A study of in vitro systems for the investigation of axonal transport

Acutely dissected adult myelinated axons can provide an in vitro model for the direct microscopical observation of axonal transport and for investigating related aspects of nerve-fibre physiology. A series of iso-osmotic immersion media were employed as acute culture media for isolated, non-permeabilized myelinated axons from the sciatic nerve of Xenopus laevis. Their general suitability as acute culture media, and their specific effects on the axon and its associated cells were investigated. Fibres mounted in conventional Ringer solution showed gross structural changes including axonal collapse, formation of large cavities at or near the adaxonal surface of the Schwann cell, dilation of the Schmidt-Lanterman incisures and cessation of particle transport. Media based on 120 mM potassium aspartate or 120 mM potassium glutamate improved the structural stability both of the axon and the Schwann cell. Incisural dilation was greatly reduced, periaxonal cavities did not form and axonal collapse did not occur. Axoplasmic function, as assessed by the persistence of particle transport, was also maintained. Media based on the amino acids glycine and taurine but with low K+ and Na+ levels caused gross structural changes distinct from those caused by Ringer solution and including dissociation of the myelin. The effects associated with Ringer solution must therefore be regarded as relatively specific and not a simple response to general trauma. Inclusion of 60mM KCl in the glycine and taurine based media reduced structural disruption and allowed particle transport to persist, although incisural dilation was recorded. The effects are discussed with reference to the structure of the myelinated fibre and the ionic environments within it. Direct microscopical observation of intra-axonal particle movement was carried out. Findings were analysed and additionally used to give an indication of the viability of the acutely isolated fibre preparations. Retrograde particle velocity was found to be variable and the selection of typical axons was necessary. In typical fibres immersed in potassium aspartate or potassium glutamate medium particle velocity ranged from 0 to 3.15 μm/sec with mean speeds of 1.05 μm and 1.2 μm. In vitro HRP uptake by acutely isolated nerve preparations from Xenopus laevis was investigated. Diffuse entry was demonstrated. Possible applications of the findings in the investigations using in vitro and in vivo nerve and nerve-ganglion preparations are discussed. (D82769)