Brain endothelial cell TRPA1 channels initiate neurovascular coupling
- Department of Pharmacology, Center for Molecular and Cellular Signaling in the Cardiovascular System, University of Nevada, Reno School of Medicine, United States
- Department of Pharmacology, College of Medicine, University of Vermont, United States
- Department of Physiology, College of Medicine, University of Arizona, United States
- Institute of Cardiovascular Sciences, University of Manchester, United Kingdom
- Department of Molecular Physiology and Biological Physics, University of Virginia, United States
- Robert M. Berne Cardiovascular Research Center, University of Virginia, United States
- Department of Physiology & Cell Biology, Center for Molecular and Cellular Signaling in the Cardiovascular System, University of Nevada, Reno School of Medicine, United States
Figures
Figure 1 with 1 supplement
TRPA1 channels are functionally expressed in native brain capillary endothelial cells.
(A and B) Representative current versus time trace (A) and I-V relationship (B) from a whole-cell patch-clamp electrophysiology experiment demonstrating that the TRPA1 activator 4-HNE (100 nM) …
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Figure 1—source data 1
Individual data points and analysis summaries for datasets shown in Figure 1.
- https://cdn.elifesciences.org/articles/63040/elife-63040-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Lack of functional IK and SK channels in native capillary endothelial cells.
(A and B) Representative current-time trace (A) and I-V relationship (B) from a whole-cell patch-clamp electrophysiology experiment demonstrating that the IK and SK channel activator NS309 (10 µM) …
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Figure 1—figure supplement 1—source data 1
Individual data points and analysis summaries for datasets shown in Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/63040/elife-63040-fig1-figsupp1-data1-v2.xlsx
Figure 2 with 2 supplements
Capillary TRPA1 channels initiate conducted vasodilation in the cerebral microcirculation.
(A) Experimental illustration. Microvascular preparations were obtained from the subcortical region that is supplied by the middle cerebral artery (MCA). Representative image of an ex vivo …
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Figure 2—source data 1
Individual data points and analysis summaries for datasets shown in Figure 2.
- https://cdn.elifesciences.org/articles/63040/elife-63040-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
KCl-induced dilation of upstream arterioles is blocked by BaCl2.
(A) Representative traces (A) and summary data (B) showing that application of a solution containing elevated KCl (10 mM; blue box) directly onto capillary extremities induced a dilation of the …
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Figure 2—figure supplement 1—source data 1
Individual data points and analysis summaries for datasets shown in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/63040/elife-63040-fig2-figsupp1-data1-v2.xlsx
Figure 2—figure supplement 2
Severing the connection between capillaries and the arteriole segment of an ex vivo microvascular preparation.
(A) Representative images showing an intact microvascular preparation (left) and the same preparation after severing the connection between the arteriole and capillary (right). Scale bar = 50 µm. (B …
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Figure 2—figure supplement 2—source data 1
Individual data points and analysis summaries for datasets shown in Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/63040/elife-63040-fig2-figsupp2-data1-v2.xlsx
Figure 3
Biphasic velocity of conductive vasodilation following activation of capillary TRPA1 channels.
(A to D) Representative fluorescence image of an ex vivo microvascular preparation obtained from a wild-type mouse. Microvascular preparations were treated with Alexa Fluor 488 conjugated isolectin …
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Figure 3—source data 1
Individual data points and analysis summaries for datasets shown in Figure 3.
- https://cdn.elifesciences.org/articles/63040/elife-63040-fig3-data1-v2.xlsx
Figure 4 with 2 supplements
Activation of capillary TRPA1 channels produces a purinergic signal that travels through the capillary bed.
(A) Representative traces showing that application of AITC (30 µM; red box) or 4-HNE (1 µM; green box) onto capillary extremities did not dilate the upstream arteriole in microvascular preparations …
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Figure 4—source data 1
Individual data points and analysis summaries for datasets shown in Figure 4.
- https://cdn.elifesciences.org/articles/63040/elife-63040-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
ATP-induced dilation persisted in preparations from Trpa1-ecKO mice.
(A and B) Representative traces (A) and summary data (B) showing that application of elevated KCl (10 mM; blue box) and ATP (10 µM; orange box) directly onto capillary extremities induced a dilation …
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Figure 4—figure supplement 1—source data 1
Individual data points and analysis summaries for datasets shown in Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/63040/elife-63040-fig4-figsupp1-data1-v2.xlsx
Figure 4—figure supplement 2
Average length of a cerebral capillary endothelial cell.
