Abstract
Proper brain function depends on neurovascular coupling: neural activity rapidly increases local blood flow to meet moment-to-moment changes in regional brain energy demand1. Neurovascular coupling is the basis for functional brain imaging2, and impaired neurovascular coupling is implicated in neurodegeneration1. The underlying molecular and cellular mechanisms of neurovascular coupling remain poorly understood. The conventional view is that neurons or astrocytes release vasodilatory factors that act directly on smooth muscle cells (SMCs) to induce arterial dilation and increase local blood flow1. Here, using two-photon microscopy to image neural activity and vascular dynamics simultaneously in the barrel cortex of awake mice under whisker stimulation, we found that arteriolar endothelial cells (aECs) have an active role in mediating neurovascular coupling. We found that aECs, unlike other vascular segments of endothelial cells in the central nervous system, have abundant caveolae. Acute genetic perturbations that eliminated caveolae in aECs, but not in neighbouring SMCs, impaired neurovascular coupling. Notably, caveolae function in aECs is independent of the endothelial NO synthase (eNOS)-mediated NO pathway. Ablation of both caveolae and eNOS completely abolished neurovascular coupling, whereas the single mutants exhibited partial impairment, revealing that the caveolae-mediated pathway in aECs is a major contributor to neurovascular coupling. Our findings indicate that vasodilation is largely mediated by endothelial cells that actively relay signals from the central nervous system to SMCs via a caveolae-dependent pathway.
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Code availability
The source code to run the pial arteriolar dilation analysis is available at https://github.com/gulabneuro/Pial-Vasodilation-Analysis. The source code to run the parenchymal arterial dilation analysis is available at https://github.com/gulabneuro/divingArterioleTracking.
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Acknowledgements
We thank C. Harvey for his help in the design and construction of the two-photon microscope; O. Mazor and P. Gorelik at the HMS Research Instrumentation Core Facility and T. LaFratta and J. LeBlanc of the Harvard Neuroengineering and Imaging Machine Shop for their help in the construction of the two-photon microscope; S. Ashrafi, J. Cohen, D. Ginty, C. Weitz., G. Yellen and members of the Gu laboratory for comments on the manuscript; C. Lahmann for protocol advice and assistance on cranial window surgery; P. Scherer, R. Adams and B. Zhou for the Cav1-floxed, BMXcreER and Mfsd2acreER mice, respectively; D. Silver for the MFSD2A antibody; and the HMS Electron Microscopy Core Facility, HMS Neurobiology Imaging Facility and HMS NeuroDiscovery Center for consultation and instrument availability. This work was supported by Quan Fellowship (B.W.C), NIH T32 and the Mahoney Postdoctoral Fellowship (V.N.), Jane Coffin Childs Fund (A.J.G.), K99 NS102429 (A.J.G), R37 NS046579 (B.L.S.), P30NS072030 (HMS Neurobiology Imaging Facility), the NIH DP1 NS092473 Pioneer Award (C.G.), Fidelity Biosciences Research Initiative (C.G.). The research of C.G. was also supported in part by a Faculty Scholar grant from the Howard Hughes Medical Institute.
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Contributions
B.W.C. and C.G. conceived the project. B.W.C, V.N. and C.G. designed experiments. B.W.C., V.N., A.J.G., K.B. and H.L.Z. performed experiments. B.W.C., V.N., A.J.G., L.K. and P.K. analysed all data. B.W.C. and C.G. wrote the manuscript, with feedback from all authors.
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Peer review information Nature thanks Dritan Agalliu, Brian MacVicar and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data figures and tables
Extended Data Fig. 1 In vivo two-photon imaging of neurovascular coupling in the barrel cortex and retrosplenial cortex.
