Figure 2.
Microglia contribute to neurovascular coupling in a P2Y12R-mediated manner. (a) Schematic showing the outline of the experiment. (b) Difference images show CBF changes in the right barrel cortex relative to baseline in response to contralateral whisker stimulation before, during, and after stimulus (stim.; white rectangle indicates the barrel field). Time course of stimulus-evoked CBF responses is shown in the right of b. Scale bar, 1 mm. (c) Absence of microglia or acute blockade of P2Y12R reduces the maximum of evoked CBF responses compared with controls. n = 7 control, n = 7 depleted, and n = 6 PSB-0739 injected mice; *, P < 0.05, one-way ANOVA followed by Dunnett’s multiple comparison test (control versus depleted, P = 0.0191; control versus PSB-0739, P = 0.0243). (d) Protocol of manually and electromechanically controlled whisker stimulation. (e and f) Representative CBF traces and quantification show impaired neurovascular coupling response in the absence of microglia and in P2Y12R KO mice. n = 7 control, n = 6 depleted, and n = 7 P2Y12R KO mice (e and f); n = 10 control, n = 11 depleted, and n = 6 P2Y12R KO mice; **, P = 0.0075 (e); **, P = 0.0058 (f), one-way ANOVA followed by Dunnett’s multiple comparison test (e: *, P = 0.0378, control versus depleted; **, P = 0.0052 control versus P2Y12R KO; f: *, P = 0.0311 control versus depleted; **, P = 0.0047 control versus P2Y12R KO). (g) Representative CBF traces and graph show changes in neurovascular coupling response in L-NAME–treated mice in both the presence and the absence of microglia. n = 9 control, n = 10 depleted, n = 8 L-NAME–treated, n = 9 L-NAME–treated depleted; P < 0.0001, one-way ANOVA followed by Tukey’s multiple comparison test (**, P = 0.005, control versus depleted; **, P = 0.0049, control versus L-NAME; **, P = 0.0026, L-NAME versus depleted + L-NAME; ***, P = 0.0008, depleted versus depleted + L-NAME). (h) fUS imaging reveals reduced CBV responses compared with controls in the ipsilateral (ipsi) and contralateral (contra) barrel cortex. Representative traces of 10 subsequent stimulations (30 s each) are shown for control and microglia-depleted mice. (i) Peak trace averages of the contralateral side in control and depleted mice, with 95% confidence intervals. (j) Averaged AUC distribution for each group, as shown in pink window in i. Data are presented as mean ± SEM; n = 30 and n = 40 stimulations from three control and four depleted mice, respectively (j); **, P = 0.0093, two-way ANOVA followed by Sidak’s multiple comparisons test. Data are presented as mean ± SEM. LSCI data have been pooled from two to three independent experiments.

Microglia contribute to neurovascular coupling in a P2Y12R-mediated manner. (a) Schematic showing the outline of the experiment. (b) Difference images show CBF changes in the right barrel cortex relative to baseline in response to contralateral whisker stimulation before, during, and after stimulus (stim.; white rectangle indicates the barrel field). Time course of stimulus-evoked CBF responses is shown in the right of b. Scale bar, 1 mm. (c) Absence of microglia or acute blockade of P2Y12R reduces the maximum of evoked CBF responses compared with controls. n = 7 control, n = 7 depleted, and n = 6 PSB-0739 injected mice; *, P < 0.05, one-way ANOVA followed by Dunnett’s multiple comparison test (control versus depleted, P = 0.0191; control versus PSB-0739, P = 0.0243). (d) Protocol of manually and electromechanically controlled whisker stimulation. (e and f) Representative CBF traces and quantification show impaired neurovascular coupling response in the absence of microglia and in P2Y12R KO mice. n = 7 control, n = 6 depleted, and n = 7 P2Y12R KO mice (e and f); n = 10 control, n = 11 depleted, and n = 6 P2Y12R KO mice; **, P = 0.0075 (e); **, P = 0.0058 (f), one-way ANOVA followed by Dunnett’s multiple comparison test (e: *, P = 0.0378, control versus depleted; **, P = 0.0052 control versus P2Y12R KO; f: *, P = 0.0311 control versus depleted; **, P = 0.0047 control versus P2Y12R KO). (g) Representative CBF traces and graph show changes in neurovascular coupling response in L-NAME–treated mice in both the presence and the absence of microglia. n = 9 control, n = 10 depleted, n = 8 L-NAME–treated, n = 9 L-NAME–treated depleted; P < 0.0001, one-way ANOVA followed by Tukey’s multiple comparison test (**, P = 0.005, control versus depleted; **, P = 0.0049, control versus L-NAME; **, P = 0.0026, L-NAME versus depleted + L-NAME; ***, P = 0.0008, depleted versus depleted + L-NAME). (h) fUS imaging reveals reduced CBV responses compared with controls in the ipsilateral (ipsi) and contralateral (contra) barrel cortex. Representative traces of 10 subsequent stimulations (30 s each) are shown for control and microglia-depleted mice. (i) Peak trace averages of the contralateral side in control and depleted mice, with 95% confidence intervals. (j) Averaged AUC distribution for each group, as shown in pink window in i. Data are presented as mean ± SEM; n = 30 and n = 40 stimulations from three control and four depleted mice, respectively (j); **, P = 0.0093, two-way ANOVA followed by Sidak’s multiple comparisons test. Data are presented as mean ± SEM. LSCI data have been pooled from two to three independent experiments.

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