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

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

Published: 2020-12-08 DOI: 10.1038/s41596-020-00425-w

vascular mural cells

optogenetic modulation

intravital imaging

blood–brain barrier

neurodegenerative diseases

AI 解读

Generation of mouse–human chimeric embryos

Genome-wide quantification of transcription factor binding at single-DNA-molecule resolution using methyl-transferase footprinting

Generation of CRISPR–Cas9-mediated genetic knockout human intestinal tissue–derived enteroid lines by lentivirus transduction and single-cell cloning

A puromycin-dependent activity-based sensing probe for histochemical staining of hydrogen peroxide in cells and animal tissues

Decoding personal biotic and abiotic airborne exposome

A clinically relevant model of focal embolic cerebral ischemia by thrombus and thrombolysis in rhesus monkeys

Bioengineering human intestinal mucosal grafts using patient-derived organoids, fibroblasts and scaffolds

Biomimetic tail-to-head terpene cyclizations using the resorcin[4]arene capsule catalyst

Neuroscience robotics for controlled induction and real-time assessment of hallucinations

Scanorama: integrating large and diverse single-cell transcriptomic datasets

Single-cell RNA sequencing (scRNA-seq)（单细胞RNA测序）

Data integration（数据集成）

Heterogeneity（异质性）

查看全部

Sections

Figures

References

Mural cells (smooth muscle cells and pericytes) are integral components of brain blood vessels that play important roles in vascular formation, blood–nobreakbrain barrier maintenance, and regulation of regional cerebral blood flow (rCBF). These ce
Introduction
Materials
Procedure 1: Mural cell labeling and imaging
Procedure 2: Mural cell physiology
Procedure 3: Mural cell optogenetics
Troubleshooting
Timing
Anticipated results
References
ACKNOWLEDGMENTS