a, Setup of the in vivo microscopy. Awake mice with cranial windows over the barrel cortex are head-fixed and allowed to move on a foam ball. Whisker stimulator (arrow) is used for brushing whiskers to evoke neural activity in the barrel cortex. b–g, Imaging in the barrel cortex. b, Hydrazide injection in Thy1-GCaMP6s mice enables simultaneous imaging of neural activity (green) and arteriolar dilation (magenta). Two-photon imaging of arterioles and neural activity before (left) and after (right) whisker stimulation. Hashes indicate the baseline diameter at time = 0 s. c, Time course of change in arteriolar dilation (magenta) and GCaMP6s fluorescence (green). Orange bar signifies the period of whisker stimulation. d, Two-photon imaging of arterioles (magenta) and capillary blood flow (blue). After intravenous injection of quantum dots, the plasma is bright whereas the red blood cells are dark. e, High magnification of a capillary outlined by the red box in d. Minimizing the image size increases the temporal resolution to about 610 Hz or 1.6 ms per frame. f, Kymographs of capillary blood flow during baseline (left) and whisker stimulation (right). Kymographs were generated from the parallel line scan (red line) of the capillary blood flow in e. g, Time course of change in red blood cell velocity. h, Time course of change in arteriolar dilation in the barrel cortex (black, n = 78 arterioles, 3 mice) and in the retrosplenial cortex (red, n = 54 arterioles, 3 mice). i, Maximum percentage change in arteriolar dilation upon whisker stimulus in these two brain regions upon whisker stimulus. The orange bar signifies the period of whisker stimulation. Data are mean ± s.e.m.; nested unpaired, two-tailed t-test for i.
Extended Data Fig. 2 Cav1-knockout mice have impaired vasodilation in both pial arteries and penetrating arterioles diving deep into the parenchyma.
a, Three-dimensional volume rendering of a two-photon-imaged site in Cav1+/+ mouse barrel cortex, from the pial surface to a depth of about 400 μm. The lumen of all vessels is filled with quantum dots (blue) and arterioles are labelled with hydrazide (magenta). The deepest imaged bin is at 300 μm because we see the appearance of the hydrazide start at 300 μm, indicating that this is at the start of the arteriolar vessels. This observation is also consistent with a previous study5, which characterized hydrazide as an arteriolar vessel marker. Grey slices correspond to z cross-sections shown per depth. Independent replicates for a were performed in five wild-type mice. b, c, Time course of change in arteriolar dilation in the barrel cortex from Cav1+/+ (n = 5 mice, 10–15 arterioles per depth) (b) and Cav1−/− mice (n = 5 mice, 10–15 arterioles per depth) (c). d, Maximum percentage change in arteriolar dilation upon whisker stimulation between in Cav1+/+ and Cav1−/− mice at the indicated depth. Statistical significance was determined by two-way ANOVA with a post hoc Bonferroni multiple comparison adjustment for d. All data are mean ± s.e.m. We compared the maximum percentage change in arteriolar dilation upon whisker stimulation between Cav1+/+ and Cav1−/− mice at each depth and also compared the responses across depth within the same genotype.
Extended Data Fig. 3 Cav1-knockout mice have attenuated vasodilation but normal neural activity and neurovascular coupling kinetics.
a–d, Maximum percentage change in dilation response (a) and baseline diameter (b) latency to maximum change in arteriolar dilation (c), time to peak dilation (d) in Cav1+/+ (n = 193 arterioles, 40 capillaries, 5 mice), Cav1+/− (n = 123 arterioles, 40 capillaries, 5 mice), and Cav1−/− mice (n = 153 arterioles, 31 capillaries, 5 mice). e, f, Latency to maximum red blood cell flow velocity (e) and time to peak red blood cell flow (f) in Cav1+/+ (n = 193 arterioles, 40 capillaries, 5 mice), Cav1+/− (n = 123 arterioles, 40 capillaries, 5 mice) and Cav1−/− mice (n = 153 arterioles, 31 capillaries, 5 mice). g, h, Maximum percentage change in GCaMP6s (g) and latency to peak change in GCaMPs (h) in Cav1+/+;Thy1-GCaMP6s (n = 78 field of views of the neuropils, 5 mice) and Cav1−/−;Thy1-GCaMP6s (n = 78 neuropils, 5 mice). Each circle represents an individual trial of GCaMP6s signal. i, Baseline diameter to absolute maximum diameter response during whisker stimulation in Cav1+/+ and Cav1−/− mice. j, Tail-cuff blood pressure measurements between Cav1+/+ (n = 5 mice) and Cav1−/− mice (n = 5 mice). Statistical significance was determined by a one-way nested ANOVA with a post hoc Bonferroni multiple comparison adjustment for a–f, a nested unpaired, two-tailed t-test for g, h, and two-tailed Mann–Whitney U test for j. All data are mean ± s.e.m.
