Optical monitoring of neural networks evoked by focal electrical stimulation on microelectrode arrays using FM dyes

Patch-clamping or microelectrode arrays (MEA) are conventional methods to monitor the electrical activity in biological neural networks in vitro. Despite the effectiveness of these techniques, there are disadvantages including the limited number of electrodes and the predetermined location of electrodes in MEAs. In particular, these drawbacks raise a difficulty in monitoring a number of neurons outnumbering the electrodes. Here, we propose an optical technique to determine the effective range of focal electrical stimulation using FM dyes in neural networks grown on planar-type MEAs. After 3 weeks in culture, electrical stimulation was delivered to neural networks via an underlying electrode in the presence of FM dyes. The stimulation induced the internalization of the dye into the neurons around the stimulating electrodes. Fluorescent images of dye distribution successfully showed the effects of focal stimulation. A range of stimulus amplitudes and frequencies were examined to collect fluorescence images. FM-dye uptake after electrical stimulation resulted in the labeling of cells up to approximately 300 mu m away from the stimulating electrode. Fluorescence intensity increased proportionally to stimulation amplitude. Tetrodotoxin was shown to inhibit the labeling of neurons except those located immediately adjacent (within 40 mu m) from the stimulating electrode. In the presence of AMPA and NMDA receptors antagonists, the FM-dye labeling appeared within 80 mu m from the electrode, indicating directly evoked neural networks via blocking of glutamatergic synaptic transmission. These results showed that FM dyes can be a useful tool for monitoring activity-dependent synaptic events and determining the effect of focal stimulation in cultured neural networks.