(A) Representative image of an isolated cerebral capillary network. Endothelial cells and nuclei were stained with Alexa Fluor 568 isolectin B4 (IB4) (red) and DAPI (blue), respectively. Scale bar = …
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Figure 4—figure supplement 2—source data 1
Individual data points and analysis summaries for datasets shown in Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/63040/elife-63040-fig4-figsupp2-data1-v2.xlsx
Figure 5 with 1 supplement
Activation of capillary TRPA1 channels and purinergic receptors produces a Ca2+ response that travels through the capillary bed.
(A) Representative time course images demonstrating the fractional increase in fluorescence (F/F0) of the Ca2+ signal in a capillary segment in microvascular preparations from transgenic mice …
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Figure 5—source data 1
Individual data points and analysis summaries for datasets shown in Figure 5.
- https://cdn.elifesciences.org/articles/63040/elife-63040-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Kinetics of the Ca2+ response in capillaries following activation of TRPA1 channels purinergic receptors.
(A to C) Duration (A), rise time [half-time (t1/2, s)] (B) and decay time (t1/2, s) (C) of the Ca2+ response following focal application of AITC (30 µM) and ATP (10 µM) to distal capillaries in …
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Figure 5—figure supplement 1—source data 1
Individual data points and analysis summaries for datasets shown in Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/63040/elife-63040-fig5-figsupp1-data1-v2.xlsx
Figure 6 with 2 supplements
Rapid propagation of TRPA1-induced vasodilation is dependent on Kir, IK, and SK channels.
(A) Representative image of a cerebral microvascular preparation in which the capillary bed was removed while leaving the post-arteriole transitional segment (TS) intact. The transitional segment is …
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Figure 6—source data 1
Individual data points and analysis summaries for datasets shown in Figure 6.
- https://cdn.elifesciences.org/articles/63040/elife-63040-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
ATP does not evoke dilation of upstream arterioles when applied to the post-arteriole transitional segment.
(A) Representative trace showing that application of elevated KCl (10 mM; blue box) or AITC (30 µM; red box) onto the post-arteriole transitional segment increased the lumen diameter of the upstream …
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Figure 6—figure supplement 1—source data 1
Individual data points and analysis summaries for datasets shown in Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/63040/elife-63040-fig6-figsupp1-data1-v2.xlsx
Figure 6—figure supplement 2
Application of NS309 to capillary extremities has no effect on the upstream arteriole.
(A) Representative images of an intact microvascular preparation showing the drug-administering cannula positioned adjacent to capillary extremities (left) and arteriole segment (right). Scale bar = …
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Figure 6—figure supplement 2—source data 1
Individual data points and analysis summaries for datasets shown in Figure 6—figure supplement 2.
- https://cdn.elifesciences.org/articles/63040/elife-63040-fig6-figsupp2-data1-v2.xlsx
Figure 7 with 1 supplement
In vivo stimulation of capillary TRPA1 channels increases RBC flux within the capillary bed.
(A) Experimental illustration. Mice were injected with FITC-conjugated dextran to allow visualization of the cortical vasculature through a cranial window using in vivo two-photon laser-scanning …
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Figure 7—source data 1
Individual data points and analysis summaries for datasets shown in Figure 7.
- https://cdn.elifesciences.org/articles/63040/elife-63040-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
Kinetics of the RBC flux increase following in vivo stimulation of capillary TRPA1 channels.
(A and B) Summary data showing the rate of RBC flux increase (A) and latency of the response following local application of AITC (30 µM) directly onto a single capillary of Trpa1fl/fl mice (n = 11 …
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Figure 7—figure supplement 1—source data 1
Individual data points and analysis summaries for datasets shown in Figure 7—figure supplement 1.
- https://cdn.elifesciences.org/articles/63040/elife-63040-fig7-figsupp1-data1-v2.xlsx
Figure 8 with 3 supplements
Functional hyperemia is dependent on brain capillary TRPA1 channels.
(A and B) Representative traces (A) and summary data (B) showing the hyperemic response in the somatosensory cortex following contralateral whisker stimulation (WS) for 5 s, measured using …
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Figure 8—source data 1
Individual data points and analysis summaries for datasets shown in Figure 8.
- https://cdn.elifesciences.org/articles/63040/elife-63040-fig8-data1-v2.xlsx
Figure 8—figure supplement 1
Experimental illustration of how functional hyperemia was measured in vivo.