Extended Data Fig. 4 Cav1-mutant mice exhibit normal SMC integrity and function.
a, In vivo two-photon microscopy images of hydrazide (magenta) and DsRed (red) from Cav1+/+NG2DsRED+ and Cav1−/−NG2DsRED+ mice. b, Quantification of DsRED+ SMCs per 100 μm as shown in a, in Cav1+/+ (n = 3 mice, 27 arterioles) and Cav1−/− (n = 3 mice, 28 arterioles) mice. c, Immunostaining for SMC contractile proteins, including SMA, MYH11, TAGLN and desmin on brain arterioles from Cav1+/+ and Cav1−/− mice. d–g, Normalized fluorescence quantification of the various contractile proteins from Cav1+/+ and Cav1−/− mice. h, Still frame images of arterioles labelled with hydrazide (magenta) in ex vivo acute brain slices from Cav1+/+ and Cav1−/− mice using two-photon microscopy. Left, arterioles during baseline; middle, arterioles during U46619 (thromboxane agonist) treatment; right, arterioles during DEA NONOate (NO donor) treatment. White hashes outline the arterioles during baseline based on time = 0 min. i, j, Maximum arteriolar contraction by U46619 (i) and maximum arteriolar dilation by DEA NONOate (j) on acute brain slices from Cav1+/+ (n = 5 mice, 19 arterioles) and Cav1−/− (n = 5 mice, 22 arterioles). k, In vivo images of arterioles labelled with hydrazide (magenta) from Cav1+/+ and Cav1−/− mice using two-photon microscopy. Left, arterioles during baseline; right, arterioles during DEA NONOate superfusion. White hashes outline the arterioles during baseline based on time = 0 s. l, Quantification of maximum arteriolar dilation during DEA NONOate superfusion in vivo (n = 5 mice for both genotypes). Statistical significance was determined by nested, unpaired, two-tailed t-test for b, d–g, i, j, and by two-tailed Mann–Whitney U test for l. Data shown as mean ± s.e.m.
Extended Data Fig. 5 Caveolae in CNS aECs are abolished in aEC conditional Cav1-knockout mice.
a, Immunostaining of adult brain sections for ECs (ICAM2, green), SMCs (SMA, magenta) and CAV1 (red) from control (BMXcreER−;Cav1+/fl) and aEC-specific conditional CAV1 mutant (BMXcreER+;Cav1−/fl) mice. Arrows point to aECs. b, Transmission electron microscopy images of CNS aECs and SMCs from control and aEC-specific conditional Cav1-mutant mice. Arrowheads point to caveolae. L, Lumen. c, Quantification of mean normalized immunofluorescence of CAV1 in aECs from control (n = 5 mice) and aEC-specific conditional Cav1-mutant mice (n = 5 mice). d, Quantification of the mean vesicular density in aECs and SMCs between control (n = 4 mice, 20 arterioles) and aEC-specific conditional Cav1-mutant mice (n = 5 mice, 22 arterioles). e, f, Quantification of baseline diameter (e) and baseline velocity in control (BMXcreER−;Cav1+/fl; n = 7 mice, 260 arterioles, 122 capillaries) and aEC conditional Cav1-knockout mice (BMXcreER+;Cav1−/fl; n = 5 mice, 193 arterioles, 94 capillaries). Statistical significance was determined by Mann–Whitney test for (c) and nested, unpaired, two-tailed t-test for (d–f). Data are shown as mean ± s.e.m.
