Tracing neuronal connections with radioisotopes applied extracellularly

Methodological and technical problems of the autoradiographic-neuroanatomical tracing (ARNT) technique are discussed. The size of the labeled area after a tritiated amino acid injection varies directly with the volume of isotope, the rate of injection, and the length of exposure of the secretion to the emulsion. Frozen sections can be used for autoradiography if they are mounted on subbed slides, dehydrated in ethanol, defatted for 1 hour in xylene, rehydrated through ethanol and water, and dried before coating with emulsion. Brushes used for mounting frozen sections should be used only for this purpose and dipped in boiling distilled water before use to avoid chemoreduced streaks in the emulsion due to contamination from the brushes. Excessive dilution of Kodak emulsion can leave less than a monolayer of grains over certain types of sections; the emulsion thickness should be checked by exposing coated test sections to a brief flash of light and developing immediately. A high intensity safelight recommended for the ARNT darkroom is the Thomas Duplex Super monochromatic sodium vapor bulb safelight; for Kodak NTB-2, -3 and Ilford L4 emulsions the red-banded and yellow-banded filters are used. A useful stain combination for ARNT is Luxol fast blue stained before coating and cresyl violet stained after developing which demonstrates both neuronal cell bodies and myelinated tracts in the same section.