Astrocytes mediate neurovascular signaling to capillary pericytes but not to arterioles

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.

Figure 1. Neuronal activity evokes capillary dilation.

(a) A cortical capillary response to 200 nM U46619 and superimposed neuronal stimulation (stim). Lines show lumen diameters plotted in b. (b) U46619 evoked constriction and stimulation-evoked dilation at regions indicated in a (in this and subsequent example traces, a large response is shown for illustrative purposes). (c) 500 nM TTX blocks stimulation-evoked capillary dilation. (d) 10 µM NBQX blocks stimulation-evoked dilation. (e) 25 µM D-AP5 did not reduce stimulation-evoked dilation. (f-h) Mean data showing the block of capillary dilation by TTX (f) and NBQX (g) but not by D-AP5 (h). Numbers on bars are capillary regions (putative pericytes) studied. Data are shown as box and whisker plots as defined in the Statistics part of the Methods.

Figure 2. P2X₁-evoked astrocyte Ca²⁺ signalling mediates capillary-level neurovascular coupling.

(a) A DIC image of a cortical capillary (left panel) and the AlexaFluor 488 fill of the astrocyte network after whole-cell patch-clamp dialysis for 1 min (middle) and 10 mins (right) after breaking into the cell. Whole-cell patch-clamped cell is indicated by arrowhead and endfoot indicated by arrow. (b) Example traces and (c) mean data demonstrating that stimulation-evoked capillary dilation is intact when the astrocyte network is dialyzed with a control internal solution containing 1 mM EGTA but significantly reduced when filled with 30 mM BAPTA, a fast Ca²⁺-chelator. (d-f) An inhibitor of group I and II mGluRs, S-MCPG (1 mM; d), the P2Y₁ blocker MRS2179 (25 μM; e) and the TRPA1 blocker A967079 (10 μM; f) do not block stimulation-evoked capillary dilation. (g) The P2X₁ blocker NF449 (100 nM) significantly reduced stimulation-evoked capillary dilation. (h) Puff-application of the P2X₁ agonist α,β-methylene ATP (α,β-meATP, 100 µM) to the neuropil downstream of the vessel induces capillary dilation. (i-l) Quantification of the effect of S-MCPG (i), MRS2179 (j), A967079 (k) and NF449 (l) on capillary dilation. (m) Mean response of capillaries in experiments like those in h, puffing external solution (control) or α,β-methylene ATP. Change in diameter in control experiments was measured as a 30 s average centred around the largest response seen between 30 and 120 s after puff of α,β-methylene ATP. Data are shown as box and whisker plots as defined in the Statistics part of the Methods.

Figure 3. Neuronally-evoked astrocyte [Ca²⁺]_i rise depends on P2X₁ receptors.

(a) An astrocyte patch-loaded with AlexaFluor 594 (top) and Fluo4 (bottom) showing the processes, endfoot and soma. White dashed lines delineate the vessel lumen ensheathed by the endfoot process. (b) Neuronal stimulation evoked a [Ca²⁺]_i rise in astrocyte endfeet in control conditions (left); the rise was smaller in the presence of the P2X₁ blocker NF449 (100 nM, right). Arrow indicates a spontaneous Ca²⁺ transient, demonstrating that Fluo-4 could still detect [Ca²⁺]_i transients with NF449 present. (c) Mean change in neuronally-evoked astrocyte [Ca²⁺]_i in somata, processes and endfeet in the absence and presence of different blockers. NF449 (black bars), a blocker of P2X1 channels, significantly reduced the [Ca²⁺] rise in processes and endfeet, but S-MCPG, a blocker of groups I and II mGluRs had no effect on astrocyte [Ca²⁺] in any compartment (grey bars). Time to 10% of the peak and to the peak [Ca²⁺] change in endfeet were 5.27+1.15 s and 11.57+2.39 s respectively. Data are shown as box and whisker plots as defined in the Statistics part of the Methods.

Figure 4. AA metabolites mediating stimulation-evoked capillary dilation.

