Background

In the postnatal brain, neural stem cells reside in the ventricular-subventricular zone (V-SVZ) lining the lateral walls of lateral ventricles, and continuously generate immature new neurons (neuroblasts) (Obernier and Alvarez-Buylla, 2019). These neuroblasts form chain-like cell aggregates and migrate along each other through the rostral migratory stream (RMS) toward the olfactory bulb (OB) (Luskin, 1993; Lois and Alvarez-Buylla, 1994; Lois et al., 1996), where they are integrated into the neuronal circuits and involved in the processing of odor information (Gheusi et al., 2000; Breton-Provencher et al., 2009; Moreno et al., 2009; Sakamoto et al., 2011 and 2014). Following brain injuries such as stroke and trauma, V-SVZ-derived neuroblasts migrate toward the lesion and differentiate into mature neurons, contributing to the recovery of impaired gait behaviors (Arvidsson et al., 2002; Parent et al., 2002; Yamashita et al., 2006; Yang et al., 2007 and 2008; Kreuzberg et al., 2010; Jinnou et al., 2018; Kaneko et al., 2018). Altering the migration of neuroblasts affects their final positioning and functions in the postnatal brain under both physiological and pathological conditions (Belvindrah et al., 2011; Ota et al., 2014; Petri et al., 2017; Kaneko et al., 2018; Sawada et al., 2018), suggesting that regulation of neuroblast migration is critical for the plasticity of postnatal neuronal circuits.

V-SVZ-derived migrating neuroblasts show typical immature morphology, with a long leading process and a short trailing process (Schaar and McConnell, 2005). These neuroblasts show saltatory migration, which is executed by repeated extension of a leading process and subsequent somal translocation (Schaar and McConnell, 2005). Recently, accumulating evidence suggests that the primary cilium, a tiny cellular protrusion (Guemez-Gamboa et al., 2014; Malicki and Johnson, 2017), and its associated cytoskeletal network play pivotal roles in neuronal migration (Baudoin et al., 2012; Higginbotham et al., 2012; Trivedi et al., 2014; Guo et al., 2017). Therefore, time-lapse imaging of these components is useful for analyzing their subcellular localization and understanding the mechanisms of neuroblast migration (Trivedi et al., 2014 and 2017; Matsumoto et al., 2019).

Here, we describe a detailed protocol that we have recently used for in vitro time-lapse confocal/superresolution imaging of the primary cilium in migrating neuroblasts (Matsumoto et al., 2019). Images of whole-cell morphology and the primary cilium in cultured V-SVZ-derived neuroblasts could be recorded in a four-dimensional (x, y, z, and time) manner. This technique is widely applicable not only to migrating immature neurons but also to migrating nonneuronal cells in various species.