TRPV4 channels stimulate Ca2+-induced Ca2+ release in astrocytic endfeet and amplify neurovascular coupling responses

Abstract

In the CNS, astrocytes are sensory and regulatory hubs that play important roles in cerebral homeostatic processes, including matching local cerebral blood flow to neuronal metabolism (neurovascular coupling). These cells possess a highly branched network of processes that project from the soma to neuronal synapses as well as to arterioles and capillaries, where they terminate in "endfeet" that encase the blood vessels. Ca(2+) signaling within the endfoot mediates neurovascular coupling; thus, these functional microdomains control vascular tone and local perfusion in the brain. Transient receptor potential vanilloid 4 (TRPV4) channels--nonselective cation channels with considerable Ca(2+) conductance--have been identified in astrocytes, but their function is largely unknown. We sought to characterize the influence of TRPV4 channels on Ca(2+) dynamics in the astrocytic endfoot microdomain and assess their role in neurovascular coupling. We identified local TRPV4-mediated Ca(2+) oscillations in endfeet and further found that TRPV4 Ca(2+) signals are amplified and propagated by Ca(2+)-induced Ca(2+) release from inositol trisphosphate receptors (IP3Rs). Moreover, TRPV4-mediated Ca(2+) influx contributes to the endfoot Ca(2+) response to neuronal activation, enhancing the accompanying vasodilation. Our results identify a dynamic synergy between TRPV4 channels and IP3Rs in astrocyte endfeet and demonstrate that TRPV4 channels are engaged in and contribute to neurovascular coupling.

Fig. 1.

Activation of TRPV4 with the agonist GSK1016790A (GSK) increases Ca²⁺ oscillations in astrocytic endfeet. (A) Pseudocolor Fluo-4 AM fluorescence representing [Ca²⁺]_i in astrocytic endfeet surrounding a parenchymal arteriole in a brain slice (field of view = 62 × 62 µm). (Scale bar, 10 µm.) In the image shown, Fluo-4 AM fluorescence can be observed in the astrocyte soma as well as the endfeet. (B) Time course of a propagating Ca²⁺ oscillation within an endfoot in response to GSK (100 nM). (Scale bar, 10 µm.) Total duration of the oscillation was 8.2 s. GSK stimulated fast, high-amplitude Ca²⁺ oscillations in astrocytic endfeet, mainly in the form of Ca²⁺ waves. (C, a; D, a; and E, a) Representative fractional fluorescence (ΔF/F_o) traces from ROIs on perivascular astrocytic endfeet in one brain slice before and after addition of GSK. Each color represents one ROI. (C, b and C, c) GSK increased the frequency and maximum amplitude of endfoot Ca²⁺ oscillations (P < 0.05; n = 7 slices from six animals). (D) GSK had no effect in TRPV4^−/− mice (n = 4 slices from two animals). (E, b and E, c) The effect of GSK on the frequency and amplitude of endfoot Ca²⁺ oscillations was blocked by the selective TRPV4 antagonist, HC-067047 (10 µM; n = 6 slices from from animals). Values were compared by paired t test.

Fig. 2.

The 11,12-EET increases endfoot Ca²⁺ oscillations via TRPV4 channels. (A) Time course of an 11,12-EET-stimulated Ca²⁺ oscillation in an astrocytic endfoot. (Scale bar, 10 µm.) The total duration of the oscillation was 9.3 s. (B, a and B, c) The 11,12-EET (1 µM) increased the amplitude of endfoot Ca²⁺ oscillations (P < 0.05; n = 4 slices from three animals), but did not significantly increase oscillation frequency (B, b). (C) Preincubation of brain slices with HC-067047 (1 µM) prevented the effect of 11,12-EET on endfoot Ca²⁺ oscillations (n = 5 slices from two animals). Values were compared by paired t test.

Fig. 3.

TRPV4 Ca²⁺ entry in astrocytic endfeet stimulates IP₃R Ca²⁺ release, amplifying local Ca²⁺ increases and triggering Ca²⁺ waves. (A) Time course of a Ca²⁺ oscillation in response to GSK after depletion of ER Ca²⁺ stores with the SERCA pump inhibitor, CPA (30 µM). CPA eliminated Ca²⁺ waves in response to GSK. In the presence of CPA, GSK induced slow, low-amplitude Ca²⁺ oscillations. The total duration of the oscillation was 37.1 s. (Scale bar, 10 µm.) (B, a) A representative trace of fractional fluorescence changes within ROIs on astrocytic endfeet induced by stimulation with GSK in a brain slice preincubated with CPA. The frequency (B, b) and amplitude (B, c) of endfoot Ca²⁺ oscillations was still increased by stimulation with GSK in the presence of CPA (P < 0.01; n = 6 slices from four animals). (C) CPA reduced the average spatial spread of GSK-stimulated endfoot Ca²⁺ oscillations (P < 0.001; n = 27 for Control GSK; n = 9 for GSK + CPA). (D) Locally, Ca²⁺ increases to GSK were faster and of higher amplitude under normal conditions compared with when ER Ca²⁺ was depleted by CPA. Traces depict fluorescence in ROIs of corresponding color in image panels to the left. Values in B were compared by paired t test; values in C were compared by unpaired t test.

Fig. 4.

Astrocytic TRPV4 channels contribute to NVC by augmenting the endfoot Ca²⁺ response to neuronal activation. (A and C) EFS-induced depolarization of neurons in brain slices caused a rise in endfoot Ca²⁺ and dilation of parenchymal arterioles under control conditions. (A and B) Preincubation of brain slices with the TRPV4 antagonist, HC-067047 (10 µM for 25 min), reduced the increase in endfoot [Ca²⁺]_i (B, a) and vasodilation (B, b) resulting from EFS (P < 0.01; n = 6 slices from five animals). (C and D) EFS-evoked increases in endfoot [Ca²⁺]_i (D, a) and vasodilation (D, b) were unchanged following a 10-min exposure to GSK (100 nM) (n = 4 slices from four animals). (Scale bar, 10 µm.) Values were compared by paired t test.

Fig. 5.

Astrocytic endfoot TRPV4 channel activation during NVC is not mediated by EETs. Brain slices were incubated with MAFP (5 μM for 20 min), an inhibitor of cytosolic and Ca²⁺-independent PLA₂, to block arachidonic acid production and the subsequent formation of cytochrome P450 (CYP) metabolites, including EETs. (A) Endfoot Ca²⁺ and parenchymal arteriole diameter responses to EFS before and after treatment with MAFP. (B, a) Incubation with MAFP dilated parenchymal arterioles before EFS (P < 0.01; n = 7 slices from four animals). In the presence of MAFP, the EFS-evoked increase in endfoot [Ca²⁺]_i did not differ from that in controls (B, b), but the evoked dilation was reduced (B, c) (P < 0.01; n = 7 slices from four animals). (Scale bar, 10 µm.) Values were compared by paired t test.

Fig. 6.

TRPV4 channels contribute to NVC in vivo. (A) Representative CBF recordings, presented in laser Doppler perfusion units (P.U.s), from the somatosensory cortex during 60 s contralateral whisker stimulation (indicated by the black bar) before (Left) and after (Right) cortical superfusion of HC-067047 (10 µM). (B) Cortical superfusion of HC-067047 (10 µM) attenuated the increase in CBF to contralateral whisker stimulation (P < 0.05; n = 7). Values were compared by paired t test.