Figure 5

VNUT-mediated ATP release from microglia is essential for Ca²⁺ signaling and IL-6 production from astrocytes in the mixed glial cultures.

(a) Dependency of of the MeHg^low-evoked ATP release on exocytosis. BoNT (5 units/ml, 24 h pretreatment) significantly suppressed the MeHg^low-induced ATP release from microglia (n = 5, **p < 0.01, Mann–Whitney U-test). (b) Dependency of the MeHg^low-evoked ATP release on VNUT. In contrast to ATP release from WT microglia, VNUTKO microglia showed no ATP release in response to MeHg^low (0.1 μM, 3 h) (n = 6–11, **p < 0.01 vs. WT microglia without MeHg, ^##p < 0.01 vs. WT microglia with MeHg^low, one-way ANOVA followed by Fisher's LSD test). (c) Microglial VNUT is essential for the MeHg^low-evoked ATP release in Ms mixed glial cultures. MeHg^low(0.1 μM, 3 h) evoked ATP release in WT microglia/WT astrocyte mixed cultures but not in VNUTKO microglia/WT astrocyte cultures (n = 8, **p < 0.01 vs. WT microglia/WT astrocytes without MeHg, ^##p < 0.01 vs. WT microglia/WT astrocytes with MeHg^low, one-way ANOVA followed by Fisher's LSD test). (d) Microglial VNUT is indispensable for MeHg^low-triggered Ca²⁺ oscillations in astrocytes in the Ms mixed glial cultures. MeHg^low (0.1 μM, 15 min) evoked a dramatic increase in the frequency of Ca²⁺ oscillations in astrocytes in WT microglia/WT astrocyte mixed cultures, but a small increase in VNUTKO microglia/WT astrocytes (n = 98, **p < 0.01 vs. WT microglia/WT astrocytes without MeHg, ^##p < 0.01 vs. WT microglia/WT astrocytes with MeHg^low, one-way ANOVA followed by Fisher's LSD test). (e) Microglial VNUT-dependent IL-6 production in Ms mixed glial cultures. MeHg^low (0.1 μM, 24 h) evoked IL-6 production in WT microglia/WT astrocyte mixed cultures, but not in VNUTKO microglia/WT astrocytes (n = 3, **p < 0.01 vs. WT microglia/WT astrocytes without MeHg, ^##p < 0.01 vs. WT microglia/WT astrocytes with MeHg^low, one-way ANOVA followed by Fisher's LSD test). Values are the means ± SEM for all groups.