Extended Data Fig. 6 Conditional aEC-specific and SMC Cav1-knockout mice have normal neurovascular coupling kinetics.
a, Baseline diameter to absolute maximum diameter response during whisker stimulation in control (BMXcreER−;Cav1+/fl) and mutant (BMXcreER+;Cav1−/fl) mice. b, Quantification of time to peak arteriolar dilation in control (BMXcreER−;Cav1+/fl; n = 7 mice, 234 arterioles) and aEC-specific conditional Cav1-mutant (BMXcreER+;Cav1−/fl; n = 5 mice; 202 arterioles) mice. c, Quantification of latency to peak red blood cell flow velocity in control (BMXcreER−;Cav1+/fl; n = 7 mice; 58 capillaries) and aEC-specific conditional Cav1-mutant (BMXcreER+;Cav1−/fl; n = 5 mice; 25 capillaries) mice. d, Quantification of time to peak red blood cell flow velocity in control (BMXcreER−;Cav1+/fl; n = 7 mice; 127 capillaries) and aEC-specific conditional Cav1-mutant (BMXcreER+;Cav1−/fl; n = 5 mice; 94 capillaries) mice. e, Baseline diameter to absolute maximum diameter response during whisker stimulation in control (Myh11creER−;Cav1+/fl) and mutant (Myh11creER+;Cav1−/fl mice. f, Quantification of time to peak arteriolar dilation in control (Myh11creER−;Cav1+/fl; n = 5 mice, 193 arterioles) and SMC conditional Cav1-mutant (Myh11creER+;Cav1−/fl; n = 5 mice; 180 arterioles) mice. g, Quantification of latency to red blood cell flow in control (Myh11creER−;Cav1+/fl; n = 5 mice; 36 capillaries) and SMC conditional Cav1-mutant (Myh11creER+;Cav1−/fl; n = 5 mice; 26 capillaries) mice. h, Quantification time to peak red blood cell flow velocity in (Myh11creER−;Cav1+/fl; n = 5 mice; 75 capillaries) and SMC conditional Cav1-mutant (Myh11creER+;Cav1−/fl; n = 5 mice; 75 capillaries) mice. Statistical significance was determined by a nested unpaired, two-tailed t-test for b–d, f–h).
Extended Data Fig. 7 Conditional SMC-specific Cav1-knockout mice have normal neurovascular coupling.
a, Immunostaining on brain sections for ECs (ICAM2, green), SMCs (SMA, magenta) and CAV1 (red) from control and SMC conditional Cav1-mutant mice. b, Transmission electron microscopy images of CNS aECs and SMCs from control and SMC conditional Cav1-mutant mice. Arrowheads point to caveolae. L, Lumen. c, Mean normalized immunofluorescence of CAV1 in SMCs from control (n = 5 mice) and SMC-specific conditional Cav1-mutant mice (n = 5 mice). d, Quantification of the mean vesicular density in aECs and SMCs in control (n = 5 mice, 23 arterioles) and SMC conditional Cav1−/− mice (n = 5 mice, 22 arterioles). e–g, Time course of change in arteriolar dilation (e), maximum percentage change in arteriolar dilation (f) and baseline diameter (g) in control (n = 7 mice, 193 arterioles) and SMC conditional Cav1-mutant mice (n = 5 mice, 176 arterioles). h–j, Time course of change in red blood cell velocity (h), maximum percentage change in red blood cell velocity (i) and baseline velocity (j) in control (n = 7 mice, 75 capillaries) and SMC conditional Cav1-mutant mice (n = 5 mice, 64 capillaries). Statistical significance was determined by unpaired, two-tailed Mann–Whitney U test for c and a nested, unpaired, two-tailed t-test for (d, f, g, i, j). Data are mean ± s.e.m.
Extended Data Fig. 8 Cav1-mutant mice have normal levels of eNOS protein and NO in CNS aECs and Cav1 and Nos3 double knockout mice have normal baseline diameter and red blood cell flow.
