Alternatively and our data support this as

Alternatively, and our data support this as the most likely explanation, eYFP+ cells may arise from nestin expression by extant mature neurons. Nestin is an intermediate-sized cytoskeletal protein that is important for cell remodeling, particularly in developing and regenerating tissues. In the rodent nervous system it is expressed by the majority of mitotically active progenitors (Cattaneo and McKay, 1990; Lendahl et al., 1990) but it is down-regulated upon differentiation and replaced by other cell-specific intermediate filament proteins (e.g. neurofilament in neurons and glial fibrillary acidic protein or GFAP in astrocytes) (Steinert and Liem, 1990). However nestin expression persists in NPCs and vascular endothelial cells in accepted and emerging adult rodent neurogenic niches (Palmer et al., 2000; Migaud et al., 2010; Horner et al., 2000) and is re-expressed more widely throughout the CNS in reactive astrocytes, vascular endothelial cells and ependymal cells in response to inflammation, cellular stress or injury (Michalczyk and Ziman, 2005; Mokry and Nemecek, 1999; Frisén et al., 1995). Moreover we know of two reports of evidence that mature neurons express nestin. The first demonstrates nestin immunoreactivity in cells with mature neuronal morphology and immunoreactivity in the basal forebrain and closely related areas in adult rat and human monoamine transporter (Hendrickson et al., 2011). The second indicates a short isoform of nestin (NesS) exerts a cytoprotective function in mature sensory and motor neurons in adult rats (Su et al., 2013). Nestin gene expression is regulated through enhancer elements in the second intron, which are present in the NesCreER transgene employed here (Imayoshi et al., 2006). Interestingly one of these enhancer elements is midbrain specific and is thought to be regulated by nuclear hormone receptors such as Nurr1 (reviewed in (Michalczyk and Ziman, 2005)). A large proportion (49%) of our eYFP+ cells, which were verified to be mature neurons by electrophysiology and gene expression, co-expressed Nurr1 and 9% co-expressed Nes at the time they were investigated. Furthermore the fact that eYFP+ cells were immature in other respects indicates that putative nestin expression by mature midbrain neurons is functionally relevant and, if not in the classical neurogenesis sense, then perhaps in association with another form(s) of cellular plasticity such as inflammation or cellular stress mentioned above, or neurite remodeling. Prominent expression of DCX & NCAM2 by our otherwise mature eYFP+ neurons provides further evidence of plasticity here. DCX is a marker of NPC migration but its role may be more specifically in growth of neural processes (Friocourt et al., 2003). NCAM also regulates neurite outgrowth through cell adhesion. Although DCX protein can be detected (at low levels) in proliferating SNc cells following nigrostriatal degeneration (Worlitzer et al., 2013, but see Klaissle et al., 2012; Frielingsdorf et al., 2004; Van Kampen and Eckman, 2006) or FGF2 administration (Peng et al., 2008), these cells do not differentiate into neurons and so are unlikely to correspond to our eYFP+ cells. On the other hand polysialic acid (PSA, a binding partner for NCAM) and PSA-NCAM immunoreactivity have been reported in midbrain cells with mature neuronal morphology and TH immunoreactivity in rodents, monkeys and humans (Yoshimi et al., 2005; Nomura et al., 2000). Thus the DCX & NCAM2 gene expression we describe here might signify neurite growth and/or synaptic plasticity by mature midbrain neurons rather than neurogenesis. Neurite growth and/or synaptic plasticity in mature midbrain neurons is also indicated by persistent expression of the axon guidance ligand Netrin (e.g. see http://mouse.brain-map.org/experiment/siv?id=74511838&imageId=74409159&initImage=ish&coordSystem=pixel&x=5376.5&y=3752.5&z=2) and its receptor deleted in colorectal cancer or DCC (Osborne et al., 2005) in the ventral midbrain of adult rodents. See also (Frisén et al., 1995; Hendrickson et al., 2011; Su et al., 2013) for discussion on potential non-neurogenic functional roles of nestin.