Brain metabolism dictates the polarity of astrocyte control over arterioles

Abstract

Calcium signalling in astrocytes couples changes in neural activity to alterations in cerebral blood flow by eliciting vasoconstriction or vasodilation of arterioles. However, the mechanism for how these opposite astrocyte influences provide appropriate changes in vessel tone within an environment that has dynamic metabolic requirements remains unclear. Here we show that the ability of astrocytes to induce vasodilations over vasoconstrictions relies on the metabolic state of the rat brain tissue. When oxygen availability is lowered and astrocyte calcium concentration is elevated, astrocyte glycolysis and lactate release are maximized. External lactate attenuates transporter-mediated uptake from the extracellular space of prostaglandin E(2), leading to accumulation and subsequent vasodilation. In conditions of low oxygen concentration extracellular adenosine also increases, which blocks astrocyte-mediated constriction, facilitating dilation. These data reveal the role of metabolic substrates in regulating brain blood flow and provide a mechanism for differential astrocyte control over cerebrovascular diameter during different states of brain activation.

Fig. 1

Lowering O₂ converts astrocyte-mediated vasoconstrictions to vasodilations. (a) Top: astrocytes (red) loaded with Ca²⁺ indicator surround arteriole. Bottom: astrocyte Ca²⁺ signals occur coincident with dilation caused by the mGluR agonist t-ACPD in low O₂. (b) Top: uncaging astrocyte Ca²⁺ (indicated by arrow) causes vasodilation in low O₂ and is repeatable. Bottom: expanded time scale shows the Ca²⁺ signal in endfoot 1 (shown in c I) precedes the lumen diameter increase. (c) Overlay of vessel and pseudo colored endfoot Ca²⁺ changes. Images I-V correspond to times in b. I: control state and regions of interest: endfeet 1-3. Image II: endfoot 1 shows first Ca²⁺ rise (star) before lumen diameter starts to increase. Vertical dotted line (III-V) indicates previous position of vessel wall. (d) Summary data.

Fig. 2

Low O₂ facilitates lactate and PGE₂ release and enhances astrocyte glycolysis. (a and b) Immunohistochemistry showing the astrocyte marker GFAP and the neuron marker MAP2 colocalize with PGT labeling. (c) Lactate release is elevated in low O₂. (d) t-APCD increases PGE₂ release most in low O₂. (e and f) Lactate dilates an arteriole. Images I-III correspond to time points in f, which shows the lumen diameter increase. (g) Summary showing percent dilation by lactate and block by indomethacin. (h) Colocalization of the astrocyte maker SR-101 (left stack) and the NADH signal (middle stack) from perivascular glia somas and endfeet. (i) Top: arteriole NADH fluorescence from a single image plane showing astrocyte compartments, SMCs/endothelial cells and empty lumen of the blood vessel. Middle and Lower: pseudo colored image of NADH fluorescence in high and low O₂. (j) Summary of NADH changes in astrocyte compartments caused by low O₂. (k and l) Astrocyte NADH in response to glycolysis inhibition with iodoacetate (IA) (k) and LDH inhibition with Oxamate (l). Left: single experiment; right: summary.

Fig. 3

Glycolysis and lactate release is critical for astrocyte-mediated dilations. (a) Arteriole and astrocyte NADH image in low O₂; bottom shows ROIs for astrocyte compartments (1 and 2) and vessel (box). (b) Pseudo color NADH (top) and diameter (bottom) changes caused by t-ACPD at time points I-III in c. Vertical dotted line (II and III) indicates previous position of vessel wall. (c) Astrocyte NADH (top) and lumen diameter (bottom) in response to t-ACPD; same experiment as a and b. (d) Summary of NADH increase from mGluR activation in low O₂. (e) Top: NADH image of an arteriole and astrocyte. Lower: soma close-up showing t-ACPD causes a diffuse increase in NADH. (f) t-ACPD increases extracellular lactate most in low O₂. (g) t-ACPD decreases astrocyte NADH during glycolysis inhibition; top: single experiment; bottom: summary. (h) mGluR activation increases astrocyte NADH during LDH inhibition; top: single experiment; bottom: summary. (i) Summary showing t-ACPD fails to dilate vessels in oxamate or IA and PGE₂ rescues vasodilation in these compounds. (j and k) The increase in lactate (j) and PGE₂ (k) caused by t-ACPD is significantly less in oxamate and IA.

Fig. 4

Raising PGE₂ levels by inhibiting PGT changes the polarity of astrocyte-mediating vasomotion. (a) PGE₂ levels were further elevated by mGluR activation in low O₂ when PGT was inhibited by U46619 or T34. (b) Summary data of vasomotion during PGT manipulation. (c) Uncaging Ca²⁺ in high O₂ causes vasodilation in exogenous lactate. Top panels show the astrocyte Ca²⁺ signal change (pseudo colour) from uncaging. Lower panels show close up of vessel lumen. (d) In high O₂, astrocyte-mediated vasoconstriction is converted to vasodilation during PGT blockade. Left: astrocytes and endfeet circumscribing an arteriole. Ca²⁺ is uncaged in 3 astrocytes separated in time; box indicates the vessel region examined on the right. Right: vasomotions corresponding to the separate uncaging events. Small pseudo colour images show the Ca²⁺ signal change from uncaging in each astrocyte. Lower images of the vessel and endfeet (red) show that the vasomotion switches polarity when PGTs are blocked. (e) Diagram of the supported model.