Figure 7.

Effect of TAT-Cx43_266–283 on astrocyte hemichannel activity and gap junctional intercellular communication (GJIC) in astrocyte cultures and in neuron-astrocyte co-cultures. (A,B) Astrocyte cultures were incubated with 10 μg/ml lipopolysaccharide (LPS) for 24 h or with 100 μM KA or conditioned medium from KA-treated neuron-astrocyte co-cultures (CM) for 8 h. Then, 5 μM ethidium bromide (EtBr) was added for 10 min. Two-hundred micromolar Carbenoxolone (CBX), 50 μM TAT-Cx43_266–283, 1 μM dasatinib and 1 μl/ml dimethyl sulfoxide were applied in the presence of LPS, 15 min before and during EtBr uptake. (A) Representative photomicrographs showing EtBr uptake in astrocytes after LPS treatment and the inhibition promoted by CBX and TAT-Cx43_266–283. (B) Quantification of EtBr uptake intensity in arbitrary units (AU). Dotted line represents the basal level of EtBr fluorescence found in untreated astrocytes. The results are the means ± SEM (n = 3; at least 300 cells were counted per condition). **p < 0.01, ANOVA (post-test Holm-Sidak). (C) Astrocyte cultures were incubated in the absence or presence of 50 μM TAT-Cx43_266–283 24 h before and during the scrape-loading experiment. Photomicrographs obtained after Lucifer yellow (LY) scrape loading and quantification of the number of fluorescent cells per field. The results are the means ± SEM (n = 3). (D,E) Neuron-astrocyte co-cultures were incubated with 100 μM KA for 8 h and then 5 μM EtBr was added for 10 min. Two-hundred micromolar CBX, 50 μM TAT-Cx43_266–283 or 1 μM dasatinib were applied in the presence of KA, 15 min before and during EtBr uptake. (D) Representative photomicrographs showing EtBr uptake in neuron-astrocyte co-cultures after KA treatment and the inhibition promoted by CBX and TAT-Cx43_266–283. (E) Quantification of EtBr uptake intensity in AU. The results are the means ± SEM (n = 3; at least 300 cells were counted per condition). ***p < 0.001 ANOVA (post-test Holm-Sidak). Bar, 50 μm.