Extracellular application of cobalt: a fast and simple method for delineating invertebrate neurosecretory pathways

The extracellular cobalt backfilling technique was shown to be an excellent method to obtain cobalt backfills of invertebrate neurosecretory cells (NSCs). Aqueous cobalt was placed in an extracellular suction electrode into which a portion of a neurohaemal (NH) region containing the axons and/or terminals of NSCs was drawn. Spontaneously discharging extracellular action potentials were recorded as the cobalt was applied to the NH region, and the greater the electrical activity, the more extensive the cobalt backfilling. The greatest success occurred with 0.5 M cobalt chloride dissolved in physiological saline. No backfills were obtained in the absence of electrical activity. This technique was shown to backfill NSCs terminating in NH regions of the insect, Rhodnius prolixus, the isopod crustacean, Oniscus asellus, and the freshwater pulmonate snail, Helisoma. Combined with a light insensitive silver intensification method, this paper describes a relatively fast and simple method for delineating invertebrate neurosecretory pathways.