Electroporation of neurons and growth cones in Aplysia californica

Specific labeling of individual neurons and neuronal processes is virtually an everyday task for neuroscientists. Many traditional ways for delivery of intracellular dyes have limitations in terms of speed, efficiency and reproducibility. Electroporation is a fast, reliable and efficient method to deliver microscopic amounts of polar and charged molecules into neurons and their compartments such as individual neurites and growth cones. Here, we present a simple and highly effective procedure for intracellular labeling of individual Aplysia neurons both in intact ganglia and in cell culture. Pleural mechanoreceptor neurons have been used as illustrative examples to demonstrate applicability of direct and local labeling of the smallest individual neurites (< 2 microm) and single growth cones. Specifically, a 3-s train of 1.0 V hyperpolarizing pulses at 50 Hz effectively filled discrete neurites in contact with the tip of the micropipette with no dye transfer visible to other, non-contacted neurites. Application of this localized dye labeling technique to single neurites reveals a surprisingly complex morphology for patterns of axonal branching in culture. The protocol can be easily applied to a variety of models in neuroscience including accessible nervous systems of invertebrate animals.