a, Immunostaining on adult brain sections for ECs (PECAM1, green), arterioles (SMA, magenta) and eNOS (cyan) from Cav1+/+Nos3+/+, Cav1−/−Nos3+/+ and Cav1+/+Nos3−/− mice. Independent replications were performed on three mice per genotype. b, Immunostaining for (PECAM1, green) and arterioles (SMA, magenta) on brain sections from Cav1+/+Nos3+/+, Cav1−/−Nos3+/+ and Cav1+/+Nos3−/− mice after in vivo perfusion of NO-sensitive dye; DAF-2, yellow. Independent replicates were performed on four mice per genotype. c, Quantification of eNOS immunofluorescence intensity as shown in a in aECs from Cav1+/+Nos3+/+ (n = 3 mice, 35 images), Cav1−/−Nos3+/+ (n = 3 mice, 35 images) and Cav1+/+Nos3−/− (n = 3 mice, 37 images). d, Quantification of DAF-2 intensity in aECs as shown in (b) from Cav1+/+Nos3+/+ (n = 4 mice, 73 images), Cav1−/−Nos3+/+ (n = 4 mice, 71 images), and Cav1+/+Nos3−/− (n = 4 mice, 64 images). e, f, Quantification of baseline diameter (e) and baseline velocity (f) in Cav1+/+Nos3+/+ (n = 5 mice, 148 arterioles, 76 capillaries), Cav1−/−Nos3+/+ (n = 5 mice, 128 arterioles, 68 capillaries), Cav1+/+Nos3−/− (n = 5 mice, 137 arterioles, 73 capillaries) and Cav1−/−Nos3−/− mice (n = 5 mice, 139 arterioles, 74 capillaries). Statistical significance was determined by nested, unpaired, two-tailed t-test for c, d, and nested, one-way ANOVA with a post hoc Bonferroni multiple-comparison adjustment for e, f. Data are mean ± s.e.m.
Extended Data Fig. 9 MFSD2A is not detected in CNS arterioles in brain and retina.
a, b, Immunostaining on postnatal day (P)5 (a) and adult (b) brain sections for ECs (PECAM1, green), SMCs (SMA, magenta) and MFSD2A (white) from wild-type mice. Blue hashes outline SMA+ arterioles. c, d, Immunostaining on P5 (c) and adult (d) retina for ECs (claudin 5, green), SMCs (SMA, magenta) and MFSD2A (white) from wild-type mice. A, arterioles. MFSD2A is absent in nascent, distal vessel (arrows) in P5 retina in c. e, f, Tamoxifen-treated, adult knock-in Mfsd2acreER+;Ai14+/fl reporter mice demonstrates that tdTomato is absent in SMA+ arterioles but present in SMA− capillaries in brain (e) and retina (f). Blue hashes and A indicate SMA+ arterioles. Independent replicates for a–f were performed on five wild-type mice.
Extended Data Fig. 10 Generation of a Cre-dependent MFSD2A-overexpression transgenic mouse (R26LSL-Mfsd2a).
a, Construct for Cre-dependent MFSD2A overexpression knocked-in to the ROSA26 locus. Mating with ROSA26: ΦC31 recombinase mice removes the neomycin selection cassette. Subsequent mating with BMXcreER and tamoxifen injection enables ectopic overexpression of Mfsd2a in aECs. b, PCR genotyping of Cre-dependent MFSD2A-overexpression mice. c, Quantification of latency to changes in arteriolar dilation in control (BMXcreER−;R26LSL-Mfsd2a/+; n = 5 mice, 149 arterioles) and aEC-specific MFSD2A overexpression (BMXcreER+;R26LSL-Mfsd2a/+; n = 5 mice; 138 arterioles) mice. d, Quantification of time to peak arteriolar dilation in control (BMXcreER−;R26LSL-Mfsd2a/+; n = 5 mice, 149 arterioles) and aEC-specific conditional Cav1-mutant (BMXcreER+;R26LSL-Mfsd2a/+; n = 5 mice, 138 arterioles) mice. e, Baseline diameter to absolute maximum diameter response during whisker stimulation in control (BMXcreER−;R26LSL-Mfsd2a/+; n = 5 mice, 149 arterioles) and aEC-specific conditional Cav1-mutant (BMXcreER+;R26LSL-Mfsd2a/+; n = 5 mice; 138 arterioles) mice. f, Immunostaining on adult retinas for ECs (isolectin, green), SMCs (SMA, magenta) and MFSD2A (white) from control and aEC-specific MFSD2A-overexpression mice. Independent replications for f were performed on three mice per genotype. Statistical significance was determined by a nested unpaired, two-tailed t-test for c, d.
Supplementary information
Supplementary Table 1
Statistical test results.
Supplementary Video 1 | Simultaneous in vivo imaging of neural activity and vasodynamics.
(Left panel) Video recording of the in vivo imaging paradigm. A glass cranial window is surgically implanted to optically access the brain of an awake mouse. (Right panel) Two-photon recording of GCaMP6s and arterial dilation in response to whisker stimulation. As the whisker stimulator (green foam pad) brushes the facial vibrissae, neural activity (Thy1:GCaMP6s) is robustly increased (green) followed by arterial dilation (magenta). Dotted white line outlines the interior of the artery during baseline period.
