Local potassium signaling couples neuronal activity to vasodilation in the brain

Jessica A Filosa^1, 2, Adrian D Bonev¹, Stephen V Straub¹, Andrea L Meredith^3, 4, M Keith Wilkerson¹, Richard W Aldrich^3, 4 & Mark T Nelson¹

¹ Department of Pharmacology, 89 Beaumont Avenue, College of Medicine, University of Vermont, Burlington, Vermont 05405, USA.

² Department of Psychiatry, University of Cincinnati, Cincinnati, Ohio 45237, USA.

³ Department of Molecular and Cellular Physiology and the Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA.

⁴ Present addresses: Department of Physiology, University of Maryland School of Medicine, 655 West Baltimore Street (BRB5), Baltimore, Maryland 21201, USA (A.L.M.) and Section of Neurobiology, 1 University Station C7000, University of Texas at Austin, Austin, Texas 78712, USA (R.W.A.).

Elevation of the concentration of external potassium ions ([K⁺]_o) to levels sufficient to depolarize SMCs (20 mM) induces vasoconstriction of cerebral arteries¹⁰ and arterioles¹¹. Paradoxically, a modest elevation of [K⁺]_o (<20 mM) is one of the most potent vasodilatory signals in the cerebrovasculature^{10, 12, 13}. Elevation of [K⁺]_o activates strong inward rectifier K⁺ channels^{10, 12}, in particular the Kir2.1 channel, in SMCs isolated from cerebral arteries^{14, 15}. Increasing intravascular pressure from 10 mm Hg to 60 mm Hg depolarizes SMCs in cerebral (pial) arteries from about –63 mV to –45 mV, and elevation of [K⁺]_o to 16 mM results in Kir channel–mediated hyperpolarization of SMCs from –45 mV to about –58 mV, lowers average Ca²⁺ in SMCs in the arterial wall from 192 nM to 126 nM, and causes an approximately 60% vasodilation^{10, 16}. Although it is well established that extracellular cerebral (pial) arteries dilate to activation of SMC Kir channels, it is not known if these channels are expressed in SMCs of intracerebral (parenchymal) arterioles, and whether activation of these channels constitutes a mechanism through which active neurons induce vasodilation.

To establish whether functional Kir channels are expressed in SMCs of parenchymal arterioles, we isolated single SMCs from these arterioles and measured K⁺ currents using the whole-cell, perforated-patch-clamp technique. These currents exhibited characteristic properties of Kir2 channels: namely, steep activation with membrane potential hyperpolarization, shift of the activation curve to more positive potentials with an elevation of [K⁺]_o, potent inhibition by external barium (Ba²⁺) ions, and strong rectification (Fig. 1). In fact, the current density of Kir currents at -100 mV (-16.4 pA pF^-1 in 60 mM [K⁺]_o) in arteriolar myocytes was greater than that in myocytes derived from pial arteries (-11.1 pA pF^-1 in 60 mM [K⁺]_o)¹⁷. Elevation of [K⁺]_o from 3 mM to 6 mM or 10 mM shifted the whole-cell and Ba²⁺-sensitive (Kir) currents to more positive potentials (Fig. 1a,b) and increased Kir channel conductance at all membrane potentials (Fig. 1c). The half-activation voltage (V_0.5) of Kir channel conductance shifted with the K⁺ equilibrium potential (E_K; (V_0.5 was -111.3 0.1 mV in 3 mM [K⁺]_o,-82.4 0.1 mV in 6 mM [K⁺]_o, and –68.0 0.1 mV in 10 mM [K⁺]_o; n = 3–4), and the equivalent gating charge was 2.5 0.6, properties consistent with strong inward rectifier potassium channels¹⁸. Indeed, elevation of [K⁺]_o from 3 mM ([K⁺]_o in cerebrospinal fluid) to 10 mM increased the Kir current density 6.3-fold (P < 0.05) at -45 mV.

