A new methodological approach for studying axonal transport: cytofluorometric scanning of nerves.

Abstract

A new technique for studying axonal transport has been developed. The technique, which is based on histofluorescence techniques, enables the measurement of several different accumulated substances and parameters within a single nerve in relation to a nerve crush or local cooling. Any substance that can be made to fluoresce can be measured. The tissue is treated according to the formaldehyde-induced fluorescence method of Hillarp and Falck for visualization of monoamines, or according to the indirect immunofluorescence method. For immunofluorescence the nerve is cryostat-sectioned and various sections can be incubated with primary antisera against different antigens. After incubation and mounting the sections are placed in a cytofluorimeter (Leitz MPV II). They are passed under a measuring slit at a steady speed by a motor driven cross-table. The fluorescence intensity passing through the measuring slit is continuously registered by a recording unit with an integrator. This recorder produces a graphical nerve accumulation profile, and the area under the profile, relating to the fluorescence, is expressed in arbitrary units. This article presents data on the accumulation of noradrenaline, dopamine beta-hydroxylase, and tyrosine hydroxylase in crush-operated rat sciatic nerve. The time-course accumulations for noradrenaline (visualized by the Falck and Hillarp method) and dopamine beta-hydroxylase (visualized by immunofluorescence) demonstrated a striking similarity, which is to be expected since the two substances are stored in the same organelle. Tyrosine hydroxylase (visualized by immunofluorescence) showed a slower accumulation with time, but faster than would be expected had the enzyme been 100% soluble. Colchicine but not lumi-colchicine blocked the transport of noradrenaline organelles. With the new scanning technique we have the potential to study accumulation profiles of several different substances within a single nerve. Morphometric data, morphological observations, and photograph documentation of the same nerve section are also available.