain and during brain aging. Neuron 68, 409–427 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Sagare, A. P. et al. Pericyte loss influences Alzheimer-like neurodegeneration in mice. Nat. Commun. 4, 2932 (2013).

    Article  PubMed  CAS  Google Scholar 

  11. He, L. et al. Analysis of the brain mural cell transcriptome. Sci. Rep. 6, 35108 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. He, L. et al. Single-cell RNA sequencing of mouse brain and lung vascular and vessel-associated cell types. Sci. Data 5, 180160 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Smyth, L. C. D. et al. Markers for human brain pericytes and smooth muscle cells. J. Chem. Neuroanat. 92, 48–60 (2018).

    Article  CAS  PubMed  Google Scholar 

  14. Vanlandewijck, M. et al. A molecular atlas of cell types and zonation in the brain vasculature. Nature 554, 475–480 (2018).

    Article  CAS  PubMed  Google Scholar 

  15. Dieguez-Hurtado, R. et al. Loss of the transcription factor RBPJ induces disease-promoting properties in brain pericytes. Nat. Commun. 10, 2817 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Dubrac, A. et al. NCK-dependent pericyte migration promotes pathological neovascularization in ischemic retinopathy. Nat. Commun. 9, 3463 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Jenny Zhou, H. et al. Endothelial exocytosis of angiopoietin-2 resulting from CCM3 deficiency contributes to cerebral cavernous malformation. Nat. Med. 22, 1033–1042 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Hall, C. N. et al. Capillary pericytes regulate cerebral blood flow in health and disease. Nature 508, 55–60 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Raichle, M. E. & Gusnard, D. A. Appraising the brain’s energy budget. Proc. Natl Acad. Sci. USA 99, 10237–10239 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Iadecola, C. The pathobiology of vascular dementia. Neuron 80, 844–866 (2013).

    Article  CAS  PubMed  Google Scholar 

  21. Girouard, H. & Iadecola, C. Neurovascular coupling in the normal brain and in hypertension, stroke, and Alzheimer disease. J. Appl. Physiol. 100, 328–335 (2006).

    Article  CAS  PubMed  Google Scholar 

  22. Iadecola, C. The neurovascular unit coming of age: a journey through neurovascular coupling in health and disease. Neuron 96, 17–42 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Busse, R. [Contraction and relaxation mechanisms of the smooth vascular musculature]. Z Kardiol. 73, 63–69 (1984).

    CAS  PubMed  Google Scholar 

  24. Peppiatt, C. M., Howarth, C., Mobbs, P. & Attwell, D. Bidirectional control of CNS capillary diameter by pericytes. Nature 443, 700–704 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Fernandez-Klett, F. & Priller, J. Diverse functions of pericytes in cerebral blood flow regulation and ischemia. J. Cereb. Blood Flow Metab. 35, 883–887 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Wei, H. S. et al. Erythrocytes are oxygen-sensing regulators of the cerebral microcirculation. Neuron 91, 851–862 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Fernandez-Klett, F., Offenhauser, N., Dirnagl, U., Priller, J. & Lindauer, U. Pericytes in capillaries are contractile in vivo, but arterioles mediate functional hyperemia in the mouse brain. Proc. Natl Acad. Sci. USA 107, 22290–22295 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Mishra, A. et al. Astrocytes mediate neurovascular signaling to capillary pericytes but not to arterioles. Nat. Neurosci. 19, 1619–1627 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Grutzendler, J. & Nedergaard, M. Cellular control of brain capillary blood flow: in vivo imaging veritas. Trends Neurosci. 42, 528–536 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Attwell, D., Mishra, A., Hall, C. N., O’Farrell, F. M. & Dalkara, T. What is a pericyte? J Cereb. Blood Flow Metab. 36, 451–455 (2016).

    Article  CAS  PubMed  Google Scholar 

  31. Kisler, K., Nelson, A. R., Montagne, A. & Zlokovic, B. V. Cerebral blood flow regulation and neurovascular dysfunction in Alzheimer disease. Nat. Rev. Neurosci. 18, 419–434 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Kisler, K. et al. Pericyte degeneration leads to neurovascular uncoupling and limits oxygen supply to brain. Nat. Neurosci. 20, 406–416 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Schaffer, C. B. et al. Two-photon imaging of cortical surface microvessels reveals a robust redistribution in blood flow after vascular occlusion. Plos Biol. 4, e22 (2006).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Shih, A. Y. et al. Two-photon microscopy as a tool to study blood flow and neurovascular coupling in the rodent brain. J. Cereb. Blood Flow Metab. 32, 1277–1309 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Kasischke, K. A. et al. Two-photon NADH imaging exposes boundaries of oxygen diffusion in cortical vascular supply regions. J. Cereb. Blood Flow Metab. 31, 68–81 (2011).