Figure 1. Kir channel currents in SMCs isolated from parenchymal arterioles. (a) Average leak-subtracted membrane current densities from SMCs in response to a 140-ms-long voltage ramp from –130 mV to 0 mV in the presence of 3 mM (green), 6 mM (blue) and 10 mM (red) [K+]o. (b) Average Ba2+ (100 M)-sensitive current densities recorded in the presence of 3 mM, 6 mM and 10 mM [K+]o. In a and b, the data represent the averaged I-V relationships from three cells for 3 mM [K+]o and four cells for both 6 mM and 10 mM [K+]o. (c) Hyperpolarization of the membrane potential and elevation of [K+]o increased Kir conductance. Chord conductance is plotted on a log scale to illustrate the Kir channel activation by [K+]o at physiological membrane potentials (-50 mV to -40 mV) for SMCs. The chord conductance traces were generated from the Ba2+-sensitive current densities displayed in b according to the following equation: chord conductance = current density/(V – Vrev), where V is the membrane potential and Vrev is the reversal potential for the current density. The current densities were first subjected to a fast Fourier transform (FFT) low-pass filter, which removes Fourier components with frequencies higher than 50 Hz. (d) Elevation of external [K+]o from 3 mM to 15 mM caused a membrane potential hyperpolarization of SMCs in the wall of a pressurized (40 mm Hg) parenchymal arteriole from the cortex. (e) Summary data of the membrane potential of SMCs in pressurized (40 mm Hg) parenchymal arterioles in the presence of 3 mM, 8 mM and 15 mM [K+]o. Data represent mean s.e.m. The membrane potentials in 8 mM and 15 mM [K+]o were significantly hyperpolarized relative to those in 3 mM [K+]o (six arterioles, P < 0.05). Full Figure and legend (37K)

The relevant ionic currents that regulate the membrane potential of arterial SMCs are small (<1 pA; Fig. 1 and refs. 18, 19, 20) and generally less than leak currents from the electrode seal (Methods). Furthermore, enzymatically isolated SMCs lose cell-cell coupling and are not subjected to constricting stimuli. Therefore, to determine the effect of [K⁺]_o on SMC membrane potential, we measured the membrane potential of SMCs in the wall of isolated intracerebral arterioles, using sharp microelectrodes. Parenchymal arterioles were pressurized to approximately physiological levels (40 mm Hg). Elevation of [K⁺]_o from 3 mM to 15 mM hyperpolarized the membrane potential of SMCs in isolated, pressurized parenchymal arterioles from about –40 mV to –60 mV (Fig. 1d), close to the estimated new E_K. This response would predict that elevation of [K⁺]_o to a level less than 15 mM might cause a greater hyperpolarization, as E_K would be more negative. Indeed, elevation of [K⁺]_o from 3 mM to 8 mM caused a membrane potential hyperpolarization from -44 2 mV to -80 7 mV (Fig. 1e), which is close to E_K (-76 mV) in 8 mM [K⁺]_o.

Given the relatively high Kir channel current density of arteriolar SMCs and the observation that [K⁺]_o induces hyperpolarization of SMCs in isolated parenchymal arterioles (Fig. 1), we predict that elevation of [K⁺]_o should dilate parenchymal arterioles in the brain. To evaluate the physiological response to [K⁺]_o, we examined the effects of elevating [K⁺]_o to 10 mM on arteriolar diameter and SMC Ca²⁺ oscillations in brain slices, using infrared differential interference contrast (IR-DIC) imaging and fluorescence confocal microscopy, respectively. As is the case with other resistance arterioles, parenchymal arterioles in vivo are partially constricted because of intravascular pressure and other contractile stimuli. These arterioles often exhibit smooth muscle Ca²⁺ oscillations and vasomotion in brain slices², an observation that is supported by in vivo measurements^{21, 22}. To maintain arteriolar tone over the course of the experiments at a level observed in isolated, pressurized parenchymal arterioles, we applied low concentrations (100–150 nM) of the thromboxane A₂ receptor agonist (9,11-dideoxy-11, 9-epoxymethanoprostaglandin F₂ (U46619)) to cortical brain slices²³. This approach permits the detection of processes that lead either to an increase or decrease of smooth muscle Ca²⁺ and arteriolar diameter.

