Live-Cell Visualization of Neurons

Live-cell visualization of whole neurons was performed by transfection using pLV-hSyn-RFP plasmid from Edward Callaway (Addgene plasmid # 22909) and Lipofectamine 2000 (Thermo Fisher Scientific) in accordance with the manufacturer’s protocol. Transfections were carried out in serum-reduced OptiMEM media (Thermo Fisher Scientific). A Leica DM6000 FS microscope, Leica DFC 345 FX camera, and the Leica Application Suite software were used to produce micrographs. We performed live-cell visualization of 2 neurons from two different preparations in order to illustrate our experimental approach (Figures 3a, 4a,e and 5a and Figure 2—figure supplement 1a,b) as well as to demonstrate that the spatiotemporal distribution of the recorded signals matches with neuronal morphology (Figures 1a, 2, 3a, 4e and 5a and Figure 2—figure supplement 1a,c). The latter has also been demonstrated in previous studies, where live-cell visualization was used to verify axonal AP propagation tracking by means of HD-MEA-based monitoring of extracellular APs (Bakkum et al., 2013 (link); Radivojevic et al., 2016 (link)).