Armulik, A., Genove, G. & Betsholtz, C. Pericytes: developmental, physiological, and pathological perspectives, problems, and promises. Dev. Cell 21, 193–215 (2011).
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).
Gerhardt, H. & Betsholtz, C. Endothelial–pericyte interactions in angiogenesis. Cell Tissue Res. 314, 15–23 (2003).
Armulik, A., Abramsson, A. & Betsholtz, C. Endothelial/pericyte interactions. Circ. Res. 97, 512–523 (2005).
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).
Armulik, A. et al. Pericytes regulate the blood–brain barrier. Nature 468, 557–561 (2010).
Winkler, E. A., Bell, R. D. & Zlokovic, B. V. Central nervous system pericytes in health and disease. Nat. Neurosci. 14, 1398–1405 (2011).
Farkas, E. & Luiten, P. G. Cerebral microvascular pathology in aging and Alzheimer’s disease. Prog. Neurobiol. 64, 575–611 (2001).
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).
Sagare, A. P. et al. Pericyte loss influences Alzheimer-like neurodegeneration in mice. Nat. Commun. 4, 2932 (2013).
He, L. et al. Analysis of the brain mural cell transcriptome. Sci. Rep. 6, 35108 (2016).
He, L. et al. Single-cell RNA sequencing of mouse brain and lung vascular and vessel-associated cell types. Sci. Data 5, 180160 (2018).
Dana, H. et al. Sensitive red protein calcium indicators for imaging neural activity. eLife http://doi.org/10.7554/eLife.12727 (2016).
Smyth, L. C. D. et al. Markers for human brain pericytes and smooth muscle cells. J. Chem. Neuroanat. 92, 48–60 (2018).
Vanlandewijck, M. et al. A molecular atlas of cell types and zonation in the brain vasculature. Nature 554, 475–480 (2018).
Dieguez-Hurtado, R. et al. Loss of the transcription factor RBPJ induces disease-promoting properties in brain pericytes. Nat. Commun. 10, 2817 (2019).
Dubrac, A. et al. NCK-dependent pericyte migration promotes pathological neovascularization in ischemic retinopathy. Nat. Commun. 9, 3463 (2018).
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).
Hall, C. N. et al. Capillary pericytes regulate cerebral blood flow in health and disease. Nature 508, 55–60 (2014).
Raichle, M. E. & Gusnard, D. A. Appraising the brain’s energy budget. Proc. Natl Acad. Sci. USA 99, 10237–10239 (2002).
Iadecola, C. The pathobiology of vascular dementia. Neuron 80, 844–866 (2013).
Girouard, H. & Iadecola, C. Neurovascular coupling in the normal brain and in hypertension, stroke, and Alzheimer disease. J. Appl. Physiol. 100, 328–335 (2006).
Iadecola, C. The neurovascular unit coming of age: a journey through neurovascular coupling in health and disease. Neuron 96, 17–42 (2017).
Busse, R. [Contraction and relaxation mechanisms of the smooth vascular musculature]. Z Kardiol. 73, 63–69 (1984).
Peppiatt, C. M., Howarth, C., Mobbs, P. & Attwell, D. Bidirectional control of CNS capillary diameter by pericytes. Nature 443, 700–704 (2006).
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).
Wei, H. S. et al. Erythrocytes are oxygen-sensing regulators of the cerebral microcirculation. Neuron 91, 851–862 (2016).
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).
Mishra, A. et al. Astrocytes mediate neurovascular signaling to capillary pericytes but not to arterioles. Nat. Neurosci. 19, 1619–1627 (2016).
Grutzendler, J. & Nedergaard, M. Cellular control of brain capillary blood flow: in vivo imaging veritas. Trends Neurosci. 42, 528–536 (2019).
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).
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).
Kisler, K. et al. Pericyte degeneration leads to neurovascular uncoupling and limits oxygen supply to brain. Nat. Neurosci. 20, 406–416 (2017).
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).
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).
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).
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).
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).
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).
Zhu, X., Bergles, D. E. & Nishiyama, A. NG2 cells generate both oligodendrocytes and gray matter astrocytes. Development 135, 145–157 (2008).
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).
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).
Zhu, X. et al. Age-dependent fate and lineage restriction of single NG2 cells. Development 138, 745–753 (2011).
Cuervo, H. et al. PDGFRbeta-P2A-CreER(T2) mice: a genetic tool to target pericytes in angiogenesis. Angiogenesis 20, 655–662 (2017).
Snippert, H. J. et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 143, 134–144 (2010).
Livet, J. et al. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 56–62 (2007).
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).
Lacar, B. et al. Neural progenitor cells regulate capillary blood flow in the postnatal subventricular zone. J. Neurosci. 32, 16435–16448 (2012).
Madisen, L. et al. Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance. Neuron 85, 942–958 (2015).
Tong, L. et al. Single-cell in vivo brain optogenetic stimulation by two-photon excitation fluorescence transfer. Preprint at bioRxiv http://doi.org/10.1101/2020.06.29.179077 (2020).
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 http://doi.org/10.3389/fnene.2010.00005 (2010).
Khennouf, L. et al. Active role of capillary pericytes during stimulation-induced activity and spreading depolarization. Brain 141, 2032–2046 (2018).
Fenno, L., Yizhar, O. & Deisseroth, K. The development and application of optogenetics. Annu. Rev. Neurosci. 34, 389–412 (2011).
Ellis-Davies, G. C. Caged compounds: photorelease technology for control of cellular chemistry and physiology. Nat. Methods 4, 619–628 (2007).
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).
Longden, T. A. et al. Capillary K+-sensing initiates retrograde hyperpolarization to increase local cerebral blood flow. Nat. Neurosci. 20, 717–726 (2017).
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).
Prakash, R. et al. Two-photon optogenetic toolbox for fast inhibition, excitation and bistable modulation. Nat. Methods 9, 1171–1179 (2012).
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).
Andermann, M. L. et al. Chronic cellular imaging of entire cortical columns in awake mice using microprisms. Neuron 80, 900–913 (2013).
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).
Nikolakopoulou, A. M. et al. Pericyte loss leads to circulatory failure and pleiotrophin depletion causing neuron loss. Nat. Neurosci. 22, 1089–1098 (2019).
Holm, A., Heumann, T. & Augustin, H. G. Microvascular mural cell organotypic heterogeneity and functional plasticity. Trends Cell Biol. 28, 302–316 (2018).
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).
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).
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).
Ratelade, J. et al. Reducing hypermuscularization of the transitional segment between arterioles and capillaries protects against spontaneous intracerebral hemorrhage. Circulation http://doi.org/10.1161/CIRCULATIONAHA.119.040963 (2020).
Nishiyama, A., Suzuki, R. & Zhu, X. NG2 cells (polydendrocytes) in brain physiology and repair. Front. Neurosci. 8, 133 (2014).
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).
Longa, E. Z., Weinstein, P. R., Carlson, S. & Cummins, R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20, 84–91 (1989).
Grutzendler, J. et al. Angiophagy prevents early embolus washout but recanalizes microvessels through embolus extravasation. Sci. Transl. Med. 6, 226ra231 (2014).
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).
Condello, C., Schain, A. & Grutzendler, J. Multicolor time-stamp reveals the dynamics and toxicity of amyloid deposition. Sci. Rep. 1, 19 (2011).
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).
Nortley, R. et al. Amyloid beta oligomers constrict human capillaries in Alzheimer’s disease via signaling to pericytes. Science http://doi.org/10.1126/science.aav9518 (2019).
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).
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).
Clapham, D. E. Calcium signaling. Cell 131, 1047–1058 (2007).
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).
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).
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).
Rickgauer, J. P. & Tank, D. W. Two-photon excitation of channelrhodopsin-2 at saturation. Proc. Natl Acad. Sci. USA 106, 15025–15030 (2009).
Papagiakoumou, E. et al. Scanless two-photon excitation of channelrhodopsin-2. Nat. Methods 7, 848–854 (2010).
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).
Choi, M., Yoon, J. & Choi, C. Label-free optical control of arterial contraction. J. Biomed. Opt. 15, 015006 (2010).
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).
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. http://doi.org/10.1101/pdb.prot065474 (2011).
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).
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).
Holtmaat, A. et al. Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window. Nat. Protoc. 4, 1128–1144 (2009).
Isshiki, M. & Okabe, S. Evaluation of cranial window types for in vivo two-photon imaging of brain microstructures. Microscopy 63, 53–63 (2014).
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).
Hill, R. A. & Grutzendler, J. In vivo imaging of oligodendrocytes with sulforhodamine 101. Nat. Methods 11, 1081–1082 (2014).
Kamoun, W. S. et al. Simultaneous measurement of RBC velocity, flux, hematocrit and shear rate in vascular networks. Nat. Methods 7, 655–660 (2010).
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).
Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L. & Luo, L. A global double-fluorescent Cre reporter mouse. Genesis 45, 593–605 (2007).
Madisen, L. et al. A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing. Nat. Neurosci. 15, 793–802 (2012).
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).

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

Supplementary information

Supplementary Information

Reporting Summary

推荐阅读