Elevation of [K⁺]_o in the bath (artificial cerebrospinal fluid, aCSF) solution to 10 mM induced a reversible vasodilation (60%) of parenchymal arterioles, and suppressed SMC Ca²⁺ oscillations, as measured by changes in fluo-4 fluorescence (Fig. 2, and Supplementary Video 1 online). Ba²⁺ ions, which are relatively selective for Kir2 channels at low concentrations (<100 M; ref. 17) blocked the [K⁺]_o-induced vasodilation and suppression of SMC Ca²⁺ oscillations (percent inhibition of oscillation frequency = 68.7 15.8% control versus 12.6 6.0% in 100 M Ba²⁺). In fact, in the presence of Ba²⁺, 10 mM [K⁺]_o constricted arterioles by an average of 9.8 7.5% of resting diameter (n = 5; P < 0.05; Fig. 2a,d). Notably, 10 mM [K⁺]_o did not affect astrocytic endfoot Ca²⁺ (percent change in fractional fluorescence = 1.1 2.3%; n = 3). In addition, tetraethylammonium ions (TEA, 1 mM), which act rapidly and are relatively selective for BK channels²⁴, did not alter the dilation to 10 mM [K⁺]_o (dilation = 58.2 15.5% in control versus 52.6 9.5% in TEA; n = 5; Fig. 2b,d), indicating that SMC BK channels are not directly involved in the arteriolar response to [K⁺]_o. Furthermore, arteriolar diameter was not significantly altered by treatment with Ba²⁺ or TEA suggesting that, under these experimental conditions, neither Kir nor BK channels contribute substantially to the tone of parenchymal arterioles. These results support an important role for perivascular K⁺ signaling through the activation of arteriolar SMC Kir channels in the dynamic regulation of cerebrovascular tone.

Figure 2. Elevation of [K+]o to 10 mM dilates parenchymal arterioles and suppresses Ca2+ oscillations in arteriolar SMCs in brain slices. (a) Elevation of [K+]o from 3 mM to 10 mM induced vasodilation of an arteriole within a cortical brain slice (Supplementary Video 1). This vasodilation was prevented by treatment with the Kir channel blocker Ba2+ (100 M). (b) The BK channel blocker TEA (1 mM) did not prevent [K+]o induced vasodilation of an arteriole within the slice. (c) Representative trace illustrating the reversible suppression of arteriolar SMC Ca2+ oscillations from a parenchymal arteriole exposed to 10 mM [K+]o. (d) Mean data ( s.e.m) illustrating that the Kir channel blocker Ba2+, but not the BK channel blocker TEA, inhibits [K+]o-induced dilations of arterioles in brain slices (n = 5 each). In addition, treatment of arterioles with Ba2+ or TEA did not significantly alter arteriolar diameter. Mean diameter of arterioles in brain slices after 15 min incubation in 100 M Ba2+ was 96 3% of control diameter (n = 9). Mean diameter of arterioles in brain slices after 15 min incubation in 1 mM TEA was 100 4% of control diameter (n = 14). Full Figure and legend (28K)

[K⁺]_o can act through smooth muscle Kir channels to lower SMC Ca²⁺ and induce dilation. However, the contribution of this mechanism to the rapid coupling between neuronal activity and vasodilation is not known. Using high spatial- and temporal-resolution confocal Ca²⁺ imaging of cortical brain slices, we previously determined that neuronal activity induced via field or focal electrical stimulation causes an elevation of astrocytic Ca²⁺ and a rapid suppression of SMC Ca²⁺ oscillations (ref. 2). Electrical stimulation for 3–5 s induced a 40–50% vasodilation of cerebral arterioles in brain slices with a latency (from the beginning of electrical stimulation to the onset of dilation) of 1.8 0.4 s (n = 10; Fig. 3a). The rapid coupling of electrical stimulation and vasodilation is consistent with in vivo measurements of functional hyperemia^{25, 26}. Notably, this electrical stimulation–induced vasodilation was greatly reduced (71%) by inhibition of Kir channels with 100 M Ba²⁺ (46.3 11.9% dilation in control versus 13.5 2.6% dilation in Ba²⁺, n = 5; P < 0.05; Fig. 3a,b). In time-matched control experiments in the absence of Ba²⁺ or TEA, the second electrical stimulation–induced vasodilation did not differ significantly from the first (vasodilation induced by the second electrical stimulation was 96 5% of that induced by the first, n = 11; P < 0.05). Ba²⁺ did not affect electrical stimulation–induced astrocytic Ca²⁺ signals, consistent with fact that the locus of Ba²⁺ action is the arteriolar smooth muscle. These results support the hypothesis that SMC Kir channels are active participants in the process of neurovascular coupling.