(A) Functional hyperemia was assessed in the somatosensory cortex through a thinned skull. Relative changes in blood flow in response to contralateral whisker stimulation were recorded using …
Figure 8—figure supplement 2
Lack of a hyperemic response following ipsilateral whisker stimulation.
(A and B) Summary data showing the lack of a hyperemic response in the somatosensory cortex following 5 s ipsilateral whisker stimulation, measured using laser-Doppler flowmetry in mice prior to and …
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Figure 8—figure supplement 2—source data 1
Individual data points and analysis summaries for datasets shown in Figure 8—figure supplement 2.
- https://cdn.elifesciences.org/articles/63040/elife-63040-fig8-figsupp2-data1-v2.xlsx
Figure 8—figure supplement 3
Functional hyperemic response following 2 s whisker stimulation.
(A and B) Representative traces (A) and summary data (B) showing the hyperemic response was unchanged following a 2 s whisker stimulation in animals treated with HC-030031 (100 mg/kg, i.p. for 30 …
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Figure 8—figure supplement 3—source data 1
Individual data points and analysis summaries for datasets shown in Figure 8—figure supplement 3.
- https://cdn.elifesciences.org/articles/63040/elife-63040-fig8-figsupp3-data1-v2.xlsx
Videos
Video 1
Localized application of Evans Blue dye onto capillary extremities of a microvascular preparation.
Representative time-series images of a microvascular preparation demonstrating that application of Evans Blue dye (1% w/v) is localized to the region of the capillary tree and does not spread to the …
Video 2
Localized application of AITC onto capillary extremities of a microvascular preparation dilates the upstream arteriole.
Representative time-series images of a microvascular preparation demonstrating that localized application of AITC (30 µM) onto capillary extremities dilates the upstream arteriole. AITC was applied …
Video 3
Focal application of AITC onto capillary extremities induces an increase in intracellular [Ca2+].
Representative time-series images of a microvascular preparation demonstrating that localized application of AITC (30 µM) onto distal capillary extremities produces a propagative Ca2+ signal. AITC …
Video 4
Increase in intracellular [Ca2+] initiated by AITC is blocked by the selective TRPA1 antagonist HC-030031.
Representative time-series images of a microvascular preparation demonstrating that the propagative Ca2+ signal produced by AITC (30 µM) is blocked by superfusing the preparation with HC-030031 (10 …
Video 5
Focal application of ATP onto capillary extremities induces an increase in intracellular [Ca2+].
Representative time-series images of a microvascular preparation demonstrating that localized application of ATP (10 µM) onto distal capillary extremities produces a propagative Ca2+ signal. ATP was …
Video 6
Increase in intracellular [Ca2+] initiated by AITC is blocked by the pan-P2X inhibitor PPADS.
Representative time-series images of a microvascular preparation demonstrating that the propagative Ca2+ signal produced by ATP (10 µM) is blocked by superfusing the preparation with PPADS (10 µM). …
Video 7
ATP-induced Ca2+ signal under normal conditions.
Representative time-series images of a microvascular preparation demonstrating that localized application of ATP (10 µM) onto distal capillary extremities produces a propagative Ca2+ signal in …
Video 8
ATP-induced Ca2+ signal is abolished in extracellular Ca2+-free conditions.
Representative time-series images of a microvascular preparation demonstrating that localized application of ATP (10 µM) onto distal capillary extremities failed to induced a propagative Ca2+ signal …
Video 9
ATP-induced Ca2+ signal is restored following reintroduction of extracellular Ca2+.
Representative time-series images of a microvascular preparation demonstrating that the propagative Ca2+ signal following localized application of ATP (10 µM) onto distal capillary extremities …
Video 10
Localized application of Evans Blue dye onto the post-arteriole transitional segment of a microvascular preparation.
