Supplementary Figure 1 Relative expression of K IR2.1 transcript to enos was reduced 29-fold in capillaries from knockout animals. Relative expression of K IR2.1 transcript to enos was reduced 29-fold in capillaries from knockout animals (n = 6 capillaries from 6 mice), compared to controls (n = 5 capillaries from 5 mice) indicating successful knockout of K IR2.1 (*P = 0.0108 (t 9 = 3.204); unpaired t-test). Error bars represent s.e.m.
Supplementary Figure 2 K IR currents in pial artery SM cells were unaffected by knockout of K IR2.1 in ECs. (A) Typical experiment in a WT pial artery SM cell in which control currents (black trace) are characterized by an inwardly rectifying current at potentials negative to E K (-23 mv) and an outwardly rectifying component at positive potentials. Application of 100 µm Ba 2+ inhibited the inward rectifier component (red). Currents were recorded in response to a 200-ms ramp from -140 to +50 mv with 60 mm [K + ] o and 300 nm [Ca 2+ ] i. (B) Typical experiment in a pial artery SM cell from an EC K IR2.1 -/- mouse under the same conditions. Ba 2+ (blue trace) inhibited the inward rectifier component that was evident under control conditions (black trace). (C) Summary data at -140 mv, indicating no difference in SM cell K IR current density between WT and EC K IR2.1 -/- mice (WT, n = 14 cells from 3 mice; EC K IR2.1 -/-, n = 13 cells from 4 mice; P = 0.5176 (t 25 = 0.6564) Student s unpaired t-test). (D) The density of voltage-dependent currents at +50 mv was also unchanged between SM cells from WT and EC K IR2.1 -/- mice (WT, n = 14 cells from 3 mice; EC K IR2.1 -/-, n = 13 cells from 4 mice; P = 0.2002 (t 25 = 1.316) Student s unpaired t-test). All error bars represent s.e.m.
Supplementary Figure 3 SK and IK channel activation in pial artery ECs. (A) Typical experiment in a pial artery EC in which a small inwardly rectifying current (black line) is observed in response to a 200-ms ramp from -140 to +50 mv under control conditions (6 mm [K + ] o, 300 nm [Ca 2+ ] i). Subsequent application of 1 µm NS309 (green) produced large SK and IK currents. (B) Typical experiment in a pial artery EC dialyzed with 3 µm Ca 2+, which caused the immediate development of prominent K + currents.
Supplementary Figure 4 The presence of capillaries did not affect the properties of attached, pressurized (40 mmhg) parenchymal arterioles. (A) Side-by-side comparison of vasomotor responses in parenchymal arteriole (PA) (left) and CaPA (right) preparations taken from the same mouse. Nearly identical responses were observed to bath application of 1 M NS309, 10 mm K +, and 100 nm U46619. (B) Summary data for seven paired experiments (7 mice) indicating that PA and CaPA preparations develop the same degree of myogenic tone and have identical vasomotor properties (n = 7; tone: P = 0.7549, (t 6 = 0.3268); NS309: P = 0.5995, (t 6 = 0.5542); 10 mm K + : P = 0.2616, (t 6 = 1,239); U46619: P = 0.7666, (t 6 = 0.3107); paired Student s t-test). All error bars represent s.e.m.
Supplementary Figure 5 K + concentrations lower than 10 mm also dilated upstream arterioles when applied onto capillaries, with a threshold for activation between 6 and 7 mm K + followed by moderately graded responses between 7 and 10 mm. (A) Typical intraluminal arteriolar diameter at Zone 1, located at the point from which the primary capillary initially branches out, during stimulation of attached downstream capillaries with 6, 7, 8, 9 and 10 mm K +. (B) Summary data showing peak diameter changes in Zone 1 in response to the different K + concentrations (n = 5 preparations from 5 mice; 6 mm K + vs 7 mm K + : ***P = 0.0005, (t 5 = 10.48); 7 mm K + vs 8 mm K + : *P = 0.0385, (t 5 = 3.038); 8 mm K + vs 9 mm K + : *P = 0.0330, (t 5 = 3.196); 9 mm K + vs 10 mm K + : P = 0.0538, (t 5 = 2,706), paired Student s t-test). All error bars represent s.e.m.
