Fetal Lateral Ganglionic Eminence Attracts One of Two Morphologically Different Types of Tyrosine Hydroxylase-Positive Nerve Fibers Formed by Cultured Ventral Mesencephalon

Abstract

The purpose of this study was to investigate the influence of fetal lateral ganglionic eminence (LGE) on nerve fiber outgrowth formed by fetal ventral mesencephalon (VM). Organotypic tissue cultures of fetal VM and LGE plated as single or cocultures were employed. Survival time was 3–21 days in vitro. Nerve fiber outgrowth and migration of astrocytes were analyzed using immunohistochemistry for tyrosine hydroxylase (TH) and S100. In addition, cultures were labeled with the TUNEL technique and with antibodies directed against neurofilament (NF) in order to study apoptosis and retraction of nerve fibers, respectively. The results revealed two morphologically different types of TH-positive outgrowth growing into the substrate. The initially formed TH-positive outgrowth radiated continuously without changing direction, while a second wave of TH-positive outgrowth became obvious when the initial growth already had reached a distance of approximately 1000 μm. The second wave of TH-positive outgrowth radiated from the tissue, but at a certain distance changed direction and formed a network surrounding the culture. The initially formed TH-positive growth was not associated with the presence of S100-positive astrocytes and avoided to grow into the LGE. At longer time points the first wave of TH-positive nerve fibers appeared dotted, with disrupted NF-immunoreactive fibers and in most cultures these long distance growing fibers had disappeared at 21 days in vitro. The second wave of TH-positive nerve fibers was growing onto a layer of glia and never reached the distance of the first wave. LGE became innervated by TH-positive fibers at the time point for when the second wave of TH-positive growth had been initiated, and the innervation appeared in TH-dense patches that also showed a high density of S100-positive astrocytes. Significantly increased TUNEL activity within LGE portion of cocultures was observed when TH-positive fibers entered the LGE and formed patches. In conclusion, two morphologically different types of TH-positive outgrowth were found and the initially formed fibers neither targeted the LGE nor were they guided by glial cells, but their potential to grow for long distances was high.