Figure 3. Neuronal activity–induced dilation of arterioles is largely inhibited by blockers of Kir or BK channels in brain slices. (a) Representative traces illustrating the inhibition of electrical stimulation–induced vasodilation of an arteriole in a cortical slice by Ba2+ (100 M) (Supplementary Videos 2 and 3). (b) Mean data ( s.e.m) illustrating the block of electrical stimulation–induced vasodilation by Ba2+ and TEA (n = 5 each). (c) Mean data ( s.e.m) illustrating that Ba2+ and TEA completely block electrical stimulation–induced vasodilation in the presence of the COX inhibitor indomethacin (n = 3–4). Full Figure and legend (21K)

On the basis of the effects of COX inhibition in neonatal rat brain slices, it has been suggested that an elevation of astrocytic Ca²⁺ leads to the generation of a COX product, namely PGE₂, which contributes to vasodilation presumably through activation of vascular SMC prostaglandin receptors⁷. More recently, in vivo studies demonstrated that the release of caged Ca²⁺ in endfeet causes a rapid vasodilation that is attenuated by COX inhibition⁶. To elucidate the role of Kir channels in the absence of COX activity, we examined the effects of channel blockers in the presence of the COX inhibitor indomethacin (10 M, 20–25 min incubation). In the presence of indomethacin, electrical stimulation still caused a significant vasodilation (45.8 5.2% increase in diameter; n = 4; Fig. 3c) that was completely inhibited by the subsequent addition of the Kir channel blocker Ba²⁺ (100 M; –1.5 0.1% diameter change; n = 4; P < 0.05), suggesting that the role of Kir channels is independent of COX activity.

BK channels are uniquely suited to deliver high local K⁺ concentrations by virtue of their Ca²⁺ sensitivity and the high flux of K⁺ ions through the open channel, even with a small driving force. Based on immunocytochemistry, BK channels appear to be clustered in astrocytic endfeet⁹. Furthermore, the close proximity of astrocytic endfoot BK channels to parenchymal smooth muscle suggests that only a small increase in BK channel activity would be required to release sufficient K⁺ into the perivascular space to cause vasodilation. Indeed, the opening of a single BK channel for 0.2 s should be sufficient to provide enough K⁺ ions (about 10⁶) in the restricted space between astrocytic endfeet and SMCs to elevate the local K⁺ concentration to 10 mM (using the unitary current measured at resting potential, and assuming that the distance from the endfoot to smooth muscle is 20 nm (ref. 27) and that the endfoot surface area is about 80 m² (ref. 30)).

Astrocytes exhibit a characteristic high K⁺ conductance³¹. Although BK channels are preferentially expressed in astrocytic endfeet⁹ and functional BK channels have been demonstrated in cultured astrocytes³², BK currents have not been previously measured in the endfeet of native astrocytes in brain slices. We therefore examined the contribution of BK channels to astrocytic K⁺ currents, using the perforated-patch configuration of the whole-cell patch-clamp technique on astrocytic endfeet. On the basis of the effects of the specific BK channel blocker iberiotoxin (IbTX, 200 nM), we found that at least 38.1 7.7% (n = 5) of the K⁺ current at 0 mV was carried by BK channels (Fig. 4a). To determine the effect of neuronal activation on BK channels, we measured single-channel currents from astrocytic endfeet using the on-cell configuration. Electrical stimulation increased the open probability (NPo) of single channels on astrocytic endfeet 160 71 times (n = 4), with a unitary conductance (slope conductance, 225.6 20.0 pS, n = 3 endfeet) consistent with that previously determined for BK channels (Fig. 4b). Collectively, these results support the concept that neuronal stimulation induces an elevation of intracellular Ca²⁺ and activates BK channels in astrocytic endfeet, leading to the release of K⁺ ions into the restricted space between the endfeet and the adjacent SMCs of the arterioles (Supplementary Figure 1 online).

Figure 4. BK channel currents in astrocytic endfeet in brain slices. (a) I-V relationship of whole-cell astrocytic currents before and after 7 min exposure to the specific BK channel inhibitor IbTX (200 nM). Currents were recorded in response to 200-ms-long voltage ramps from -120 mV to 100 mV. Holding potential was -80 mV. The IbTX-sensitive (difference) current is shown in gray. Inset, positioning of the patch pipette on an endfoot in a brain slice. (b) Representative traces showing single BK channel currents in cell-attached patches from an astrocytic endfoot before (upper trace) and immediately after (lower trace) electrical stimulation (ES). C, closed; O, open. Holding potential was 0 mV. The [K+]o was 6 mM in these experiments. Full Figure and legend (31K)