Supplementary Figure 6 Pressure ejection of agents onto capillaries did not directly stimulate upstream arterioles. (A) Pipette positions (tip indicated by black arrowheads) for capillary stimulation (left) and arteriole stimulation (right). (B) Pressure ejection of 1 M NS309 onto capillaries (P1, purple) had no effect on upstream arteriolar diameter, whereas direct arteriole stimulation (P2, orange) with this agent caused substantial dilation, indicating that solutions ejected onto capillaries do not spill over onto the arteriole.
Supplementary Figure 7 Endothelial function and vasomotor properties of parenchymal arterioles were unaffected by EC K IR2.1 knockout. (A) Typical diameter traces of pressurized parenchymal arterioles from WT (top) and EC K IR2.1 -/- (bottom) mice. Myogenic tone and responses to the SK and IK channel opener NS309 were essentially identical between groups. (B) Summary of myogenic tone at 40 mm Hg intravascular pressure (top) and dilation evoked by NS309 (bottom) in WT and EC K IR2.1 -/- mice (WT, n = 7 mice; EC K IR2.1 -/-, n = 9 mice); tone: P = 0.2580, (t 13 = 1.183); NS309: P = 0.7969, (t 13 = 0.2627); unpaired Student s t-test). All error bars represent s.e.m.
Supplementary Figure 8 Surgically removing the capillary tree from its upstream arteriole eliminated arteriolar dilation following capillary stimulation with 10 mm K +. (A) A CaPA preparation with capillaries attached (top) or severed (middle and bottom) with a pipette positioned for capillary (top and middle) or arteriole (bottom) stimulation by pressure ejection. Red arrow indicates the tip of the pipette. Diameter was recorded in Zone 1 (black box) where the primary capillary branches from the arteriole. (B) Arteriolar diameter at Zone 1 in response to 10 mm K + stimulation of attached capillaries (top) or severed capillaries (middle). Application of 10 mm K + to capillaries failed to produce upstream arteriolar dilation after surgical separation of the capillary tree from the arteriole, while direct stimulation of the arteriole with 10 mm K + still led to vasodilation (bottom). This observation confirms the spatial restriction of the pressure ejected solution and indicates that the observed phenomenon relies on inherent conducted signaling from capillaries to the arteriole. (C) Summary data (n = 6 preparations, 6 mice) showing peak diameter changes in Zone 1 in the different configurations: Capillary tree attached (top) or severed (middle and bottom), induced by 10 mm K + applied onto capillaries (top and middle) or the arteriole (bottom). Error bars represent s.e.m.
Supplementary Figure 9 Stimulation of capillaries in vivo with 10 mm K + increased RBC velocity in WT mice, but not in EC K IR2.1 / mice or in the presence of Ba 2+. (A) Typical velocity-time trace for pressure ejection of 10 mm K + (300 ms, 8 psi; purple arrow) onto a capillary in a WT mouse, showing a rapid and sustained increase in RBC velocity. Gray circles represent the velocities of individual RBCs, and the blue line is a running average. (B) Same as in A for an experiment performed on a capillary in an EC K IR2.1 -/- mouse, showing the lack of a substantial increase in RBC velocity to 10 mm K +. (C, D) Typical RBC flux-time trace (C) and corresponding RBC velocity time-course (D) for pressure ejection of 10 mm K + (200 ms, 6 psi; purple arrow) onto a WT mouse capillary in vivo after a 25-min incubation of the cortex with 100 µm Ba 2+. (E) Summary data showing RBC velocity before and after capillary application of 10 mm K + in WT mice (n = 11 paired experiments, 11 mice; ***P = 0.0004 (t 10 = 5.244), paired Student s t-test). (F) Summary data showing RBC velocity before and after capillary application of 10 mm K + in EC K IR2.1 -/- mice (n = 9 paired experiments, 9 mice; P = 0.88 (t 8 = 0.1558), paired Student s t- test). (G) Summary data showing RBC velocity before and after capillary application of 10 mm K + in WT mice following cortical application of 100 M Ba 2+ (n = 6 paired experiments, 6 mice; P = 0.4367 (t 5 = 0.8448) Student s paired t-test). All error bars represent s.e.m.
