Figure 8

Laminin engagement alters functional hemichannel but not GJIC activity. Functional hemichannel activity was assessed by anionic LY dye uptake (a) of cultures exposed to laminin for 1 DIV (prior to any changes in connexin expression) or 6 DIV (following changes in connexin expression). Spontaneous LY uptake was observed at low levels after 1 and 6 DIV. Open hemichannel activity could be induced by mechanical stimulation with glass microbeads within 1 DIV but not 6 DIV. Dye uptake was inhibited by the hemichannel/chloride channel inhibitor FFA but not the chloride channel blocker DIDS. LY⁺/RD^- cells is expressed as a percentage of the total number of cells per microscopic field ± standard error of the mean (SEM) counted in n = 5 fields per experiment conducted in triplicate experiments. GJIC was assayed using the scrape-loading method (b). Significant LY⁺/RD^- dye transfer was not observed after 1 or 6 DIV when NPCs were cultured in the presence of mitogens (proliferative conditions). As a positive control, we performed the same experiment in a condition known to promote glial cell differentiation and obtained robust GJIC that was significantly inhibited by the gap junction blocker GRA but not the inactive analog GZA (glial differentiation). In GJIC assays, the number of LY⁺/RD^- cells along the scrape line was established in serial photographs taken along the entire length of the scrape (9–14 photos/coverslip) over triplicate cultures. Two coverslips were assessed per culture for a total of n = 54–84 measurements per condition. Data are expressed as the mean number of LY⁺/RD^- cells ± SEM. *p < 0.05, **p < 0.01, ANOVA, post-hoc Tukey test.