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Astrocytes mediate neurovascular signaling to capillary pericytes but not to arterioles

Nature Neuroscience volume 19, pages 1619–1627 (2016)Cite this article

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A Publisher Correction to this article was published on 13 July 2020

A Corrigendum to this article was published on 26 July 2017

This article has been updated

Abstract

Active neurons increase their energy supply by dilating nearby arterioles and capillaries. This neurovascular coupling underlies blood oxygen level–dependent functional imaging signals, but its mechanism is controversial. Canonically, neurons release glutamate to activate metabotropic glutamate receptor 5 (mGluR5) on astrocytes, evoking Ca2+ release from internal stores, activating phospholipase A2 and generating vasodilatory arachidonic acid derivatives. However, adult astrocytes lack mGluR5, and knockout of the inositol 1,4,5-trisphosphate receptors that release Ca2+ from stores does not affect neurovascular coupling. We now show that buffering astrocyte Ca2+ inhibits neuronally evoked capillary dilation, that astrocyte [Ca2+]i is raised not by release from stores but by entry through ATP-gated channels, and that Ca2+ generates arachidonic acid via phospholipase D2 and diacylglycerol lipase rather than phospholipase A2. In contrast, dilation of arterioles depends on NMDA receptor activation and Ca2+-dependent NO generation by interneurons. These results reveal that different signaling cascades regulate cerebral blood flow at the capillary and arteriole levels.

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Figure 1: Neuronal activity evokes capillary dilation.
Figure 2: P2X1-evoked astrocyte Ca2+ signaling mediates capillary-level neurovascular coupling.
Figure 3: Neuronally evoked astrocyte [Ca2+]i rise depends on P2X1 receptors.
Figure 4: AA metabolites mediating stimulation-evoked capillary dilation.
Figure 5: PLD2, not PLA2, initiates neurovascular coupling at the capillary level.
Figure 6: Neurovascular signaling to arterioles is mediated by NMDAR and NOS activity, and not by astrocyte Ca2+.
Figure 7: Neurovascular signaling to capillaries in vivo is mediated by P2X1 receptors.

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Change history

  • 05 December 2016

    In the version of this article initially published, the abstract referred to diacylglycerol kinase; this should have been diacylglycerol lipase. The error has been corrected in the HTML and PDF versions of the article.

  • 13 July 2020

    A Correction to this paper has been published: https://doi.org/10.1038/s41593-020-0680-0

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Acknowledgements

We thank K. Zheng for help with two-photon microscopy, and M. Carandini, I. Christie, S. Cockcroft, M. Ford, R. Jolivet, S. Mastitskaya and S. Sullivan for comments on the manuscript. Supported by the European Research Council (BRAINPOWER to D.A. and NETSIGNAL to D.A.R.), a Fondation Leducq Transatlantic Network grant (to D.A.), a Wellcome Trust Programme Grant and Senior Investigator Award (075232 and 099222 to D.A.), a Wellcome Trust Senior Research Fellowship (095064 and 200893 to A.V.G.), a Wellcome Trust Principal Research Fellowship (101896 to D.A.R.), an EU FP7 grant (ITN EXTRABRAIN 606950 to D.A.R.) and an RSF grant (15-14-30000 to D.A.R.).

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Authors and Affiliations

  1. Department of Neuroscience, Physiology & Pharmacology, University College London, London, UK

    Anusha Mishra, Yang Chen, Alexander V Gourine & David Attwell

  2. Institute of Neurology, University College London, London, UK

    James P Reynolds & Dmitri A Rusakov

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Contributions

A.M. and D.A. conceived the study. A.M. carried out all brain slice experiments, some immunocytochemistry, and analysis of brain slice and in vivo data; J.P.R. performed in vivo experiments and analyzed the resulting data; Y.C. carried out immunocytochemistry; A.V.G. and D.A.R. provided in vivo imaging expertise; A.M. and D.A. wrote the paper; all authors revised the paper.

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Correspondence to David Attwell.

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Integrated supplementary information

Supplementary Figure 1 Schematic diagram of the mechanisms underlying neurovascular signalling to capillaries and arterioles.

