Figure 5. MFA, a gap junction inhibitor, blocked the tracer coupling but not electrical coupling and was unable to block the mitoK_ATP channels’ effects on electrical coupling of directly coupled astrocytes.

The Loading of LY into cells for 10 min resulted in transcellular diffusion (A1, bar  = 10 µm) but not when cells were pre-treated with MFA (A2, bar  = 20 µm). Furthermore, the separate loading of LY (green, C1) and Alexa Fluor® 594 (red, C2) into cells via dual patch recording electrodes for 10 min also did not lead to transcellular diffusion of the two tracers upon pre-treatment of the slices with MFA 1 h prior to recording. However, when the slices were not pre-treated with MFA, we observed transcellular diffusion of LY (green, B1) but not Alexa Fluor® 594 (red, B2) (bar  = 10 µm). (D) Bar graph shows that DIZ significantly increased the transcellular diffusion of LY (DIZc1, n = 9), MFA inhibited the transcellular diffusion of LY (n = 7), and DIZ increased the transcellular diffusion of LY even after pre-treatment of the slices with MFA 1 h prior to recording (n = 10). (E1) Representative electrical coupling trace of recorded astrocyte pairs after incubation with DIZ for 5 min under conditions with or without MFA pre-treatment. (E2) Bar graph shows that MFA inhibited the coupling ratio by 17% (n = 8), while at 5 min after loading the cell with DIZ, the MFA inhibited the coupling ratio only by 7% (n = 12). These results were determined from paired recordings from astrocytes at interastrocytic distances of 20 – 40 µm. Mean ± SEM; * P<0.05. ^# P<0.05 compared to the control. MFA, Meclofenamic acid; DIZ, Diazoxide; 5-HD, 5-hydroxydecanoate.