Supplementary Figure 10 Stimulation of capillaries in vivo with 3 mm K + had no effect on RBC flux or velocity. (A) Typical RBC flux-time trace for pressure ejection of acsf (3 mm K + ; 300 ms, 6 psi, black arrow) onto a capillary in vivo. (B) Corresponding RBC velocity-time trace for the experiment shown in A (gray circles, individual RBC velocities; blue line, running average). (C) Summary data for RBC flux (n = 6 paired experiments, 6 mice; P = 0. 5464 (t 5 = 0.6466) Student s paired t-test) and (D) velocity (n = 6 paired experiments, 6 mice; P = 0.9199 (t 5 = 0.1057) Student s paired t-test) and before and after acsf delivery, indicating that pressure ejection alone produced no change in either parameter. Error bars represent s.e.m.
Supplementary Figure 11 Capillary hyperemia to 10 mm K + persisted in the presence of tetrodotoxin (TTX), a blocker of voltage-dependent Na + channels. (A) Contralateral whisker stimulation increased capillary flux under basal conditions, and this response was eliminated by application of 3 M TTX to the cranial surface (n = 5 paired experiments, 5 mice; **P = 0.0039, two-way ANOVA with post hoc Tukey s multiple comparisons test). (B) In the presence of TTX, application of 10 mm K + to the capillary still caused an increase in RBC flux and velocity, as evidenced by the increased number and steeper angle of RBCs (black streaks against the green FITC-loaded plasma) passing through the line-scanned region. Left: baseline distance-time line scan plot; right: after application of 10 mm K +. (C) Typical RBC fluxtime plot indicating marked hyperemia after in vivo application of 10 mm K + to a capillary, after pre-treatment with 3 M TTX to silence neuronal activity. (D) Summary data for the peak increase in capillary RBC flux evoked by 10 mm K + in the presence of TTX (n = 5 paired experiments, 5 mice; *P = 0.0354 (t 4 = 3.123) paired Student s t-test). Error bars represent s.e.m.
Supplementary Figure 12 Whisker stimulation-evoked neural activity was unaffected by 100 µm Ba 2+ superfusion. (A) Representative 8 s LFP epoch (top) and accompanying 0-20 Hz spectrogram (bottom) recorded from the whisker barrel cortex in response to contralateral whisker stimulation under control conditions. Stimulation reliably entrained large oscillations at a frequency of ~5 Hz. (B) Exactly as in A for the same mouse, after superfusion of 100 µm Ba 2+ over the cranial surface. (C) Signal spectrum plot for the recording in A illustrating the predominance of 5 Hz oscillations evoked by whisker stimulation. (D) Signal spectrum plot for the recording in B. (E) Summary data showing the peak signal amplitude before and after 100 µm Ba 2+ superfusion for the 0 4 Hz band of the LFP spectrum (n = 5 paired experiments, 5 mice; P = 0.3778 (t 4 = 0.991) Student s paired t-test). (F) Summary data showing the frequency of peak LFP oscillations before and after 100 µm Ba 2+ superfusion for the 0 4 Hz band of the LFP spectrum (n = 5 paired experiments, 5 mice; P = 0.4804 (t 4 = 0.777) Student s paired t-test). (G) Summary data showing the peak signal amplitude before and after 100 µm Ba 2+ superfusion for the 4 6 Hz band of the LFP spectrum (n = 5 paired experiments, 5 mice; P = 0.5627 (t 4 = 0.6303) Student s paired t-test). (H) Summary data showing the frequency of peak LFP oscillations before and after 100 µm Ba 2+ superfusion for the 4 6 Hz band of the LFP spectrum (n = 5 paired experiments, 5 mice; P = 0.1202 (t 4 = 1.969) Student s paired t-test). (I) Summary data showing the peak signal amplitude before and after 100 µm Ba 2+ superfusion for the 6 20 Hz band of the LFP spectrum (n = 5 paired experiments, 5 mice; P = 0.755 (t 4 = 0.3337) Student s paired t-test). Note that the scale is one order of magnitude smaller than for the data in other bands, reflecting lower activity in this frequency range. (J) Summary data showing the frequency of peak LFP oscillations before and after 100 µm Ba 2+ superfusion for the 6 20 Hz band of the LFP spectrum (n = 4 paired experiments, 4 mice; P = 0.907 (t 3 = 0.1233) Student s paired t-test). All error bars represent s.e.m.
