Background

Cortical development is a highly dynamic and precisely orchestrated process (Rakic and Caviness, 1995; Kolk and Rakic, 2022). During development, neural progenitor cells from the dorsal telencephalon proliferate to sequentially generate neurons in the deeper cortical layers, neurons in the upper cortical layers, and then glial cells (Gao et al., 2014). Nascent pyramidal neurons migrate radially to the external border of the developing cortex and generate new cortical layers in an inside-out fashion. How the migrating neurons stop their radial migration and consequently locate at their proper positions in the cerebral cortex is poorly understood (Rakic, 1974; Gongidi et al., 2004; Ohtaka-Maruyama and Okado, 2015). Ex vivo time-lapse imaging can directly reveal the cellular processes occurring during cortical development (Yang et al., 2012). Compared with radial migration and proliferation, which continuously occur during neural development and only require 2–3 h of imaging, terminating radial migration takes much longer. Monitoring termination requires prolonged (≥12 h) time-lapse imaging of neuronal migration at single-cell resolution in well-maintained developing brain slices. However, the stage of a standard microscope is not specifically designed to maintain a living organotypic slice, rendering it less feasible to monitor a highly active live brain slice over a long period of time. Previously published methods of time-lapse imaging can extend several hours (Tsai et al., 2007), which is not sufficient to observe the whole process of radial migration termination.

In our recent paper, Migrating pyramidal neurons require DSCAM to bypass the border of the developing cortical plate, we described the termination process of neuron radial migration and reported a molecular mechanism that underlies this process (Yang et al., 2022). We found that the nascent pyramidal neuron bypassing the last preceding neuron is important for expanding the cortex and thus determines the thickness of the upper cortical layers. Down syndrome cell adhesion molecule (DSCAM) was involved in this process by reducing the strength of N-cadherin-mediated cell-to-cell adhesion in the upper cortical plate, which allowed migrating neurons to traverse the cortical plate border and terminate at the outermost position of the developing cortical plate. Since DSCAM aberrations have been associated with autism spectrum disorder (Wang et al., 2016; Turner et al., 2016; Narita et al., 2020) and Down syndrome (Yamakawa et al., 1998; Agarwala et al., 2000), DSCAM may contribute to these brain disorders by impairing normal cortical development.

In our study, we applied live cell time-lapse imaging on organotypic brain-slice cultures to observe the termination of radial migration in detail (Yang et al., 2022). Organotypic brain slice cultures preserve brain development within an ex vivo environment. The confocal microscope is necessary for imaging fluorescently labeled neurons at single-cell resolution in thick brain slices. Since the brain slice is 300 µm in thickness, 50–100 z-frames of confocal scanning are needed to cover 100–150 µm of tissue in depth to collect z-axis information. Confocal microscopes equipped with high-speed scanners, such as the resonance scanner of Leica SP5 confocal systems, can overcome the speed limit of regular confocal microscopes and minimize laser damage. Together, this system generates single-cell resolution images for long-term (≥12 h) observations.

As we realized that our approach could be used as a standard method for studying cortical development and be extended to other brain regions or tissues, we decided to report our protocol in detail here, so that other researchers can reference, modify, and optimize their own imaging system. For the sake of completeness, we include brief protocols for neuronal labeling and brain slice sectioning in the “animal preparation” section, although these techniques have been previously described (Yang et al., 2022).