Figure 6.

Proinflammatory cytokine-mediated influence of hμG on hRGC function involves the mTOR pathway

A temporal representation of number of active electrodes (A) and mean firing rate (B) revealed by MEA recordings of hRGCs cultured in four different conditions over 22 days. Analysis of MEA recordings at DIV17 after plating revealed that the increase observed in the number of active electrodes (C) and mean firing rate (D) in the presence of hμG CM decreased significantly when rapamycin was included in the culture demonstrating the involvement of AKT/mTOR pathway in proinflammatory cytokine-mediated influence of hμG on hRGC function, which is displayed in the Raster plots of representative hRGC recordings showing the effects of four different culture conditions on the spontaneous activities (E–H). Representative extracellular spike waveforms revealed the facilitatory effects of hμG CM on the neural function versus control (E and F), which is abrogated in antibody-neutralized hμG CM (F and G) or CM with rapamycin (F and H). Western blot analysis carried out on the cell lysates of hRGCs in different cultured conditions revealed that the expression of sodium channel subunit, SCN2A, which increased significantly in the presence of hμG CM versus controls, was abrogated in the presence of neutralizing antibodies and rapamycin (I and J). Experiments were carried out from 6 technical replicates per group. Values expressed as mean ± SEM.