In vivo local dye electroporation for Ca²⁺ imaging and neuronal-circuit tracing

A major technical challenge for using optical imaging to analyze neuronal circuit functions is how to effectively load synthetic Ca(2+) dyes or neural tracers into the brain. We introduce here a simple but versatile approach to label many neurons and clearly visualize their axonal and dendritic morphology. The method uses a large-tip patch pipette filled with dextran-conjugated Ca(2+) dyes or fluorescent tracers. By inserting the pipette into a targeted brain area and passing microampere current pulses, dyes or tracers are electroporated into dendrites and axons near the pipette tip. The dyes are then transported to reveal the entire cell morphology, suitable for both functional Ca(2+) imaging and neuronal circuit tracing. This process requires basic physiological equipment normally available in a physiological laboratory.