(a) The COX1 blocker SC-560 (1 μM) blocks the dilation. (b,c) The COX2 blocker NS-398 (10 μM; b) and the epoxygenase inhibitor PPOH (25 μM; c) had no effect on the dilation. (d-f) Mean data for the effect of SC-560 (d), NS-398 (e) and PPOH (f) on capillary dilation. (g) Blocking the PGE₂ receptor EP₄ with L-161,982 (1 μM) inhibits dilation. (h,i) Blocking the PGI₂ receptor IP with CAY10441 (1 μM; h) or NO synthase with L-NNA (100 μM; i) has no effect on capillary dilation. (j-l) Mean data showing that capillary dilation is blocked by L-161,982 (j), but not by CAY10441 (k) or L-NNA (l). Data are shown as box and whisker plots as defined in the Statistics part of the Methods.

Figure 5. PLD2, not PLA₂, initiates neurovascular coupling at the capillary level.

(a) The PLA₂ inhibitor MAFP (10 μM) does not block stimulation-evoked capillary dilation. (b) The PLC blocker, U73122 (10 μM), does not reduce dilation. (c) The PLD blocker FIPI (1 μM) inhibits dilation. (d-f) Mean data showing the effects of MAFP (d), U73122 (e) and FIPI (f) on capillary dilation. (g) Blocking PLD1 with VU0155069 (500 nM) had no effect on the dilation. (h-i) The PLD2 blocker CAY10594 (1 μM; h) and the DAGL blocker RHC80267 (50 μM; i) significantly reduce the capillary dilation. (j-l) Mean data showing the effect of VU0155069 (j), CAY10594 (k) and RHC80267 (l) on capillary dilation. Data are shown as box and whisker plots as defined in the Statistics part of the Methods.

Figure 6. Neurovascular signalling to arterioles is mediated by NMDAR and NOS activity, and not by astrocyte Ca²⁺.

(a) Example traces and (b) mean data demonstrating that stimulation-evoked arteriole dilation is not altered when the astrocyte network is dialyzed with 30 mM BAPTA, a fast Ca²⁺-chelator, compared to a control internal solution containing 1 mM EGTA. (c-e) The P2X₁ blocker NF449 (100 nM; c), the PLA₂ inhibitor MAFP (10 μM; d) and the PLD2 blocker CAY10594 (1 μM; e) do not block stimulation-evoked arteriole dilation. (f-g) The NMDA receptor blocker D-AP5 (25 μM; f) and the NO synthase blocker L-NNA (100 μM; g) abolished the arteriole dilation. Mean data showing the effect of NF449 (h), MAFP (i), CAY10594 (i), D-AP5 (j) and L-NNA (k) on arteriole dilation. Data are shown as box and whisker plots as defined in the Statistics part of the Methods.

Figure 7. Neurovascular signalling to capillaries in vivo is mediated by P2X₁ receptors.

(a) Two-photon stack (90 µm thick, maximum intensity projection) of FITC-dextran-filled vessels (green) in the somatosensory cortex of an anaesthetised rat. (b) Enlarged image showing a penetrating arteriole with a capillary branching off it. (c-d) Example traces of forepaw stimulation-evoked dilation of arterioles (c) and capillaries (d) in the presence of vehicle (aCSF, black traces) or the P2X₁ blocker NF449 (5 μM; red traces). Capillary dilations occurred faster than arteriole dilations (time to 10% dilation was 3.0±0.8s for 12 capillaries and 5.1±1.7s for 9 arterioles), as previously reported⁶; however, reflecting the smaller number of vessels studied, this effect did not reach significance in this study (p=0.3). (e) The percentage of arterioles (Art) and capillaries (Cap) that dilated in response to stimulation was not significantly different in NF449 and vehicle-treated animals. (f) Forepaw stimulation-evoked mean arteriolar dilation was similar in vehicle and NF449 treated animals, but capillary dilation was significantly inhibited by NF449. Data are shown as box and whisker plots as defined in the Statistics part of the Methods.