Cobalt mapping of the nervous system: How to avoid artifacts

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Abstract
Infusion of cobalt ions into cut axons is an established method for tracing neuron projections in the central nervous system. Artifacts, where unintended neurons are stained, however, have been reported, leading to difficulties in interpretation. Experiments in the locust Schistocera gregaria Forskål show that such artifacts can be induced through damage to axons caused by cutting peripheral nerves and by using high cobalt chloride concentrations (0.4M and above). Mixtures of cobalt and nickel chlorides and nickel chloride alone were introduced into different branches of the same nerve and developed with rubeanic acid to give precipitates of different colors in the two sets of axons. Preparations were examined with the light microscope, where mixing of ions would appear as intermediate colors, and by x‐ray probe microanalysis. No evidence for leakage of metal ions from the filled axons or for ion uptake by other axons could be detected, provided that low concentrations of cobalt and nickel chlorides were used and nerve cutting was reduced to a minimum by making preparations in vivo. If extreme conditions are avoided when making the preparation, the risk of producing artifacts is minimized, thus enabling the cobalt method to be used with greater confidence for describing neuronal projections.