To determine whether astrocytic BK channels are involved in neurovascular coupling, we tested the effects of the BK channel inhibitor TEA on electrical stimulation–induced vasodilation and suppression of arteriolar Ca²⁺ oscillations in rat cortical brain slices. TEA reduced electrical stimulation–induced vasodilation by 69% (37.8 5.0% dilation control versus 11.9 1.6% dilation in TEA; n = 5; P < 0.05; Fig. 3b), and completely prevented vasodilation in the presence of indomethacin (–1.0 0.1% diameter change; n = 3; P < 0.05; Fig. 3c). In the presence of the BK channel blockers TEA or IbTX (200 nM), electrical stimulation also failed to suppress Ca²⁺ oscillations in the SMCs of the arterioles (Ca²⁺ oscillation frequency in TEA: 0.42 0.08 Hz baseline versus 0.47 0.08 Hz following electrical stimulation, n = 11; in IbTX: 0.38 0.05 Hz baseline versus 0.37 0.03 Hz following electrical stimulation, n = 12; Fig. 5). These results indicate that functional BK channels are required for this mode of rapid neurovascular coupling. Furthermore, the observation that TEA had an inhibitory effect on electrical stimulation–induced vasodilation that is comparable (70% reduction) to that of the Kir channel blocker Ba²⁺ (compare Fig. 3b,c) provides further evidence that these two channels act in series.

Figure 5. Blockade of BK channels inhibits electrical stimulation–induced suppression of arteriolar smooth muscle Ca2+ oscillations in brain slices. (a) Representative traces illustrating the suppression of smooth muscle arteriolar Ca2+ oscillations after electrical stimulation (upper trace), and inhibition of the electrical stimulation–induced suppression of arteriolar Ca2+ oscillations in the presence of the BK channel blocker TEA (1 mM) (lower trace). (b) Electrical stimulation reduced mean ( s.e.m) arteriolar Ca2+ oscillation frequency (Hz) in control by approximately 65% (left), but had no effect in the presence of TEA (right). n = 11–12. TEA elevated the mean frequency about twofold. (c) Electrical stimulation did not affect mean ( s.e.m) arteriolar Ca2+ oscillations in slices preincubated for 20 min with the specific BK channel blocker IbTX (200 nM, n = 12). Inhibition of SMC Ca2+ oscillation frequency was reproducible over multiple electrical stimulations (inhibition due to first electrical stimulation was 58.7 6.9%; that due to second stimulation was 62.8 6.7; n = 10). The [K+]o was 6 mM in these experiments. Full Figure and legend (28K)

To provide additional evidence for the role of BK channels in neurovascular coupling, we used brain slices from mice lacking the gene for the pore-forming alpha subunit of the BK channel (that is, Kcnma1^-/- mice). These mice exhibit a number of pathologies including ataxia, an overactive bladder and incontinence³³. Parenchymal arterioles in brain slices from both Kcnma1^+/+ and Kcnma1^-/- mice exhibited smooth muscle Ca²⁺ oscillations and vasomotion. As expected, electrical stimulation significantly suppressed arteriolar smooth muscle Ca²⁺ oscillations in Kcnma1^+/+ mice (Ca²⁺ oscillation frequency = 0.44 0.05 Hz in control versus 0.22 0.05 Hz following electrical stimulation; n = 17; P < 0.05; Fig. 6a,b). In contrast, arteriolar smooth muscle Ca²⁺ oscillations in brain slices from Kcnma1^-/- mice were unaffected by neuronal stimulation (0.57 0.07 Hz control versus 0.59 0.08 Hz following electrical stimulation; n = 12; Fig. 6a,b). This failure of astrocyte–to–smooth muscle communication in Kcnma1^-/- mice could not be attributed to a disruption of neuron-to-astrocyte communication, as the electrical stimulation–induced astrocytic Ca²⁺ elevation was maintained (Fig. 6a). Electrical stimulation–induced astrocytic Ca²⁺ elevations were similarly unaffected by simultaneous block of BK and Kir channels in rat cortical brain slices (Fig. 6c). Electrical stimulation–induced increases in peak astrocytic Ca²⁺, as measured by changes in fractional fluorescence, were 3.97 0.68 in the astrocytic soma and 2.80 0.30 in endfeet under control conditions, and 3.57 0.26 in the soma and 2.63 0.14 in endfeet in the presence of 200 nM IbTX, 100 M BaCl₂ and 1 mM TEA (n = 3; Fig. 6c). These results strongly suggest that the activation of BK channels involved in mediating neuron-to-arteriole communication occurs downstream of the astrocytic Ca²⁺ elevation induced by neuronal stimulation.

