Fig. 5: Microglial CD39 regulates basal CBF, neurovascular coupling and whisker stimulation-induced local adenosine concentration increase.

a Real-time CBF changes after ICM-injection of ARL67156 (1 mM, 3 μl). b Does-responses of CBF changes after ICM-injection of ARL67156 (3 μl). Each dot represents an individual mouse. Sample sizes are n = 3, 3, 4 for the 0.01 mM, 0.2 mM, 1 mM conditions, respectively. Data are presented as mean ± s.e.m. Source data are provided as a Source Data file. c The CBF responses to ICM injection of ARL67156 (1 mM, 3 μl) in control and PLX-treated conditions. Each dot represents an individual mouse. Sample sizes are n = 8, 6 for control and PLX conditions, respectively. Data are presented as mean ± s.e.m. p-values were determined by unpaired t-test two sided. Source data are provided as a Source Data file. d, e Comparison of the CBF baseline (d) and the CBF change to whisker stimulation (e) in control and at 30 min after ICM injection of ARL67156 (1 mM, 3 μl). Each line denotes an individual mouse. n = 7 mice for each group. p-values were determined by unpaired t test two sided. Source data are provided as a Source Data file. f–i Comparison of the CBF baseline (f), CBF change to whisker stimulation (g), CBF plateau to whisker stimulation (h), CBF change to ICM-ATP injection (i), and CBF change to ICM-adenosine injection (j) in tamoxifen-induced Cx3cr1-CreER^-;Entpd1 (CD39)^fl/fl and Cx3cr1-CreER⁺; Entpd1 (CD39)^fl/fl mice. Note that microglial CD39-deleted mice showed elevated basal CBF, and reduced CBF change to whisker stimulation and ICM-ATP injection, but no clear changes in the CBF change to ICM-adenosine injection. Each dot represents one individual mouse. n = 6, 6 for Entpd1^fl/flCre⁺ and Entpd1^fl/flCre⁻ groups, respectively. Data are presented as mean ± s.e.m. P-values were determined by unpaired t-test two sided. Source data are provided as a Source Data file. k, l A 3-D color plot of whisker stimulation-induced cyclic voltammogram signals in contralateral barrel cortex (k). The 3-D color plot depicts the time on the x-axis, potential on the y-axis, and current in false color. Background subtracted cyclic voltammogram showed the primary oxidation at 1.4 V and the secondary oxidation at 1.0 V (l), which are typical for adenosine. m–o Current-vs-time plot shows the whisker stimulation-induced adenosine currents on the contralateral (m) and ipsilateral (n) barrel cortex. Whisker stimulation was started in the 30 s and ended at 50 s. Blue dish line marked the 20 s stimulation window. o Summary of whisker stimulation-induced adenosine release in different conditions. Each dot represents one individual mouse. Sample sizes are n = 11, 12, 6, 6 for control, PLX, control + ARL, PLX + ARL groups, respectively. p-values were determined by one-way ANOVA with Tukey post hoc test; *P < 0.05, **P < 0.01, ***P < 0.0001. Data are presented as mean ± s.e.m. Source data are provided as a Source Data file. p Schematics of the deduced mechanism in this study by which microglia modulate neurovascular coupling (Created in BioRender. Kuan, A. (2025) https://BioRender.com/h84u232). Upon neuronal excitation such as whisker stimulation, ATP is released from neurons or glia, and undergo microglia CD39-initiated hydrolysis, followed by CD73 or tissue-nonspecific alkaline phosphatase (TNAP)-mediated hydrolysis to form adenosine, leading to vasodilation.