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

Abstract

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair. Video LinkThe video component of this article can be found at

RalA promotes a direct exocyst-Par6 interaction to regulate polarity in neuronal development

“…The empty vector (EV), or pCX-EGFP (Oudin et al, 2011) served as controls. Electroporation in the lateral ventricle of P2 mouse pups allows transfection of a neuroblast subpopulation in the intact SVZ (Boutin et al, 2008;Sonego et al, 2013a). Brains were immunostained for GFP 5 days later to examine SVZ-derived neuroblasts in the RMS.…”

“…Animals were subjected to five electrical pulses of 100 V for 50 milliseconds with 850-millisecond intervals using a CUY21SC electroporator (Nepagene) (Sonego et al, 2013a).…”

Regional effects of endocannabinoid, BDNF and FGF receptor signalling on neuroblast motility and guidance along the rostral migratory stream

“…Electroporation of P2/P3 mouse pups was performed as previously described ( Oudin et al, 2011; Sonego et al, 2013b ). Briefly, mouse pups were anaesthetized with isofluorane (0.6 l/min).…”

“…Here we focussed our attention on eCB, FGF and BDNF signalling as there is compelling evidence for all three regulating neuroblast migration from RMS explant cultures with limited or no evidence describing their effect on neuroblast dynamics and guidance along the different areas of the intact stream ( Bagley and Belluscio, 2010; Chiaramello et al, 2007; Garcia-Gonzalez et al, 2010; Oudin et al, 2011; Snapyan et al, 2009 ). To clarify how these pathways control neuroblast migration in the post-natal mouse brain, we have used in vivo electroporation and live cell imaging to fluorescently label SVZ-derived neuroblasts and analyse their migration along the RMS ( Sonego et al, 2013b ). Our results show that eCB and BDNF signalling are required for motility and guidance throughout the RMS.…”

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 .…”