Local IP3 receptor-mediated Ca2+ signals compound to direct blood flow in brain capillaries

Abstract

Healthy brain function depends on the finely tuned spatial and temporal delivery of blood-borne nutrients to active neurons via the vast, dense capillary network. Here, using in vivo imaging in anesthetized mice, we reveal that brain capillary endothelial cells control blood flow through a hierarchy of IP₃ receptor-mediated Ca²⁺ events, ranging from small, subsecond protoevents, reflecting Ca²⁺ release through a small number of channels, to high-amplitude, sustained (up to ~1 min) compound events mediated by large clusters of channels. These frequent (~5000 events/s per microliter of cortex) Ca²⁺ signals are driven by neuronal activity, which engages G_q protein-coupled receptor signaling, and are enhanced by Ca²⁺ entry through TRPV4 channels. The resulting Ca²⁺-dependent synthesis of nitric oxide increases local blood flow selectively through affected capillary branches, providing a mechanism for high-resolution control of blood flow to small clusters of neurons.

Fig. 1. Dynamic Ca²⁺ signaling in the brain endothelium.

(A) In vivo imaging strategy. Top left: A cranial window was centered over the somatosensory region, imaged using two-photon laser scanning microscopy. Left: Vascular field mapped from luminal TRITC-dextran. Bottom left: Region around a penetrating arteriole imaged at higher resolution, selected for 4D imaging, and a single plane, within the 4D block of interest above. Magenta, TRITC-dextran; green, GCaMP8. (B to I) Characterization of cEC Ca²⁺ events (proto, n = 1184 events; unitary, n = 786; compound, n = 1257). (B) Capillary Ca²⁺ events ranging from proto events to unitary singlets and multicomponent compound events. (C) Durations for all events analyzed. Color overlay indicates event classes: red, proto; green, unitary; magenta, compound. (D) Amplitudes for all events analyzed. (E) Event mass by class [P < 0.0001 (q₃₂₂₄ = 10.76), proto versus unitary; P < 0.0001 (q₃₂₂₄ = 43.89), unitary versus compound; P < 0.0001 (q₃₂₂₄ = 61.51), compound versus proto; one-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test]. (F) Lengths of cEC Ca²⁺ events. The dashed line indicates the average length of a cEC [from (K)]. (G) Event length by class [P < 0.0001 (q₃₂₂₄ = 15.25), proto versus unitary; P < 0.0001 (q₃₂₂₄ = 34.45), unitary versus compound; P < 0.0001 (q₃₂₂₄ = 56.01), compound versus proto; one-way ANOVA and Tukey’s multiple comparisons test]. (H) Event spread velocity by class. Deviation above the line represents event expansion, whereas below the line represents contraction. (I) Relationship between cEC Ca²⁺ event mass and spread. (J) Di-8-ANEPPS (magenta) and Hoechst 33342 (yellow) staining of the capillary endothelium and cEC nuclei. (K) Internuclear distances measured using Di-8-ANEPPS and Hoechst 33342 (n = 150 measurements, 3 mice).

Fig. 2. TTX suppresses brain cEC Ca²⁺ signaling in vivo.

(A) Top left: Imaging outline. A single optical section through a field of vessels within the brain was imaged for 236 s. Top right three panels: Typical field showing TRITC-dextran–filled vasculature (magenta; left) and accumulation plots showing all Ca²⁺ activity recorded in this plane under baseline conditions (middle) and all activity in the same field after the addition of 3 μM TTX (right). Bottom: Recordings converted to ST maps, total length of all active sites are side by side on the x axis and time on the y axis (left), and the effects of TTX (right). TTX decreases in the number of active sites, which reduces the length of the x axis, and decreases in event amplitudes and durations appear as smaller individual events in the y axis. (B) Summary data showing the effects of TTX on total Ca²⁺ activity in each field [n = 6 experiments, 6 mice; P = 0.0038 (t₅ = 5.091), paired Student’s t test]. (C) Sensory stimulation engages arteriolar and capillary Ca²⁺ signals. Top: A single plane through the penetrating arteriole and the connected first- to third-order capillaries. Bottom: Electrical FPS–induced increases in Ca²⁺ in arteriolar ECs and cECs. (D) Traces from an individual experiment showing robust responses of capillaries and arterioles to FPS across two trials separated by ~1 min. (E) Summary showing Ca²⁺ signals evoked by neural activity in the penetrating arteriole [n = 19 paired experiments; P < 0.0001 (t₁₈ = 7.685), paired Student’s t test] and capillaries [n = 15 paired experiments; P = 0.0003 (t₁₄ = 4.699), paired Student’s t test].

Fig. 3. G_qPCR signaling through IP₃Rs and TRPV4 drives cEC Ca²⁺ signaling.

