Khlghatyan Jivan, Saghatelyan Armen
The Cellular Neurobiology Unit, Centre de Recherche Université Laval Robert-Giffard.
J Vis Exp. 2012 Sep 12(67):e4061. doi: 10.3791/4061.
There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB). In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes using blood vessels as a structural support and a source of molecular factors required for migration. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network. In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.
有大量证据表明，成年哺乳动物大脑特定区域内源性神经干细胞持续产生新的功能性神经元。来自脑室下区（SVZ）的新生神经母细胞沿着吻侧迁移流（RMS）迁移至嗅球（OB）的最终目的地。在RMS中，神经母细胞以链式切向迁移，被星形胶质细胞突起包裹，利用血管作为结构支撑和迁移所需分子因子的来源。在OB中，神经母细胞从链上脱离，径向迁移到不同的球层，在那里分化为中间神经元并整合到现有网络中。在本手稿中，我们描述了监测啮齿动物脑急性切片中细胞迁移的方法。使用急性切片可在与体内条件极为相似的微环境以及体内成像难以进入的脑区评估细胞迁移。此外，它避免了器官型和细胞培养中可能最终改变细胞迁移特性的长时间培养条件。急性切片中的神经元前体可用微分干涉对比（DIC）光学显微镜或荧光蛋白进行可视化。对SVZ中的神经元前体进行病毒标记、将报告基因小鼠的神经母细胞移植到野生型小鼠的SVZ以及使用在神经母细胞中表达荧光蛋白的转基因小鼠，都是可视化神经母细胞并追踪其迁移的合适方法。然而，由于标记细胞密度高，后一种方法无法长时间追踪单个细胞。我们使用配备电荷耦合器件（CCD）相机的宽场荧光直立显微镜，以实现相对快速的采集间隔（每15或30秒采集一张图像），从而可靠地识别静止期和迁移期。准确识别静止期和迁移期的持续时间对于明确解释结果至关重要。我们还进行了多次z步采集以监测神经母细胞的三维迁移。宽场荧光成像已被广泛用于可视化神经元迁移。在此，我们描述了标记神经母细胞、对成年小鼠前脑急性切片中的神经母细胞迁移进行实时视频成像以及分析细胞迁移的详细方案。虽然所描述的方案以成年RMS中神经母细胞的迁移为例，但它也可用于追踪胚胎期和出生后早期大脑中的细胞迁移。
