Fig. 6: NMDARs mediate neural activity-induced ETC Ca²⁺ activities via VEGF signaling.

a Typical traces of Ca²⁺ activities at the branch and soma of ETCs evoked by local puff application of 0.1% bovine serum albumin (BSA) or VEGF. The breaks in the right traces indicate the time used for unclogging the micropipette for puffing to the targeted ETC. Gray dashed lines indicate local puff pulses. b Summary of data showing global Ca²⁺ activities of ETCs induced by repetitive local puff application of 0.1% BSA or VEGF. Each pink square indicates a successful induction of global Ca²⁺ transients, and each gray square indicates a failed induction. The traces shown in (a) for 0.1% BSA and VEGF groups correspond to ETC 4 and ETC 5 in the relevant groups, respectively. c, d Suppression of optogenetic stimulation-evoked global Ca²⁺ activities of ETCs by application of the VEGFR2 inhibitor Ki8751. Tg(elavl3:ChrimsonR-tdTomato);Tg(kdrl:GCaMP6s) larvae at 4 dpf were used. Data were obtained under three consecutive conditions: spontaneous (“Spont.”), optogenetic stimulation (“Opto. stim.”), and optogenetic stimulation in combination with Ki8751 treatment (“Opto. stim. + Ki8751”). c Typical case from an ETC, showing Ca²⁺ activities under the three conditions. With Ki8751 application, local Ca²⁺ transients in ETC soma or branches were still observed. Gray dashed lines, global Ca²⁺ transients; open arrowheads, local Ca²⁺ transients. d Summary data from 7 ETCs, showing the number of global Ca²⁺ transients per h in groups of Spont., Opto. stim., Opto. Stim. + Ki8751. Each data point was obtained from individual larvae. e Working model. NMDARs activated by neural activity enhance neuronal VEGF expression through downstream ERK/CAMKIIα pathway and released VEGF further stimulates global Ca²⁺ activities and associated growth capability of ETCs, leading to enhanced vascular development in the brain. The number in the brackets indicates the number of larvae examined. Paired two-tailed Student’s t-test was used for d. Data are represented as mean ± s.e.m.