2.6. Calcium Imaging in Neuronal Culture

Calcium imaging was used to evaluate the effect of electrically released GLU and GABA in cultured neurons. Fluo-4 calcium imaging kit was used for the detection of calcium influx during drug release. Calcium-bound Fluo-4 dye has an excitation of 494 nm and emission of 506 nm. In addition, 20 mM glucose in 2 mL live cell imaging solution was added to the well to support the cell health in the longer term. A single drug wire (Pt/PEDOT/SNP(GLU) or Pt/PEDOT/SNP(GABA)) was inserted perpendicularly into the Petri dish cultured with live neurons. With the aid of a Leica DMIRB microscope, the drug wire was positioned immediately adjacent to or in contact with the cell layer. Electrical stimulations (2 Hz, 0 ± 0.5 V, burst mode) were then applied, and the stimulation duration was set to 5 s. The fluorescence images were acquired at 5 frame/sec for 30 s, which covers the 5 s before, 5 s onset, and 20 s after stimulation. The exposure time was set to 10 ms. The calcium signal of individual neurons was analyzed using ImageJ. The baseline fluorescence intensity F₀ was obtained by averaging the signal before stimulation. ΔF is the fluorescence intensity after F₀ subtraction.