Innovative Methodology Electroporation Loading of Calcium-Sensitive Dyes Into the CNS

Bonnot, Agnès, George Z. Mentis, Jesse Skoch, and Michael J. O’Donovan. Electroporation loading of calcium-sensitive dyes into the CNS. J Neurophysiol 93: 1793–1808, 2005. First published October 27, 2004; doi:10.1152/jn.00923.2004. Calcium imaging of neural network function has been limited by the extent of tissue labeled or the time taken for labeling. We now describe the use of electroporation—an established technique for transfecting cells with genes—to load neurons with calcium-sensitive dyes in the isolated spinal cord of the neonatal mouse in vitro. The dyes were injected subdurally, intravascularly, or into the central canal. This technique results in rapid and extensive labeling of neurons and their processes at all depths of the spinal cord, over a rostrocaudal extent determined by the position and size of the electrodes. Our results suggest that vascular distribution of the dye is involved in all three types of injections. Electroporation disrupts local reflex and network function only transiently ( 1 h), after which time they recover. We describe applications of the method to image activity of neuronal populations and individual neurons during antidromic, reflex, and locomotor-like behaviors. We show that these different motor behaviors are characterized by distinct patterns of activation among the labeled populations of cells.