Functional neurogenesis in the adult midbrain?

Neurogenesis in the adult brain occurs within the two principle neurogenic regions, the hippocampus and the SVZ of the lateral ventricles. The occurrence of adult neurogenesis in non-neurogenic regions including the midbrain remains controversial, but isolation of neural stem cells (NSCs) from several parts of the adult brain including the substantia nigra has been reported. Nevertheless it is unclear whether adult NSCs do have the capacity to produce functional dopaminergic neurons, the cell type lost in Parkinson's disease. We describe here the existence and characterization of NSC in the adult tegmentum (midbrain and hindbrain) in vivo. We developed a model of neurogenesis in the tegmetum according to Doetsch and colleagues for the SVZ of the lateral ventricle, showing the existence of type B and A cells with nearly no C cells at the fourth ventricle. Furthermore, we show here the isolation, expansion and in vitro characterization of adult mouse tegmental neural stem cells (tNSCs) and their differentiation into functional nerve cells including dopaminergic neurons. These tNSCs showed neurosphere formation, expressed high levels of early neuroectodermal markers, such as the proneural genes NeuroD1, Neurog2 and Olig2, the NSC markers Nestin and Musashi1 and the proliferation markers Ki67 and BrdU. The cells showed typical PI-FACS analysis of slowly dividing cells. In the presence of selected growth factors, tNSCs differentiated into astroglia, oligodendroglia and neurons expressing markers for cholinergic, GABAergic and glutamatergic cells. Electrophysiological analyses revealed functional properties of mature nerve cells, such as TTX-sensitive sodium channels, action potentials as well as GABA-, glutamate and NMDA-induced currents. Clonal analysis demonstrated that individual NSCs retain the capacity to generate both glia and neurons. After a multiple-step differentiation protocol using co-culture conditions with PA6 stromal cells, a small number of cells acquired morphological and functional properties of dopaminergic neurons in culture. Here we demonstrate the existence of adult tNSCs with functional neurogenic and dopaminergic potential, a prerequisite for future endogenous cell replacement strategies in Parkinson's disease.