New methods of dye application for staining motor neurons in an insect

Motor neurons innervating tiny muscles in the antennal system of the cockroach, Periplaneta americana, were stained by two new approaches to backfilling motor axons and somata. Both methods are easier, and produce less leakage of dye, than conventional methods that rely on application of dye to the distal cut end of a motor nerve or immersion of a whole muscle in dye. One approach used here was to pressure-inject nickel (or cobalt) chloride directly into a muscle from a micropipette. The other was to place filter paper soaked in nickel chloride on the muscle surface. The first approach enabled staining of motor neurons most reliably and with preservation of stain to the specific muscle of interest. The second approach had different advantages: it was easier to perform, resulted in more intense staining and frequently more complete staining of dendritic structure than the first approach. Taken together, these two approaches should prove useful for tracing motor circuits in a number of model systems.