Glial influence on nerve fiber formation from rat ventral mesencephalic organotypic tissue cultures

Rat fetal ventral mesencephalic organotypic cultures have demonstrated two morphologically different dopamine nerve fiber growth patterns, in which the initial nerve fibers are formed in the absence of astrocytes and the second wave is guided by astrocytes. In this study, the presence of subpopulations of dopamine neurons, other neuronal populations, and glial cells was determined. We used roller-drum organotypic cultures, and the results revealed that beta-tubulin-positive/tyrosine hydroxylase (TH)-negative nerve fibers were present as early as I day in vitro (DIV). A similar growth pattern produced by TH-positive neurons was present from 2 DIV. These neurites grew to reach distances over 4 mm and over time appeared to be degenerating. Thin, vimentin-positive processes were found among these nerve fibers. As the first growth was retracted, a second outgrowth was initiated and formed on migrating astrocytes. TH- and aldehyde dehydrogenase-1 (ALDH1)-positive nerve fibers formed both the nonglia-associated and the glia-associated outgrowth. In cultures with membrane inserts, only the glia-associated outgrowth was found. Vimentin-positive cells preceded migration of NG2-positive oligodendrocytes and Iba-1-positive microglia. Oligodendrocytes appeared not to be involved in guiding neuritic growth, but microglia was absent over areas dense with TH-positive neurons. In conclusion, in roller-drum cultures, nerve fibers are generally formed in two sequences. The early-formed nerve fibers grow in the presence of thin, vimentin-positive processes. The second nerve fiber outgrowth is formed on astroglia, with no correlation to the presence of oligodendrocytes or microglia. ALDH1-positive nerve fibers, presumably derived from A9 dopamine neurons, participate in formation of both sequences of outgrowth.