Supplementary Video 2 | In vivo imaging of capillary blood flow
Visualization of capillary blood flow is accomplished using two-photon microscopy and intravascular fluorescent tracers. Qdots 525 (blue) in the bloodstream enable the recording of capillary blood flow velocity. Red blood cells appear dark compared to the brightly fluorescing vessel lumen. Hydrazide positive arteriole (magenta). Video shows a 0.5 mm x 0.5 mm area of somatosensory cortex vasculature.
Supplementary Video 3 | In vivo imaging of sensory-evoked capillary blood flow changes
Capillary blood flow videos recorded using two-photon microscopy in an awake mouse during baseline period (Top panel) and during whisker stimulation period (Bottom panel). Qdots 525 (blue) fill the bloodstream, red blood cells appear as dark flowing cells. Images represent an area of 100 µm x 25 µm at pixel size = 0.04 µm2/pixel.
Supplementary Video 4 | Cav1 is required for sensory-evoked arterial dilation
Representative videos recorded using in vivo two-photon microscopy of sensory-evoked arterial dilation. Hydrazide stained arteries (magenta) in Cav1+/+ and Cav1+/- mice show robust dilation upon whisker stimulation whereas Cav1-/- littermates show significantly attenuated arterial dilation responses. White dotted lines indicate vessel dilation state at baseline.
Supplementary Video 5 | Cav1 is required for sensory-evoked capillary blood flow increases
Representative videos recorded using in vivo two-photon microscopy of sensory-evoked capillary blood flow. Capillaries filled with Qdots 525 (blue) in Cav1+/+ and Cav1+/- mice show robust increase in red blood cell velocity upon whisker stimulation whereas Cav1-/- littermates show significantly attenuated capillary blood flow velocity responses compared to baseline.
Supplementary Video 6 | Acute ex vivo slice imaging shows normal pharmacologically-evoked arterial contraction and dilation
Representative video recorded from acute brain slices from Cav1+/+ mice using two-photon microscopy combined with pharmacology. Hydrazide stained arteriole (magenta). White dotted line indicates baseline before constriction with U46619. DEA-NONOate was then added, dilating the arteries back to baseline.
Supplementary Video 7 | Acute ex vivo slice imaging shows smooth muscle cell contraction and relaxation function are unaltered in the absence of Cav1
Representative video recorded from acute brain slices from Cav1-/- mice using two-photon microscopy combined with pharmacology. Hydrazide stained arteriole (magenta). White dotted line indicates vessel dilation state before constriction with U46619. DEA-NONOate was then added, dilating the arteries back to baseline.
Supplementary Video 8 | Smooth muscle cell relaxation function assessed using in vivo pharmacology
Representative video of pial artery dilation response to acute administration of nitric oxide donor, DEA-NONOate, in vivo. Pharmacological access to pial arteries was gained acutely following an exposed cortex preparation. DEA-NONOate was topically administered to the surface of the exposed cortical tissue over the barrel cortex. Dilation responses of hydrazide stained arteries in deeply anesthetized Cav1+/+ mice were recorded using two-photon microscopy in deeply anesthetized mice. White dotted line indicates baseline diameter before addition of DEA-NONOate.
Supplementary Video 9 | Smooth muscle cell relaxation function is unaltered in the absence of Cav1 expression using in vivo pharmacology
Representative video of pial artery dilation response to acute administration of nitric oxide donor, DEA-NONOate, in vivo. DEA-NONOate was topically administered to the surface of the exposed cortical tissue over the barrel cortex. Dilation responses of hydrazide stained arteries (magenta) in deeply anesthetized Cav1-/- mice were recorded using two-photon microscopy. White dotted line indicates baseline diameter before addition of DEA-NONOate.
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Chow, B.W., Nuñez, V., Kaplan, L. et al. Caveolae in CNS arterioles mediate neurovascular coupling. Nature 579, 106–110 (2020). https://doi.org/10.1038/s41586-020-2026-1
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DOI: https://doi.org/10.1038/s41586-020-2026-1
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