No abstract

Explants of adult or 10-day-old rat sciatic and optic nerves were implanted as "bridges" through a silicon grease seal in a three-compartment chamber culture system, leading from a narrow center chamber to two adjacent side chambers. Dissociated newborn rat sympathetic or sensory neurons were plated into the center chamber and grown in the presence of optimal concentrations of nerve growth factor (NGF). By light microscopy, nerve fibers were seen to grow out of the sciatic nerve explants in the side chambers after 2 to 3 weeks. Electron microscopy showed large numbers of axons present inside the sciatic nerves, irrespective of the presence and number of living Schwann cells. Besides their tendency to fasciculate, axons grew with high preference on Schwann cell membranes and the Schwann cell side of the basal lamina, a situation identical to in vivo regeneration. In contrast to the sciatic nerves, no axons could be found under any condition in the optic nerves. This result points to the existence of extremely poor, non-permissive substrate conditions in the differentiated optic nerves which cannot be overcome by the strong fiber outgrowth-promoting effects of NGF.

We have used video time-lapse microscopy to analyze in vitro the interactions of growth cones of newborn rat dorsal root ganglion cells with dissociated young rat CNS glial cells present in the cultures at low density. To provide optimal conditions for neurite extension, cells were grown on laminin and in NGF-supplemented medium. Our initial observation showed that there are 2 subpopulations of growth cones differing in their growth rate on laminin (averages of 12 and 45 microns/hr). When these growth cones encountered astrocytes, they maintained their normal configuration and growth velocity. They subsequently grew along or on top of astrocytes. In some cases, however, fast-moving growth cones showed a slight reduction in their growth rate. When growth cones countered oligodendrocytes, however, firm filopodial contact was sufficient to induce a rapid and long-lasting arrest of the growth cone motility, often followed by a collapse of the growth cone structure. One third of the paralyzed growth cones were observed to retract. Growth arrest and growth cone collapse were strictly dependent on membrane contact between neurons and oligodendrocytes. This contact inhibition phenomenon was exclusively found with differentiated oligodendrocytes and could be prevented by the monoclonal antibody IN-1 directed against neurite growth inhibitors NI-35 and NI-250 (Caroni and Schwab, 1988b). These results confirm previous findings that the neurite growth inhibitor proteins are important in axon outgrowth. Further, the inhibition of neurite growth exerted by oligodendrocytes is a contact-mediated phenomenon that can be triggered by the tip of growth cone filopodia.

Several novel myelin-associated/oligodendrocytic basic protein (MOBP) isoforms were identified in this study by cDNA cloning. They are small, highly basic polypeptides comprising 69, 81, and 99 amino acids (8.2, 9.7, and 11.7 kDa, respectively) and show no significant homology with described proteins or domain structures. All (as yet) identified MOBP isoforms are identical in amino acids 1-68 but differ in the length and polarity of the C-terminal region. One isoform, designated MOBP81, was shown to be expressed abundantly during development. Interestingly, MOBP81 has a significant clustering of positively charged residues at positions 69-81, a feature that also has been observed for myelin basic protein (MBP) and Po. As demonstrated by in situ hybridization, MOBP gene expression occurs during development of the rat optic nerve later than that of MBP and proteolipid protein and coincides exactly with the beginning of myelin compaction. The 2.6 kb MOBP81-A transcript is localized in the processes of oligodendrocytes, whereas the 3.8 kb MOBP81-B transcript is restricted to the perinuclear region. Therefore, MOBP81-A and related mRNAs seem to be transported to the periphery of the oligodendrocytes, as is known for the transcripts of the MBP gene. The late developmental expression of the MOBP gene suggests that the MOBP proteins act at the late steps of myelin formation, possibly in myelin compaction and in the maintenance of the myelin sheath.

A very sensitive and specific method for in situ hybridization has been developed. This method detects low copy numbers of mRNA(NGF) transcripts in both tissue sections and cultured cells using 35S‐labelled cRNA and oligonucleotide probes. In order to reduce the high nonspecific background occurring with 35S‐labelled probes, prehybridization in the presence of non‐labelled thio alpha UTP at pH 5.5 proved to be essential, together with a series of additional changes in the standard procedures for in situ hybridization. With this improved method it was possible to demonstrate that in tissues densely innervated by sensory (whisker pad) or both sympathetic and sensory (iris) fibers, NGF is synthesized not only by Schwann cells ensheathing these fibers, but also‐‐and even to a much larger extent‐‐by the target cells of the sensory and sympathetic neurons, i.e. epithelial cells, smooth muscle cells and fibroblasts. Moreover, in the sciatic nerve of newborn rats (where the mRNA(NGF) levels are 15 X higher than in adults) it was demonstrated that all Schwann cells have the capacity to express mRNA(NGF), not just those ensheathing the axons of NGF‐responsive neurons.