If nestin cells do generate new neurons and DA neurons

If nestin+cells do generate new neurons and DA neurons within the microenvironment of the adult midbrain, knowledge about their ontogenesis will be crucial to identify signaling mechanisms regulating neurogenesis and DA neurogenesis here, which might help progress cell-replacement therapies for PD. Hence, the aims of this study were to: (1) assess whether cells derived from nestin+cells in the adult midbrain have a neuronal phenotype as defined by electrophysiology and gene expression; and (2) if so, assess whether they achieved this via classical neurogenesis.
Materials and methods
Results
Discussion The question of whether or not neurogenesis occurs in the adult SNc has been addressed many times, motivated by the prospect that replacing degenerated SNc DA neurons in PD will provide much longer motor symptom relief and fewer side effects than current treatments. Although the weight of evidence indicates there is little or no neurogenesis in adult SNc there remains cause for optimism because many cells in the adult rodent SNc incorporate the cell division marker BrdU (Aponso et al., 2008; Chen et al., 2005; Lie et al., 2002; Zhao et al., 2003; Frielingsdorf et al., 2004; Peng et al., 2008) but see Hermann et al., 2009) and some SNc cells appear capable of generating neurons in the presence of appropriate environmental cues such as FGF2 or FGF8 and retinoic ribosomal s6 kinase in vitro or when transplanted into an adult neurogenic niché (the hippocampus (Lie et al., 2002)). Lie et al. (Lie et al., 2002) speculated that these are multi-potent nestin+NPCs. Indeed even within the adult rodent SNc there is evidence of very low levels of neurogenesis from nestin+NPCs (Shan et al., 2006). Given the importance for progressing better therapies for PD we sought to confirm whether or not nestin+cells in the adult SNc and midbrain generate new neurons and DA neurons in situ using NesCreER/R26eYFP transgenic mice and a combination of electrophysiology combined with single-cell gene expression. We found most cells labelled with eYFP by prior (1–32weeks) administration of a ‘pulse’ of tamoxifen to adult (≥8-weeks old) NesCreER/R26eYFP mice (i.e. adult nestin+cells and their progeny and ontogeny) do indeed exhibit a mature neuronal phenotype with large amplitude fast action potentials (APs), spontaneous post-synaptic currents (sPSCs) and expression of ‘mature’ neuronal genes (NeuN, Gad1, Gad2 and/or VGLUT2). These findings imply a strong association between nestin expression and neurons in the adult midbrain. However the more important question is whether this association is through classical neurogenesis. During rodent brain development ventral mesencephalic DA neurons derive from nestin+stem cells or NPCs in the ventricular (germinal) zone of the Aq (Ling et al., 1998; Schiff et al., 2009) from where they undergo radial migration ventrolaterally to populate the VTA and SNc, differentiating into DA neurons along the way (Prakash and Wurst, 2006). In the present study the morphology and spatial distribution of eYFP+ cells suggested a similar ontogenesis might be occurring in the adult midbrain. eYFP+ cells lining the Aq were oval- or columnar-shaped and had several cilia protruding into and beating within the ventricular space. In these respects they are classical ependymal glial cells, which could be stem cells or latent stem cells (Alvarez-Buylla et al., 2001). We did not investigate glia-related genes in the present study so are unable to confirm at the transcriptional level the glial phenotype of cells lining the Aq. Note that this also introduces a bias away from glial cells more broadly in the present study. Immediately below the Aq in the ventral midline eYFP+ cells had a fusiform or bipolar shape often with a long process extending from one or both poles, which is reminiscent of migrating neuroblasts or type-3 cells. Also the predominant alignment of the long axis of these cells ventrolaterally suggested radial migration between Aq and VTA/SNc. Lastly, eYFP+ cells in the VTA and SNc had various sizes and shapes including large multipolar cells reminiscent of mature neurons. Shan et al. (Shan et al., 2006) reported decreasing proliferation of nestin+cells along this same anatomical trajectory (Aq to ventral midline to VTA/SNc) in mice, which is also consistent with birth in Aq followed by migration through ventral midline and maturation/integration in VTA and SNc.