Figure 3.

Intracellular alkalization and gap junction uncoupling on epileptiform activity. A, Left, Diagram of the experimental setup (top) and a fluorescent image of E²GFP expression the bigenic mice, Mlc1-tTA::tetO-lck-E²GFP. Right, Mean pH change (mean ± SEM) from the baseline is plotted. Values were normalized to the control. After 15 min of 0Mg²⁺-Pic perfusion (abbreviated as 0Mg²⁺), intracellular pH became higher (alkalized) compared with control at both +5 and +10 min time points (control, n = 5; 0Mg²⁺-Pic, n = 11; **p < 0.01, two-way repeated-measures ANOVA followed by Tukey's post hoc test). B, Left, Diagram of the experiments. NBC activation resulted in intracellular alkalization because of the influx of the alkaline ion HCO₃^–. Application of S0859, a NBC blocker, prevented the HCO₃^– influx. Right, Intracellular alkaline pH shifts were prevented by S0859 (0Mg²⁺-Pic, same data as in Fig. 3A, n = 11; S0859 in 0Mg²⁺-Pic, n = 10; **p < 0.01, two-way repeated-measures ANOVA followed by Tukey's post hoc test). C, Gap junction coupling evaluated by the [Na⁺]_i imaging in astrocytes. Top panels, Application of 20 mm NH₄Cl, which induces intracellular alkalization, resulted in a strong gap junction uncoupling. Bottom, Epileptiform activity induced by 0Mg²⁺-Pic perfusion normally induced intracellular alkalization in astrocytes as well as gap junction uncoupling among the astrocytes. In the presence of S0859, gap junctions were relieved from uncoupling, although the slice was exposed to epileptiform activity. Scale bars, 20 µm. D, Normalized peak ΔF_ratio plotted against the distance from the starter cell in alkalized and S0859 conditions. Gap junction coupling was significantly reduced by direct alkalization by NH₄Cl (left; control, same data as in Fig. 2E, n = 22, 23, 4; NH₄Cl, n = 13, 13, 13 for each interval, 10–40, 50–70, 80–100 µm, respectively; *p < 0.05, ****p < 0.0001, Welch's t test with Holm correction). Even when the slice was exposed to 0Mg²⁺-Pic solution, if the solution was supplemented with S0859, the gap junction was relieved from uncoupling (0Mg²⁺-Pic, same data as in Fig. 2E, n = 11, 13, 8; S0859 in 0Mg²⁺-Pic, n = 26, 13, 3 for each interval, respectively; ***p < 0.001, Welch's t test).