Synaptic activity (top) evokes ATP release from post-synaptic neurons, which activates ionotropic ATP receptors containing P2X1 subunits on astrocytes, leading to a rise in [Ca2+]i via influx from the extracellular space. This rise in [Ca2+]i activates PLD2, resulting in the formation of phosphatidic acid (PA) which is converted into diacylglycerol (DAG), which is then further metabolized by DAG lipase into arachidonic acid (AA). AA is then metabolized, by the consecutive activity of COX1 and PGES, to produce PGE2, which dilates capillaries by acting on EP4 receptors, presumably on pericytes. Synaptic activation of Ca2+ entry through NMDA receptors increases NOS activity in interneurons, resulting in NO release onto arterioles to dilate them. The neurovascular coupling pathways involved in signalling to capillaries and arterioles are highlighted in blue, while the enzymes and receptors ruled out by our experiments are shown in red with black crosses.

Supplementary Figure 2 Stimulation-evoked field potentials and identification of vessels.

(a-b) A schematic (a) and a low-magnification image (b) of a cortical slice demonstrating the placement of the stimulation and recording electrodes. Cortical layers are indicated. Inset shows a high magnification image of the recording electrode near a capillary. (c) Stimulation-evoked fibre volley and field excitatory post-synaptic currents (fEPSCs) are not affected by U46619. NBQX blocks the fEPSCs but not the fibre volley and TTX blocks both the fibre volley and the fEPSCs. (d) Mean data (peak fEPSC amplitude measured 5 to 9 ms after the stimulation) showing the effect of U46619 and NBQX on fEPSCs. (e) Arterioles can be distinguished by the thick layer of smooth muscle cells (SMC, white brackets) that surround them, shown here alongside FITC-isolectin B4 (IB4) labeling of the basement membrane. (f, g) Capillaries are identified as smaller vessels that have only occasional pericyte cell bodies (arrowheads) outlined by the IB4-labeling. IB4 also labels microglia (asterisk). Vessel lumen is marked by red brackets. Data shown as mean±s.e.m.

Supplementary Figure 3 Constriction of cortical capillaries evoked by U46619 before each experiment.

(a) Example trace of a U46619 (200 nM) induced capillary constriction lasting at least 30 minutes. (b-x) Mean constriction of capillaries to U46619 in interleaved control and drug experiments for the: (b) voltage-gated sodium channel blocker TTX, (c) AMPA/KA receptor blocker NBQX, (d) NMDA receptor blocker D-AP5, (e) fast Ca2+ chelator BAPTA, (f) inhibitor of group I and II mGluRs S-MCPG, (g) P2Y1 blocker MRS2179, (h) TRPA1 blocker A967079, (i) P2X1 blocker NF449, (j) P2X1 blocker NF023, (k) COX1 blocker SC-560, (l) COX2 blocker NS-398, (m) epoxygenase blocker PPOH, (n) EP4 receptor blocker L-161,982, (o) IP receptor blocker CAY10441, (p) NO synthase blocker L-NNA, (q) PLA2 blocker MAFP, (r) PLC blocker U73122, (s) PLD blocker FIPI, (t) PLD1 blocker VU0155069, (u) PLD2 blocker CAY10594, (v) DAGL blocker RHC80267, (w) P2X1 agonist α,β-meATP, and (x) PGE2 in the presence or absence of the PLD blocker FIPI. P-values comparing U46619-evoked constrictions for control and drug experiments were >0.05 for all panels even without correcting for multiple comparisons. For experiments involving BAPTA (e), PPOH (m) and L-161,982 (n), U-46619 was applied in the presence of the relevant drug (see Methods). For all other experiments, vessels were pre-constricted with U46619 before drug application. Data shown as mean±s.e.m.

Supplementary Figure 4 Signaling to capillary pericytes is similar for different stimulation durations in P21 rats, and in P45 rats.

Mean dilation (left panels) and time traces of the diameter change (right panels) observed in cortical capillaries following electrical stimulation of neuronal activity (at 100 Hz for 0.2 sec, repeated once/sec) in the absence and presence of P2X1 blockers. (a-c) Capillary dilation evoked by short (200 ms; a), medium (5 sec; b) or long duration (1 min; c, reproduced from Fig. 2l) stimulation in slices from P21 rats is inhibited by the P2X1 blocker NF449 (100 nM). (d) Stimulation-evoked capillary dilation in slices from P21 rats is inhibited by a second P2X1 blocker NF023 (5 μM). (e) Stimulation-evoked capillary dilation in slices from P45 rats is also inhibited by the P2X1 blocker NF449 (100 nM). Data shown as mean±s.e.m.