Representative time-series images of a modified microvascular preparation in which the capillary tree was removed. Application of Evans Blue dye (1% w/v) onto the post-arteriole transitional segment …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (M. musculus) | C57BL/6J | Jackson Laboratory | Stock #: 000664 RRID:IMSR_JAX:000664 | |
| Genetic reagent (M. musculus) | Panx1-ecKO | Dr. Brant Isakson PMID:26242575 | ||
| Genetic reagent (M. musculus) | TekCre | Jackson Laboratory | Stock #: 008863 RRID:IMSR_JAX:008863 | |
| Genetic reagent (M. musculus) | Trpa1fl/fl | Jackson Laboratory | Stock #: 008654 RRID:IMSR_JAX:008654 | |
| Genetic reagent (M. musculus) | Cdh5-GCaMP8 | CHROMus(https://chromus.vet.cornell.edu/cdh5gcamp8/) PMID:23240011 | ||
| Antibody | Cy3 conjugated anti-α-smooth muscle actin (Mouse monoclonal) | Sigma-Aldrich, Inc | Cat. #: C6198 RRID:AB_476856 | (1:200, 1.0–1.5 mg/ml) |
| Other | Alexa Fluor 488 conjugated isolectin B4 | ThermoFisher Scientific | Cat. #: I21411 | (1:200, 1.0 mg/ml) |
| Other | Alexa Fluor 568 conjugated isolectin B4 | ThermoFisher Scientific | Cat. #: I21412 | (1:200, 1.0 mg/ml) |
| Other | Alexa Fluor 633 conjugated hydrazide | ThermoFisher Scientific | Cat. #: A30634 | (1:1000, 1.0 mg/ml) |
| Other | DAPI Fluoroshied mounting medium | Abcam plc. | Cat. #: ab104139 | |
| Other | Fluorescein isothiocyanate (FITC)-dextran | Sigma-Aldrich, Inc | Cat. #: FD150S | |
| Other | Tetramethylrhodamine isothiocyanate (TRITC)-dextran | Sigma-Aldrich, Inc | Cat. #: T1287 | |
| Peptide, recombinant protein | Apamin | Tocris Bioscience | Cat. #: 1652 | |
| Peptide, recombinant protein | Apyrase | Sigma-Aldrich, Inc | Cat. #: A6535 | |
| Peptide, recombinant protein | Collagenase type I | Worthington Biochemical Corporation | Cat. #: LS004194 | |
| Peptide, recombinant protein | Elastase | Worthington Biochemical Corporation | Cat. #: LS002292 | |
| Peptide, recombinant protein | Neutral protease | Worthington Biochemical Corporation | Cat. #: LS02104 | |
| Chemical compound, drug | 4-hydroxynonenal (4-HNE) | Cayman Chemical | Cat. #: 32100 | |
| Chemical compound, drug | Adenosine 5-triphosphate (ATP) disodium salt | Sigma-Aldrich, Inc | Cat. #: A2383 | |
| Chemical compound, drug | Allyl isothiocyanate (AITC) | Sigma-Aldrich, Inc | Cat. #: 377430 | |
| Chemical compound, drug | HC-030031 | Tocris Bioscience | Cat. #: 2896 | |
| Chemical compound, drug | Indomethacin | Sigma-Aldrich, Inc | Cat. #: I7378 | |
| Chemical compound, drug | NS309 | Tocris Bioscience | Cat. #: 3895 | |
| Chemical compound, drug | Nω-Nitro-L-arginine methyl ester (L-NAME) hydrochloride | Sigma-Aldrich, Inc | Cat. #: N5751 | |
| Chemical compound, drug | PPADS tetrasodium salt | Tocris Bioscience | Cat. #: 0625 | |
| Chemical compound, drug | Tamoxifen | Sigma-Aldrich, Inc | Cat. #: T5648 | |
| Software, algorithm | pClamp software | Molecular Devices, LLC. (http://www.moleculardevices.com/products/software/pclamp.html) | RRID:SCR_011323 | Version 10.2 |
| Software, algorithm | FluoView FV1000 FV10-ASW software | Olympus (https://www.olympus-lifescience.com/en/support/downloads/) | RRID:SCR_014215 | Version 4.02 |
| Software, algorithm | GraphPad Prism software | GraphPad Software, Inc (https://www.graphpad.com/) | RRID:SCR_002798 | Version 8.2 |
| Software, algorithm | ImageJ software | National Institutes of Health (https://imagej.nih.gov/ij/) | RRID:SCR_003070 | Version 1.52 n |
| Software, algorithm | IonWizard software | IonOptix, LLC. (https://www.ionoptix.com/products/software/ionwizard-core-and-analysis/) | Version 6.4.1.73 | |
| Software, algorithm | VisiView software | Visitron Systems GmbH (https://www.visitron.de/products/visiviewr-software.html) | Version 4.5.0.7 | |
| Software, algorithm | μManager software | University of California, San Francisco (https://micro-manager.org/) | RRID:SCR_000415 | Version 1.4.22 |