(A) ST maps before/after FR900359. Scale bars apply to all maps. (B) FR900359 on cEC Ca²⁺ activity [n = 5 experiments, 5 mice; P = 0.0147 (t₄ = 4.117), paired t test], (C) total events [n = 5 experiments, 5 mice; P = 0.0282 (t₄ = 3.364), paired t test], and (D) events by class [n = 5 experiments, 5 mice; proto, P = 0.0064 (t₈ = 4.445); unitary, P = 0.023 (t₈ = 3.522); compound, P = 0.0008 (t₈ = 6.223); two-way ANOVA and Sidak’s multiple comparisons test]. (E) Focal application of PGE₂ on cEC Ca²⁺. Left: Pipette (P) position. Right: Peak cEC Ca²⁺ after PGE₂. (F) No Ca²⁺ response of a capillary to aCSF, and an increase in Ca²⁺ following PGE₂ (G). (H) Summary [n = 6 to 8 experiments, 3 to 5 mice; P = 0.0016 (t₁₂ = 4.054), unpaired t test]. (I) CPA decreased Ca²⁺ activity [n = 7 experiments, 7 mice; P = 0.0178 (t₆ = 3.233); paired t test]. (J and K) Reduction in Ca²⁺ activity by xestospongin C [n = 8 experiments, 8 mice; P = 0.0318 (t₇ = 2.675); paired t test]. (L to N) Maps and summary for EGTA-mediated changes in Ca²⁺ activity [n = 11 experiments, 11 mice; P = 0.0090 (t₁₀ = 3.230), paired t test] and spatial spread [n = 11 experiments, 11 mice; P = 0.0415 (t₁₀ = 2.337), paired t test]. (O) ST map of TRPV4 activation with GSK1016790A. (P) TRPV4 activation on event classes [n = 5 experiments, 5 mice; proto, P = 0.0001 (t₈ = 8.102); unitary, P = 0.0006 (t₈ = 6.418); compound, P = 0.0341 (t₈ = 3.262); two-way ANOVA and Sidak’s multiple comparisons test]. (Q) Inhibition of TRPV4 with GSK2193874 [n = 10 experiments, 10 mice; P = 0.0303 (t₉ = 2.567), unpaired t test].

Fig. 4. Capillary Kir2.1 channel–mediated electrical signaling shapes Ca²⁺ signaling.

(A) ST maps extracted from the same field before and after inhibition of Kir2.1 channels with topically applied Ba²⁺ (100 μM). (B) Summary data showing the in vivo effects of Ba²⁺ on total Ca²⁺ activity [n = 7 experiments, 7 mice; P = 0.0353 (t₆ = 2.706), paired Student’s t test] and total number of events observed (C) [n = 7 experiments, 7 mice; P = 0.0401 (t₆ = 2.610), paired Student’s t test].

Fig. 5. Ca²⁺ signaling tunes capillary diameter and blood flow.

(A) Flux experiment setup. (B) Line scans showing RBCs (black) passing through a capillary in fluorescent plasma (x, time; y, space). (C) Hyperemia (blue) following a spontaneous Ca²⁺ signal (green). (D) l-NNA inhibition of Ca²⁺-associated increases in RBC flux. (E) Changes in flux after a Ca²⁺ event at 1° to 3° bifurcations of the capillary network with and without l-NNA and in deeper capillaries (≥5°) [n = 18 to 24 experiments, 7 to 9 mice; P = 0.0001 (q₅₈ = 4.768) control versus l-NNA; P = 0.0001 (q₅₈ = 5.965) control versus deep; one-way ANOVA and Dunnett’s multiple comparison test]. (F) Line scan showing capillary dilation to a Ca²⁺ event (y, time; upper bar, baseline; lower bar, peak). (G) Diameter (magenta) and Ca²⁺ (green) for the experiment in (F). (H) As in (G), an experiment following cortex perfusion with l-NNA. (I) Dilation following Ca²⁺ signaling [n = 6 experiments, 5 mice; P = 0.0029 (t₅ = 5.438); paired t test]. (J) l-NNA inhibition of Ca²⁺-induced dilation [n = 6 to 11 paired experiments, 5 to 7 mice; P < 0.0001 (t₁₅ = 6.589); unpaired t test]. (K) Directional control of blood flow by capillary Ca²⁺. Top: Experimental approach. Two branches, fed by a 1° to 3° capillary, were simultaneously line-scanned. Bottom: Summary showing differential blood flow when a Ca²⁺ signal occurred in one branch only (left) and uniform blood flow through the two segments when a signal occurred in both branches (right). [one-branch Ca²⁺ event: n = 7 paired experiments; P = 0.0395 (t₆ = 2.621); paired t test; two-branch Ca²⁺ event: n = 5 paired experiments; P = 0.0837 (t₄ = 2.292); paired t test]. (L) Pipette containing spermine NONOate next to a pericyte. Flux was measured by line-scanning the capillary during ejection. (M) RBCs passing through the line-scanned region against fluorescent plasma (x, time). (N) Time course of RBC flux to NO. (O) Peak RBC flux at baseline and after NO [n = 19 experiments, 8 mice; P < 0.0001 (t₁₈ = 5.714); paired t test] or HPSS [n = 9 experiments, 5 mice; P = 0.4587 (t₈ = 0.7784); paired t test].