    Article  CAS  PubMed  Google Scholar 

  36. Tiret, P., Chaigneau, E., Lecoq, J. & Charpak, S. Two-photon imaging of capillary blood flow in olfactory bulb glomeruli. Methods Mol. Biol. 489, 81–91 (2009).

    Article  CAS  PubMed  Google Scholar 

  37. Armstrong, J. J., Larina, I. V., Dickinson, M. E., Zimmer, W. E. & Hirschi, K. K. Characterization of bacterial artificial chromosome transgenic mice expressing mCherry fluorescent protein substituted for the murine smooth muscle alpha-actin gene. Genesis 48, 457–463 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Novak, A., Guo, C., Yang, W., Nagy, A. & Lobe, C. G. Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision. Genesis 28, 147–155 (2000).

    Article  CAS  PubMed  Google Scholar 

  39. Zhu, X., Bergles, D. E. & Nishiyama, A. NG2 cells generate both oligodendrocytes and gray matter astrocytes. Development 135, 145–157 (2008).

    Article  CAS  PubMed  Google Scholar 

  40. Winkler, E. A., Bell, R. D. & Zlokovic, B. V. Pericyte-specific expression of PDGF beta receptor in mouse models with normal and deficient PDGF beta receptor signaling. Mol. Neurodegener. 5, 32 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Cuttler, A. S. et al. Characterization of Pdgfrb-Cre transgenic mice reveals reduction of ROSA26 reporter activity in remodeling arteries. Genesis 49, 673–680 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Zhu, X. et al. Age-dependent fate and lineage restriction of single NG2 cells. Development 138, 745–753 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Cuervo, H. et al. PDGFRbeta-P2A-CreER(T2) mice: a genetic tool to target pericytes in angiogenesis. Angiogenesis 20, 655–662 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Snippert, H. J. et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 143, 134–144 (2010).

    Article  CAS  PubMed  Google Scholar 

  45. Livet, J. et al. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 56–62 (2007).

    Article  CAS  PubMed  Google Scholar 

  46. Damisah, E. C., Hill, R. A., Tong, L., Murray, K. N. & Grutzendler, J. A fluoro-Nissl dye identifies pericytes as distinct vascular mural cells during in vivo brain imaging. Nat. Neurosci. 20, 1023–1032 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Lacar, B. et al. Neural progenitor cells regulate capillary blood flow in the postnatal subventricular zone. J. Neurosci. 32, 16435–16448 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Madisen, L. et al. Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance. Neuron 85, 942–958 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Tong, L. et al. Single-cell in vivo brain optogenetic stimulation by two-photon excitation fluorescence transfer. Preprint at bioRxiv https://doi.org/10.1101/2020.06.29.179077 (2020).

  50. Hamilton, N. B., Attwell, D. & Hall, C. N. Pericyte-mediated regulation of capillary diameter: a component of neurovascular coupling in health and disease. Front. Neuroenergetics https://doi.org/10.3389/fnene.2010.00005 (2010).

  51. Khennouf, L. et al. Active role of capillary pericytes during stimulation-induced activity and spreading depolarization. Brain 141, 2032–2046 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Fenno, L., Yizhar, O. & Deisseroth, K. The development and application of optogenetics. Annu. Rev. Neurosci. 34, 389–412 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Ellis-Davies, G. C. Caged compounds: photorelease technology for control of cellular chemistry and physiology. Nat. Methods 4, 619–628 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Hirase, H., Creso, J., Singleton, M., Bartho, P. & Buzsaki, G. Two-photon imaging of brain pericytes in vivo using dextran-conjugated dyes. Glia 46, 95–100 (2004).

    Article  PubMed  Google Scholar 

  55. Longden, T. A. et al. Capillary K+-sensing initiates retrograde hyperpolarization to increase local cerebral blood flow. Nat. Neurosci. 20, 717–726 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Dana, H. et al. Sensitive red protein calcium indicators for imaging neural activity. eLife https://doi.org/10.7554/eLife.12727 (2016).

  57. Lin, J. Y., Knutsen, P. M., Muller, A., Kleinfeld, D. & Tsien, R. Y. ReaChR: a red-shifted variant of channelrhodopsin enables deep transcranial optogenetic excitation. Nat. Neurosci. 16, 1499–1508 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Prakash, R. et al. Two-photon optogenetic toolbox for fast inhibition, excitation and bistable modulation. Nat. Methods 9, 1171–1179 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Dombeck, D. A., Harvey, C. D., Tian, L., Looger, L. L. & Tank, D. W. Functional imaging of hippocampal place cells at cellular resolution during virtual navigation. Nat. Neurosci. 13, 1433–1440 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Andermann, M. L. et al. Chronic cellular imaging of entire cortical columns in awake mice using microprisms. Neuron 80, 900–913 (2013).

