Figure 7

ATP-triggered trilateral communication pathways among microglia, astrocytes and neurons in organotypic hippocampal slice cultures.

(a), (b) The endogenous neuroprotection is mediated by glial cells, P2Y₁ receptors and IL-6. MeHg^low (0.1 μM, 48 h) did not cause an increase in PI incorporation in WT slices. The PI incorporation was increased by minocycline (mino, 10 μM, 24 h pretreatment) or fluorocitrate (FC, 1 μM, 24 h pretreatment), MRS2179 (MRS, 10 μM, 24 h pretreatment) and an IL-6 blocking antibody (IL6Ab, 200 pg/ml, simultaneous treatment) (n = 8–11, **p < 0.01 vs. control). (c) MeHg^low (0.1 μM, 24 h) increased IL-6 production from WT slices (n = 4, **p < 0.01 vs. control). (d) MeHg^low (0.1 μM, 3 h) increased ATP release from WT slices (n = 5, **p < 0.01 vs. control). (e), (f) MeHg^low (0.1 μM, 48 h) increased PI incorporation in VNUTKO slices, which was prevented by ATP (100 μM, 30 min pretreatment) or recombinant IL-6 protein (100 pg/ml, simultaneous treatment) (n = 17–22, **p < 0.01 vs. control, ^##p < 0.01 vs. MeHg^low). (g) VNUTKO slices did not release ATP in response to MeHg^low (0.1 μM, 3 h) (n = 6, p = 0.41). (h) MeHg^low (0.1 μM, 24 h) did not induce any increase in IL-6 production from VNUTKO slices (n = 4, p = 0.87). (i), (j) Similar to the findings in VNUTKO slices, MeHg^low (0.1 μM, 48 h) increased PI incorporation in P2Y₁KO slices; however, unlike the results in VNUTKO slices, exogenously applied ATP (100 μM, 30 min pretreatment) had no effect on MeHg^low-increased PI incorporation. Recombinant IL-6 protein (100 pg/ml, simultaneous treatment) significantly reduced the PI signals (n = 8–11, **p < 0.01 vs. control, ^##p < 0.01 vs. MeHg^low). (k) MeHg^low (0.1 μM, 24 h) did not induced IL-6 production from P2Y₁KO slices (n = 4, p = 0.19). Values are the mean ± SEM for (b–d), (f–h), (j) and (k). Student's t-test and one-way ANOVA followed by Fisher's LSD test were used for comparisons of two and multiple groups, respectively.