Dey Anupama, Farzanehfar Parisa, Gazina Elena V, Aumann Tim D
Florey Institute of Neuroscience & Mental Health, The University of Melbourne, Parkville, Victoria 3010, Australia.
Stem Cell Res. 2017 Aug;23:143-153. doi: 10.1016/j.scr.2017.07.001. Epub 2017 Jul 4.
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/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.
成体中脑新神经元的诞生，即神经发生，对于推进帕金森病的多巴胺细胞替代疗法至关重要。大多数研究表明，新生细胞在成体中脑内保持未分化状态或分化为神经胶质细胞。然而，一些研究表明巢蛋白阳性神经前体细胞（NPCs）有在此处产生新神经元的倾向。我们试图通过给成年NesCreER/R26eYFP转基因小鼠注射他莫昔芬来证实这一点，该转基因小鼠用增强型黄色荧光蛋白（eYFP）永久标记成年表达巢蛋白的细胞及其后代。然后在1 - 32周后，使用全细胞膜片钳电生理学结合单细胞RT - qPCR，对急性制备的脑片中的eYFP阳性中脑细胞进行表征。大多数eYFP阳性细胞表现出成熟的神经元表型，具有大振幅快速动作电位（APs）、自发性突触后电流（sPSCs），以及“成熟”神经元基因（NeuN、Gad1、Gad2和/或VGLUT2）的表达。即使在他莫昔芬处理后的最早时间点（即1周）也是如此。与相邻的eYFP阴性（对照）细胞相比，eYFP阳性细胞在每单位电流注入时发放更多的动作电位，并且动作电位的峰值时间更快，静息膜电位超极化，膜电容更小，自发性突触后电流的持续时间更短。eYFP阳性细胞还通过“不成熟”的神经前体基因（Pax6、Ngn2和/或Msx1）表达增加与eYFP阴性细胞区分开来。然而，进一步分析未能揭示表明经典神经发生的出生位置、神经元分化、成熟和整合的证据。因此，我们的研究结果不支持成体黑质致密部和中脑中巢蛋白阳性NPCs通过经典神经发生产生新神经元的观点。相反，它们提出了一种可能性，即成熟神经元在未知情况下表达巢蛋白，并且这与生理和基因表达的改变有关。
