Electrophysiological and gene expression characterization of the ontogeny of nestin-expressing cells in the adult mouse midbrain

Abstract

The birth of new neurons, or neurogenesis, in the adult midbrain is important for progressing dopamine cell-replacement therapies for Parkinson's disease. Most studies suggest newborn cells remain undifferentiated or differentiate into glia within the adult midbrain. However, some studies suggest nestin+neural precursor cells (NPCs) have a propensity to generate new neurons here. We sought to confirm this by administering tamoxifen to adult NesCreER^T2/R26eYFP transgenic mice, which permanently labelled adult nestin-expressing cells and their progeny with enhanced yellow fluorescent protein (eYFP). eYFP+ midbrain cells were then characterized 1-32weeks later in acutely prepared brain slices using whole-cell patch clamp electrophysiology combined with single-cell RT-qPCR. Most eYFP+ cells exhibited 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). This was the case even at the earliest time-point following tamoxifen (i.e. 1week). In comparison to neighboring eYFP- (control) cells, eYFP+ cells discharged more APs per unit current injection, and had faster AP time-to-peak, hyperpolarized resting membrane potential, smaller membrane capacitance and shorter duration sPSCs. eYFP+ cells were also differentiated from eYFP- cells by increased expression of 'immature' pro-neuronal genes (Pax6, Ngn2 and/or Msx1). However, further analyses failed to reveal evidence of a place of birth, neuronal differentiation, maturation and integration indicative of classical neurogenesis. Thus our findings do not support the notion that nestin+NPCs in the adult SNc and midbrain generate new neurons via classical neurogenesis. Rather, they raise the possibility that mature neurons express nestin under unknown circumstances, and that this is associated with altered physiology and gene expression.

Keywords: Dopamine; Parkinson's disease; Single-cell RT-qPCR; Substantia nigra; Ventral tegmental area.