Figure 6. The absence of functional BK channels prevents electrical stimulation–induced suppression of Ca2+ oscillations in SMCs of parenchymal arterioles in brain slices. (a) Representative recordings illustrating simultaneous Ca2+ changes in an astrocytic endfoot (blue) and a vascular SMC (red) in cortical brain slices from Kcnma1+/+ (left) and Kcnma1-/- (right) mice. Electrical stimulation induced a rise in astrocytic Ca2+ in brain slices from both types of mice, and suppressed smooth muscle Ca2+ oscillations in Kcnma1+/+, but not Kcnma1-/-, mice. (b) Mean ( s.e.m) arteriolar Ca2+ oscillation frequency (Hz) before and during electrical stimulation from Kcnma1+/+ mice (n = 17) and Kcnma1-/- (n = 12) mice. Electrical stimulation reduced arteriolar Ca2+ oscillations frequency by 50% in Kcnma1+/+, but had no effect in Kcnma1-/- mice. (c) Astrocytic Ca2+ transients from a rat brain slice following electrical stimulation in the absence (left) and the presence (right) of the Kir and BK channel blockers BaCl2, IbTX and TEA. The [K+]o was 6 mM in these experiments. Full Figure and legend (42K)

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This mechanism assigns an important physiological role to the well-characterized responsiveness of the cerebrovascular circulation to elevation of [K⁺]_o, and provides for fine and rapid regulation of arterial diameter. In addition, it could explain apparently paradoxical and opposing effects of astrocyte activation on arterial diameter^{2, 5, 6, 7}, as the response of arterioles would depend on the amount of K⁺ released from the astrocytic endfeet. Accordingly, higher concentrations of K⁺ (20 mM) released from the astrocytic endfeet through BK channels could lead to vasoconstriction through depolarization of arterial smooth muscle, whereas lower concentrations of astrocytic endfeet K⁺ release (<20 mM) would cause membrane hyperpolarization of arteriolar smooth muscle and vasodilation. Furthermore, activation of any hyperpolarizing process (for example, electrogenic sodium or potassium ATPase or K_ATP channels³⁶) in SMCs would also lead to an engagement of Kir channels through their steep voltage dependence.

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Cortical brain slices were prepared from juvenile (>20 d old) Sprague-Dawley rats and from adult Kcnma1^+/+ and Kcnma1^-/- mice, following protocols approved by the Office of Animal Care Management at the University of Vermont. The cortex was rapidly removed, cut into 200-m-thick coronal slices using a vibratome (Leica VT 1000S) in cold aCSF containing 3mM KCl, 125 mM NaCl, 1 mM MgCl₂, 26 mM NaHCO₃, 1.25 mM NaH₂PO₄, 10 mM glucose, 2 mM CaCl₂ and 400 M L-ascorbic acid, equilibrated with 95% O₂ and 5% CO₂. Ascorbic acid was added to reduce cell swelling associated with oxidative stress. In experiments presented in Figures 4, 5, 6, KCl and NaCl concentrations were 6 mM and 122 mM, respectively, to allow direct comparison with previously published data^{2, 10, 16}. Slices were immediately incubated at room temperature (21–24 °C) in aCSF equilibrated with 95%O₂ and 5%CO₂ (pH 7.45) until needed.

Ca²⁺ imaging was performed using a Solamere confocal unit (QLC 100) in combination with a high-sensitivity and high-resolution camera (XR MEGA-10, Stanford Photonics). Cortical slices were incubated at room temperature in aCSF containing 10 M Fluo-4 AM and pluronic acid (2.5 g ml^-1, Molecular Probes). After a 2- to 3-h incubation period, slices were washed and placed in aCSF until needed. At the time of the experiment, a slice was transferred to a perfusion chamber and continuously superfused with aCSF maintained at 35–37 °C. Parenchymal arterioles and astrocytes were visualized with a 60 water-dipping objective (NA 1.0). Fluorescence images were obtained using a krypton/argon laser at 488 nm, and emitted light was captured at >495 nm. Images were acquired at 30 or 60 frames per s for 60 s.

