Astrocytic endfoot Ca2+ and BK channels determine both arteriolar dilation and constriction

Abstract

Neuronal activity is thought to communicate to arterioles in the brain through astrocytic calcium (Ca(2+)) signaling to cause local vasodilation. Paradoxically, this communication may cause vasoconstriction in some cases. Here, we show that, regardless of the mechanism by which astrocytic endfoot Ca(2+) was elevated, modest increases in Ca(2+) induced dilation, whereas larger increases switched dilation to constriction. Large-conductance, Ca(2+)-sensitive potassium channels in astrocytic endfeet mediated a majority of the dilation and the entire vasoconstriction, implicating local extracellular K(+) as a vasoactive signal for both dilation and constriction. These results provide evidence for a unifying mechanism that explains the nature and apparent duality of the vascular response, showing that the degree and polarity of neurovascular coupling depends on astrocytic endfoot Ca(2+) and perivascular K(+).

Fig. 1.

Level of astrocytic endfoot Ca²⁺ determines arteriolar dilation and constriction. (A) An astrocytic endfoot on an arteriole in a brain slice loaded with the Ca²⁺ cage, DMNP-EDTA, before (Control) and after (Uncaging) two-photon Ca²⁺ uncaging in the region of interest (red squares). Shown are infrared differential interference contrast (IR-DIC) transmitted light images overlaid with pseudocolor-mapped [Ca²⁺]_i (based on Fluo-4 fluorescence). The slice shown on Left was loaded with DMNP-EDTA for 60 min, whereas the one shown on Right was loaded for 120 min. The vessel lumen is denoted by red dotted lines. (Scale bar: 10 μm.) (B) Traces showing the time course of changes in endfoot Ca²⁺ (red) and arteriole diameter (green) after Ca²⁺ uncaging. Normal Ca²⁺ increases led to dilation (Left), whereas high Ca²⁺ increases caused constriction (Right). Red arrows indicate the onset of Ca²⁺ uncaging. (C) Scatter plot illustrating the relationship between astrocytic endfoot [Ca²⁺]_i and the changes in the arteriole diameter after EFS (blue dots) and Ca²⁺ uncaging (red dots). Each dot represents an individual endfoot/arteriole (n = 40 arterioles in 35 brain slices from 22 animals).

Fig. 2.

BK channels are required for both vasodilations and constrictions induced by astrocytic endfoot Ca²⁺ increases. Time course of arteriolar diameter changes (green) to (A) normal and (B) high Ca²⁺ increases, measured as F/F₀ (red), after Ca²⁺ uncaging before and after BK-channel block with paxilline (1 μM). Red arrows indicate the onset of Ca²⁺ uncaging. (C) Summary of percentage diameter changes in response to astrocytic endfoot [Ca²⁺]_i increases before and after application of paxilline for 20 min. Both EFS and Ca²⁺ uncaging (DMNP-EDTA) were used to induce astrocytic [Ca²⁺]_i increases in brain slices. (P < 0.01 for all control versus paxilline groups; paired t test; n = 5–7 arterioles). (D) Paxilline alone caused a small constriction of arterioles preconstricted with 125 nM U46619 (P < 0.05; n = 4 arterioles) but did not affect the diameter of nonpreconstricted arterioles (n = 5).

Fig. 3.

Elevation of extracellular K⁺ dilates and constricts isolated pressurized arterioles. Representative traces (A) and summary data (B) illustrating the relationship between [K⁺]_o and changes in diameter of isolated, pressurized (40 mmHg) parenchymal arterioles (n = 8). The K_ir-channel blocker, Ba²⁺ (30 μM), prevented dilations but not constrictions to [K⁺]_o (n = 4).

Fig. 4.

Elevation of extracellular K⁺ converts endfoot Ca²⁺-induced dilations to constrictions. (A and B) Examples of simultaneous recording of changes in arteriolar diameter and [Ca²⁺]_i after Ca²⁺ uncaging in brain slices superfused with 3 mM or 8 mM K⁺ showing the images (A) and time course of changes (B). Ba²⁺ (100 μM) was added to 8 mM K⁺ aCSF to inhibit the vasodilation induced by elevated K⁺. Elevation of [K⁺]_o from 3 mM to 8 mM converted endfoot Ca²⁺-uncaging–induced dilation to constriction (Left) without affecting [Ca²⁺]_i. Paxilline inhibited the endfoot Ca²⁺-evoked constriction in 8 mM K⁺ and Ba²⁺ (Right). Red arrows indicate the onset of Ca²⁺ uncaging. The lumen of an arteriole is denoted by the dotted red lines. (Scale bar: 10 μm.) (C) Summary of diameter (Left) and [Ca²⁺]_i (Right) changes in response to uncaging in brain slices perfused with aCSF containing 3 mM or 8 mM [K⁺]_o in the presence or absence of Ba²⁺ and paxilline (n = 5–10 arterioles). Paxilline inhibition of constriction was statistically significant (P < 0.05; n = 5). [Ca²⁺]_i after uncaging was not different for the four groups (P < 0.05). [K⁺]_o was elevated by increasing the KCl concentration at the expense of NaCl.

Fig. 5.

Regulation of CBF responses to whisker stimulation and astrocyte activation by BK channels, Kir channels, and [K⁺]_o. (A) Local administration of the BK-channel blocker, paxilline (1 μM), or K_ir-channel blocker, Ba²⁺ (100 μM), significantly reduced cortical CBF response to whisker stimulation and astrocyte activation (50 μM t-ACPD) in wild-type mice (A, B, and F) and in mice lacking the β1 subunit of the BK channel (P < 0.05; one-way ANOVA; n = 5–6 mice/group) (D). It did not significantly reduce cortical CBF response in mice lacking the α subunit of the BK channel (n = 4–5 mice/group). (C) In all cases, the effects of paxilline and barium on CBF were not statistically different from paxilline or barium alone. Panel A shows representative traces of the effects of BK- and K_ir-channel blockers on the whisker stimulation-induced increase in CBF. Paxilline or Ba²⁺ was superfused over the cranial window for 20 min before and after whisker stimulation. (E) Representative traces show the effects of 3 mM and 15 mM KCl on the CBF responses to astrocyte stimulation with t-ACPD in the presence or absence of Ba²⁺ and paxilline; TTX (3 μM) was included to block potential neuronal effects. (F) Summary data of t-ACPD–induced changes in CBF in the presence of 3 mM and 15 mM [K⁺]_o, illustrating the ability of [K⁺]_o to convert hyperemic responses to decreases in CBF, which are also blocked by paxilline (P < 0.05; n = 5 mice/group).