Microtubule number increases locally within regions of axonal compression

Abstract

Along the length of myelinated axons, stereotyped variations in the interaction with the enveloping Schwann cell produce different amounts of compressive force on the axon and its internal elements. Comparisons of histologically distinct axonal regions indicate that compression of the axon by the Schwann cell increases in the following order: internodal regions of compact myelin < Schmidt-Lanterman (S-L) clefts < nodes of Ranvier. Quantitative morphometry has shown that regions of greater Schwann cell compression are associated with regions of smaller axon diameter and greater neurofilament (NF) packing density. By locally decreasing axon diameter and increasing NF density, Schwann cell compression (especially at the node of Ranvier) may slow the transport of those axonal structures - such as microtubules (MTs) - that move by a polymer sliding mechanism. Under this hypothesis, a local slowing of MT transport should produce a local increase in MT number, and here we have studied whether such a local increase in MT number can be quantitatively documented.A twenty-eight day old chicken was perfused by cardiac puncture with 4 1 of fixative (4% paraformaldehyde, 2% glutaraldehyde, 0.005% CaCl2, in 0.1M cacodylate buffer, pH 7.4). The somatic motor branch of the oculomotor nerve was removed (noting the proximo-distal orientation) and fixed overnight at 4°C, followed by a buffer rinse in 0.1M cacodylate buffer at room temperature and postfixation in OSO4. The nerve was then dehydrated in a graded ethanol series and embedded in PolyBed/812. Starting at the proximal end of the nerve segment, l00nm sections were cut at consecutive 2-3 um intervals over a 225 um length of nerve, stained with an LKB Ultrostainer, and photographed on a JEOL 100CX microscope. We selected two axons with normal morphology, in which the cytoskeletal polymer profiles were consistently cross-sectioned and in which nodes were located approximately 120 um distal to the first section taken. All sections from these 2 axons were photographed, and MTs were counted on 58,000X micrographs.