Video microscopy was used to determine arteriolar diameter changes, using IR-DIC and a CCD camera (Hamamatsu). Images were acquired at 30 images per s and stored for later analysis. Internal diameter changes were determined from the distance between multiple points across the arteriolar lumen. The internal diameter of arterioles investigated in this study was between 4–20 m, consistent with other studies of arterioles in cortical brain slices⁴⁴.

Parenchymal arterioles were carefully dissected from the cerebral cortex and then digested with papain (0.3 mg ml^-1 and 1 mg ml^-1 dithioerythritol for 12 min at 37 °C) and collagenase (1 mg ml^-1 collagenase, type F and type H in a 70%/30% mixture, incubated for 5 min at 37°C). The digested tissue was gently triturated with fire-polished glass Pasteur pipettes to yield single myocytes. The mean cell capacitance was 9.2 0.7 pF (n = 16).

Whole-cell (perforated-patch configuration) and single-channel currents were amplified by an Axopatch 200B and analyzed using Clampfit 9.2 software (Axon Instruments). Whole-cell currents were filtered at 1 kHz and digitized at 10 kHz. Single-channel currents were filtered at 2 kHz and digitized at 10 kHz. Open probability was determined using the half-crossing method. Pipette solution for whole-cell configuration (perforated patch) consisted of 30 mM KCl, 110 mM potassium aspartate, 10 mM NaCl, 1 mM MgCl₂, 10 mM HEPES (adjusted to pH 7.2 with NaOH) and 250 g ml^-1 amphotericin B. Pipette solution for on-cell single-channel recordings consisted of 150 mM KCl, 1 mM MgCl₂ and 10 mM HEPES (adjusted to pH 7.2 with KOH). For whole-cell and single-channel recordings from astrocyte endfeet, brain slices were continuously superfused with aCSF at 35–37°C. The bathing solution for whole-cell recordings from isolated arteriole myocytes consisted of 6 mM KCl, 134 mM NaCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES and 10 mM glucose (adjusted to pH 7.4 with NaOH), and was maintained at room temperature. KCl concentration was changed to 3 mM, 10 mM or 60 mM by substituting NaCl for KCl. Whole-cell seal resistances varied from 10 G to 20 G, corresponding to -10 pA to -5 pA of leak current at -100 mV. Therefore, the leak current was larger than relevant physiological currents (for example, see Fig. 1b). Voltage-dependent K⁺ channel activity, other than Kir channels, is minimal negative to -60 mV. Therefore, Ba²⁺-insensitive currents between -60 mV and -80 mV were considered leak currents and were extrapolated over the entire voltage range in order to subtract leak currents from the whole-cell current. The magnitude of the subtracted leak current ranged from -1.5 pA to -6.2 pA (-3.6 0.6 pA average; n = 11) at -60 mV. This linear leak current was subtracted from the whole-cell currents to obtain the data depicted in Figure 1a.

Brains were removed into MOPS-buffered saline containing 145 mM NaCl, 3 mM KCl, 1.2 mM NaH₂PO₄, 1.17 mM MgSO₄, 2.0 mM CaCl₂, 5 mM glucose, 0.02 mM EDTA, 3 mM MOPS and 10 mg ml^-1 bovine serum albumin (pH 7.4) at 4 °C. Middle cerebral arteries with attached, penetrating parenchymal arterioles were carefully dissected from the brain, and parenchymal arterioles (30–40 m diameter) were isolated from middle cerebral arteries and cannulated to resistance-matched micropipettes. Vessels were pressurized to 40 mm Hg and monitored throughout the experiment. Microelectrodes were filled with 500 mM KCl to produce electrode resistances of 100–200 M, and potential was measured using a WPI Intra 767 amplifier.

Ca²⁺ imaging and arteriolar diameter experiments were analyzed with custom software created by A. Bonev. Fractional fluorescence (F/F₀) was determined by dividing the fluorescence intensity (F) within a region of interest (ROI) by a baseline fluorescence value (F₀) determined from 50 images showing no activity. The frequency of Ca²⁺ oscillations was determined within an ROI (10 10 square pixels, or 2.5 2.5 m²) on a cell exhibiting Ca²⁺ oscillations. The number of peaks over a given time was automatically detected from oscillations crossing a set threshold value (>1.15 F/F₀). Data are expressed as mean s.e.m. Differences between two means were determined using Student's t-test. Statistical significance was tested at 95% (P < 0.05) confidence level.

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Competing interests statement:  The authors declare that they have no competing financial interests.