Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

Abstract

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) 1 . In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes 2,3 using blood vessels as a structural support and a source of molecular factors required for migration 4,5 . 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 1,6 . 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 [7][8][9][10] . 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. Video LinkThe video component of this article can be found at http://www.jove.com/video/4061/ Protocol Labeling Neuronal PrecursorsNeuroblasts can be visualized using transgenic mice that selectively express fluorescent proteins in neuroblasts (i.e., Dcx-GFP, Gad67-GFP), by stereotaxically injecting viral particles encoding fluorescent proteins into the SVZ or RMS, or by grafting neu...

<em>In vivo</em> Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

“…Spinning disk confocal microscopy is a cheaper alternative to two-photon microscopy. It allows 3D imaging at higher speed through multiple z planes, limiting photobleaching compared to standard confocal microscopy and offering a higher resolution than wide-field fluorescence imaging [31][32][33] . The majority of migrating neuroblasts have a clearly visible soma and a highly dynamic leading process tipped with a growth cone.…”

“…The main drawback consists in the fact that it is limited to early postnatal stages. Indeed, we strongly recommend using postnatal day 2 mouse pups, since we observed a substantial decrease in labeling efficiency at later times, when viral delivery methods become more suitable 31 . However, strategies like electroporation of CRE-expressing plasmids in appropriate mouse genetic models may be used to study neurogenesis in adult stages 22 .…”

“…Indeed, electroporation allows sparse labeling of neuroblasts, thus allowing detailed analysis of their morphology and migration dynamics. Compared to the stereotactic delivery of viral vectors 31 , this technique is cheaper, faster and is very well tolerated by mouse pups. The main drawback consists in the fact that it is limited to early postnatal stages.…”

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration <em>In vitro</em>

“…This experimental procedure provides an initial, fast and relatively simple method to evaluate the role of candidate regulators of neuroblast migration, which can be further validated by other approaches like in vivo postnatal electroporation and timelapse imaging of brain slice cultures 28,31,32 .…”

“…First, nucleofection can so far be used for early postnatal rodent neuroblasts, while infection with viral vectors remains the most efficient transfection method for adult neuroblasts 28 . Second, the in vitro migration assay does not fully reproduce the complex architecture of the RMS observed in vivo.…”