In vivo visualization of living flatworm neurons using Lucifer yellow intracellular injections

Turbellarian flatworms lend themselves to neurobiological investigations using intracellular iontophoresis of Lucifer yellow provided that one is able to anesthetize the animal and expose the nervous system. This paper details the methods used with the polyclad Notoplana acticola and the rhabdocoel Mesostoma ehrenbergii. Marine turbellarians can be anesthetized with equal parts of sea water and isotonic MgCl2 and fresh-water animals with an 8% ethanol in spring water. Animals can be held steady with minuten pins and spines of the cactus Opuntia basilaris or O. littoralis. Sheaths surrounding the brain can be digested away with a protease. Conventional glass microelectrode techniques are used to fill the cells with fluorescent dye, Lucifer yellow. The preparation needs to be viewed using darkfield illumination. Cells can be photographed through the microscope or traced using a camera lucida attachment to a fluorescence microscope. Tracings tend to be more useful for preserving details of the three-dimensional nature of the neuronal cytoarchitecture.