    Article  CAS  PubMed  Google Scholar 

  61. Doronina-Amitonova, L. V. et al. Implantable fiber-optic interface for parallel multisite long-term optical dynamic brain interrogation in freely moving mice. Sci. Rep. 3, 3265 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Nikolakopoulou, A. M. et al. Pericyte loss leads to circulatory failure and pleiotrophin depletion causing neuron loss. Nat. Neurosci. 22, 1089–1098 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Holm, A., Heumann, T. & Augustin, H. G. Microvascular mural cell organotypic heterogeneity and functional plasticity. Trends Cell Biol. 28, 302–316 (2018).

    Article  PubMed  Google Scholar 

  64. Wirth, A. et al. G12-G13-LARG-mediated signaling in vascular smooth muscle is required for salt-induced hypertension. Nat. Med. 14, 64–68 (2008).

    Article  CAS  PubMed  Google Scholar 

  65. Wendling, O., Bornert, J. M., Chambon, P. & Metzger, D. Efficient temporally-controlled targeted mutagenesis in smooth muscle cells of the adult mouse. Genesis 47, 14–18 (2009).

    Article  CAS  PubMed  Google Scholar 

  66. Hartmann, D. A. et al. Pericyte structure and distribution in the cerebral cortex revealed by high-resolution imaging of transgenic mice. Neurophotonics 2, 041402 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  67. Ratelade, J. et al. Reducing hypermuscularization of the transitional segment between arterioles and capillaries protects against spontaneous intracerebral hemorrhage. Circulation https://doi.org/10.1161/CIRCULATIONAHA.119.040963 (2020).

  68. Nishiyama, A., Suzuki, R. & Zhu, X. NG2 cells (polydendrocytes) in brain physiology and repair. Front. Neurosci. 8, 133 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  69. Koizumi, J.-i, Yoshida, Y., Nakazawa, T. & Ooneda, G. Experimental studies of ischemic brain edema1. A new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area. Nosotchu 8, 1–8 (1986).

    Article  Google Scholar 

  70. Longa, E. Z., Weinstein, P. R., Carlson, S. & Cummins, R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20, 84–91 (1989).

    Article  CAS  PubMed  Google Scholar 

  71. Grutzendler, J. et al. Angiophagy prevents early embolus washout but recanalizes microvessels through embolus extravasation. Sci. Transl. Med. 6, 226ra231 (2014).

    Article  Google Scholar 

  72. Saunders, N. R., Dziegielewska, K. M., Mollgard, K. & Habgood, M. D. Markers for blood-brain barrier integrity: how appropriate is Evans blue in the twenty-first century and what are the alternatives? Front. Neurosci. 9, 385 (2015).

    PubMed  PubMed Central  Google Scholar 

  73. Condello, C., Schain, A. & Grutzendler, J. Multicolor time-stamp reveals the dynamics and toxicity of amyloid deposition. Sci. Rep. 1, 19 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Kimbrough, I. F., Robel, S., Roberson, E. D. & Sontheimer, H. Vascular amyloidosis impairs the gliovascular unit in a mouse model of Alzheimer’s disease. Brain 138, 3716–3733 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  75. Nortley, R. et al. Amyloid beta oligomers constrict human capillaries in Alzheimer’s disease via signaling to pericytes. Science https://doi.org/10.1126/science.aav9518 (2019).

  76. Yoder, E. J. & Kleinfeld, D. Cortical imaging through the intact mouse skull using two-photon excitation laser scanning microscopy. Microsc. Res. Tech. 56, 304–305 (2002).

    Article  PubMed  Google Scholar 

  77. Chaigneau, E., Roche, M. & Charpak, S. Unbiased analysis method for measurement of red blood cell size and velocity with laser scanning microscopy. Front. Neurosci. 13, 644 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  78. Clapham, D. E. Calcium signaling. Cell 131, 1047–1058 (2007).