Supplementary Figure 13 Proposed mechanism for K + regulation of CBF. Neural activity (1) leads to an increase in local K + around capillaries (2). Through activation of K IR channels (3), this generates local hyperpolarization of the capillary endothelial membrane, which then spreads to adjacent ECs, presumably through gap junctions, activating K IR channels to rapidly propagate a regenerative electrical signal upstream to the feed arteriole (4). After spreading into adjacent SM cells (SMC), hyperpolarization deactivates voltage-dependent Ca 2+ channels (VDCC). The ensuing decrease in intracellular Ca 2+ causes SM relaxation and arteriolar dilation (5), promoting an increase in blood flow into the capillaries (6).
Supplementary Table 1. Comparison of electrophysiological, ex vivo and in vivo observations from KIR2.1 fl/fl (N = 5) and Tek-cre (N = 4) mice with wild-type (N = 4 to 8). All values for laser Doppler flowmetry experiments are expressed as % increase in blood flow from baseline. The presence of loxp sites around the Kcnj2 gene, or the presence of cre recombinase in ECs alone had no effect on KIR currents or KIR-mediated effects. Preparation KIR2.1 fl/fl Tek-cre Wild-type Electrophysiology Capillary EC KIR current density (pa/pf) at -140 mv 12.7 ± 2.7 13.3 ± 2.7 12.9 ± 2.6 CaPA % dilation at Zone 2 induced by capillary stimulation with 10 mm [K + ]o % inhibition by Ba 2+ (30 µm) 52.1 ± 2.6 51.7 ± 1.2 56.2 ± 3.9 96.7 ± 1.4 97.9 ± 2.3 95.4 ± 1.4 Laser-Doppler flowmetry Whisker stimulation 29.6 ± 1.8 32.8 ± 3.2 31.1 ± 3.3 Whisker stimulation + 100 µm Ba 2+ 5.9 ± 0.8 8.0 ± 1.7 6.6 ± 1.1 15 mm K + 30.1 ± 4.1 34.0 ± 4.0 32.7 ± 6.7 15 mm K + + 100 µm Ba 2+ 7.3 ± 0.5 2.7 ± 3.6 6.9 ± 2.6
Supplementary Table 2. Physiological variables for mice used for laser Doppler flowmetry experiments. There were no significant differences between groups. Age (weeks) Body weight (g) Blood gas ph pco2 (mmhg) po2 (mmhg) Body temp ( C) Blood pressure (mmhg) Mouse strain n C57BL/6 14.8 ± 2.2 27.4 ± 1.0 7.40 ± 0.01 39.9 ± 0.8 91.9 ± 1.1 36.6 ± 0.1 106.2 ± 4.6 7 EC K IR2.1 -/- 17.0 ± 2.3 27.7 ± 1.7 7.41 ± 0.01 39.4 ± 0.8 94.9 ± 1.6 36.7 ± 0.1 105.0 ± 4.8 6 K IR2.1 fl/fl 9.9 ± 1.7 23.7 ± 2.1 7.40 ± 0.02 39.8 ± 1.5 92.6 ± 2.4 36.8 ± 0.2 104.7 ± 4.0 4 Tek-cre 12.9 ± 0.2 26.6 ± 0.6 7.38 ± 0.02 41.0 ± 1.8 93.4 ± 2.4 36.7 ± 0.2 113.3 ± 6.0 5