Supplementary Figure 5 COX1 and PGES are expressed in astrocyte endfeet along vessels.

(a) AQP4-expressing astrocyte endfeet along arterioles (arrows) and capillaries (arrowheads) are immunoreactive for COX1. Vascular basement membrane is labeled with Alexa dye conjugated isolectin B4 (IB4). (b) COX2 is absent from GFAP-expressing astrocyte somata (white asterisk) and endfeet (arrowhead) but is expressed in neuronal cell bodies (black asterisk). (c) Epoxygenase (CYP2C11) is expressed in AQP4-expressing astrocyte endfeet along arterioles (arrows) but not in those along capillaries (arrowhead). Arterioles can be identified as larger vessels, often with space between layers of IB4-labeled basement membrane (white circle) where the vascular smooth muscle cells are located. (d) PGES is expressed in GFAP-expressing astrocyte cell bodies (white asterisk), processes and endfeet along vessels (arrowheads). (e) Control experiments where slices were incubated with the secondary antibody used to stain the enzyme shown in the corresponding panels in a-d, but with the primary antibody omitted, demonstrating the lack of non-specific binding. Control for the secondary antibody used to detect COX1 is shown in the top row, COX2 in the second row, epoxygenase in third row and PGES in the fourth row.

Supplementary Figure 6 PLA2 is expressed in GFAP-labeled astrocyte endfeet along the vasculature.

Arrowhead indicates a pericyte cell body.

Supplementary Figure 7 PLD is upstream of PGE2 in the signaling pathway.

(a) After preconstriction with 200 nM U46619, applying 1 μM PGE2 evokes a dilation. (b) A similar PGE2-evoked dilation was seen in the presence of 1 μM FIPI to block PLD1 and PLD2. (c) Quantification of data from experiments like those in panels a-b, showing that PLD inhibition does not affect the capillary dilation evoked by PGE2. Data shown as mean±s.e.m.

Supplementary Figure 8 PLD1 and PLD2 expression in the cortex.

(a, b) PLD1 was diffusely expressed in the cortical neuropil and appeared concentrated in endothelial cells along arterioles (arrow, a) but not capillaries (b). No PLD1 labeling was observed in AQP4-labeled endfeet (arrowheads). (c, d) PLD2 expression was detected in GFAP-expressing astrocyte endfeet along capillaries (arrowheads, c) as well as in astrocyte somata (asterisks, d). (e) Control experiments where slices were labeled with the secondary antibody used to detect the enzymes shown in the corresponding panels in a-d, but with the primary antibody omitted, demonstrating the lack of non-specific binding. Control for secondary antibodies used to detect PLD1 is shown in the top two panels and for PLD2 is shown in the last two panels (for these pictures illumination was applied appropriate for evoking fluorescence from the secondary antibody used for PLD1/PLD2 labeling and from the dye-conjugated IB4 or DAPI).

Supplementary Figure 9 Constriction of cortical arterioles evoked by U46619 before each experiment.

Mean constriction of arterioles to U46619 in interleaved control and drug experiments for the: (a) P2X1 blocker NF449, (b) PLA2 blocker MAFP and PLD2 blocker CAY10594 (experiments were on the same animals with interleaved controls in common), (c) fast Ca2+ chelator BAPTA, (d) NMDA receptor blocker D-AP5, (e) NO synthase blocker L-NNA. P-values comparing U46619-evoked constrictions for control and drug experiments were >0.05 for all panels even without correction for multiple comparisons. Data shown as mean±s.e.m.

Supplementary Figure 10 Experimental setup for puffing α,β-methylene-ATP.

(a) DIC image of slice showing a capillary. (b) DIC image of the slice surface showing position of the puff pipette. (c) Fluorescence image showing the pipette containing Alexa Fluor 594 positioned above the slice. (d-i) The spread of the Alexa Fluor 594 dye imaged at the start (d) and 5 consecutive seconds (e-i) following a 5 s puff at 5 psi. Note how the dye flows downwards and does not reach the location of the vessel at the top (demarcated by white dotted lines).

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Mishra, A., Reynolds, J., Chen, Y. et al. Astrocytes mediate neurovascular signaling to capillary pericytes but not to arterioles. Nat Neurosci 19, 1619–1627 (2016). https://doi.org/10.1038/nn.4428

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