    Article  CAS  PubMed  Google Scholar 

  79. Borysova, L., Wray, S., Eisner, D. A. & Burdyga, T. How calcium signals in myocytes and pericytes are integrated across in situ microvascular networks and control microvascular tone. Cell Calcium 54, 163–174 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Rungta, R. L., Chaigneau, E., Osmanski, B. F. & Charpak, S. Vascular compartmentalization of functional hyperemia from the synapse to the pia. Neuron 99, 362–375 e364 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Thrane, A. S. et al. General anesthesia selectively disrupts astrocyte calcium signaling in the awake mouse cortex. Proc. Natl Acad. Sci. USA 109, 18974–18979 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Rickgauer, J. P. & Tank, D. W. Two-photon excitation of channelrhodopsin-2 at saturation. Proc. Natl Acad. Sci. USA 106, 15025–15030 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Papagiakoumou, E. et al. Scanless two-photon excitation of channelrhodopsin-2. Nat. Methods 7, 848–854 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Rungta, R. L., Osmanski, B. F., Boido, D., Tanter, M. & Charpak, S. Light controls cerebral blood flow in naive animals. Nat. Commun. 8, 14191 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Choi, M., Yoon, J. & Choi, C. Label-free optical control of arterial contraction. J. Biomed. Opt. 15, 015006 (2010).

    Article  PubMed  Google Scholar 

  86. Nelson, A. R. et al. Channelrhodopsin excitation contracts brain pericytes and reduces blood flow in the aging mouse brain in vivo. Front. Aging Neurosci. 12, 108 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Grutzendler, J., Yang, G., Pan, F., Parkhurst, C. N. & Gan, W. B. Transcranial two-photon imaging of the living mouse brain. Cold Spring Harb. Protoc. https://doi.org/10.1101/pdb.prot065474 (2011).

  88. Yang, G., Pan, F., Parkhurst, C. N., Grutzendler, J. & Gan, W. B. Thinned-skull cranial window technique for long-term imaging of the cortex in live mice. Nat. Protoc. 5, 201–208 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Xu, H. T., Pan, F., Yang, G. & Gan, W. B. Choice of cranial window type for in vivo imaging affects dendritic spine turnover in the cortex. Nat. Neurosci. 10, 549–551 (2007).

    Article  CAS  PubMed  Google Scholar 

  90. Holtmaat, A. et al. Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window. Nat. Protoc. 4, 1128–1144 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Isshiki, M. & Okabe, S. Evaluation of cranial window types for in vivo two-photon imaging of brain microstructures. Microscopy 63, 53–63 (2014).

    Article  PubMed  Google Scholar 

  92. Nimmerjahn, A., Kirchhoff, F., Kerr, J. N. & Helmchen, F. Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. Nat. Methods 1, 31–37 (2004).

    Article  CAS  PubMed  Google Scholar 

  93. Hill, R. A. & Grutzendler, J. In vivo imaging of oligodendrocytes with sulforhodamine 101. Nat. Methods 11, 1081–1082 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Kamoun, W. S. et al. Simultaneous measurement of RBC velocity, flux, hematocrit and shear rate in vascular networks. Nat. Methods 7, 655–660 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Back, T., Kohno, K. & Hossmann, K. A. Cortical negative DC deflections following middle cerebral artery occlusion and KCl-induced spreading depression: effect on blood flow, tissue oxygenation, and electroencephalogram. J. Cereb. Blood Flow Metab. 14, 12–19 (1994).

    Article  CAS  PubMed  Google Scholar 

  96. Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L. & Luo, L. A global double-fluorescent Cre reporter mouse. Genesis 45, 593–605 (2007).

    Article  CAS  PubMed  Google Scholar 

  97. Madisen, L. et al. A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing. Nat. Neurosci. 15, 793–802 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. Han, X. et al. A high-light sensitivity optical neural silencer: development and application to optogenetic control of non-human primate cortex. Front. Syst. Neurosci. 5, 18 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. Department of Neurology, Yale School of Medicine, New Haven, CT, USA

    Lei Tong, Robert A. Hill, Eyiyemisi C. Damisah, Katie N. Murray, Peng Yuan & Jaime Grutzendler

  2. Department of Biological Sciences, Dartmouth College, Hanover, NH, USA

    Robert A. Hill

  3. Department of Neurosurgery, Yale School of Medicine, New Haven, CT, USA

    Eyiyemisi C. Damisah & Angelique Bordey

  4. Department of Biology, Stanford University, Stanford, CA, USA

    Peng Yuan

  5. Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT, USA

    Angelique Bordey

  6. Department of Neuroscience, Yale School of Medicine, New Haven, CT, USA

    Jaime Grutzendler

Authors
  1. Lei Tong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Robert A. Hill
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Eyiyemisi C. Damisah
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Katie N. Murray
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Peng Yuan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Angelique Bordey
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jaime Grutzendler
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding author

Correspondence to Jaime Grutzendler.

Ethics declarations

Competing interests

The authors have no competing interests.

Additional information

Peer review information Nature Protocols thanks Katerina Akassoglou and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

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

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2.

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-020-00425-w

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing