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

Abstract

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted area of the brain, neural stem cells proliferate and constantly generate neuroblasts that migrate tangentially in chains along the rostral migratory stream (RMS) to reach the olfactory bulb (OB). Once in the OB, neuroblasts switch to radial migration and then differentiate into mature neurons able to incorporate into the preexisting neuronal network. Proper neuroblast migration is a fundamental step in neurogenesis, ensuring the correct functional maturation of newborn neurons. Given the ability of SVZ-derived neuroblasts to target injured areas in the brain, investigating the intracellular mechanisms underlying their motility will not only enhance the understanding of neurogenesis but may also promote the development of neuroregenerative strategies.This manuscript describes a detailed protocol for the transfection of primary rodent RMS postnatal neuroblasts and the analysis of their motility using a 3D in vitro migration assay recapitulating their mode of migration observed in vivo. Both rat and mouse neuroblasts can be quickly and efficiently transfected via nucleofection with either plasmid DNA, small hairpin (sh)RNA or short interfering (si)RNA oligos targeting genes of interest. To analyze migration, nucleofected cells are reaggregated in 'hanging drops' and subsequently embedded in a three-dimensional matrix. Nucleofection per se does not significantly impair the migration of neuroblasts. Pharmacological treatment of nucleofected and reaggregated neuroblasts can also be performed to study the role of signaling pathways involved in neuroblast migration.

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

“…See also supplementary material Figs S1 and S2. cultured them in suspension for 48 hours, embedded them in Matrigel and let them migrate for 24 hours before fixation (Falenta et al, 2013). RalA knockdown caused a ,60% reduction in migration (Fig.…”

“…For migration assays, cells were re-suspended in 30 ml of DMEM + 10% FCS, pipetted as a drop inside a p35 dish lid, and inverted over a dish containing Neurobasal complete medium. Hanging drops were transferred into the medium 5 hours later and cultured in suspension for 48 hours before embedding in growth-factor-reduced Phenol-Red-free Matrigel (BD) on glass coverslips (Falenta et al, 2013). Neuroblasts were allowed to migrate for 24 hours in Neurobasal complete medium at 37˚C under 5% CO 2 before fixation.…”

Drebrin Regulates Neuroblast Migration in the Postnatal Mammalian Brain

“…After 5 hours at 37°C/5% CO 2 , the cell reaggregate was transferred into the medium, and cultured in suspension at 37°C/5% CO 2 for either 24 or 48 hours. Cell reaggregates were then embedded in growth factor-reduced, phenol red-free Matrigel (Becton Dickinson) diluted 3:1 in Neurobasal complete medium as previously described [ 21 ]. Explants were left to migrate for up to 24 hours at 37°C/5% CO 2 .…”

“…Pictures of explants were taken on a Zeiss Axioplan 2 microscope equipped with an ApoTome module and a Zeiss MRm Axiocam CCD camera using A-Plan 10x/0.25 and Plan Apo 20x/0.75 objectives and Axiovision software. The migration distance was measured from the edge of the explants to the nucleus of the furthest migrated cell (identified by Hoechst staining) for at least 6 different positions around the explants using ImageJ as described [ 21 ]. Data were collected from 3 independent experiments, analysing at least 15 explants per condition in each experiment.…”

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

“…To quantify migration out of the RMS explants, pictures were taken on an Apotome microscope (Zeiss). Using ImageJ, we measured the distance between the edge of the explant and the furthest cell (identified by Hoechst staining) for at least 10 different positions around the explant to obtain the average migration distance out of each explant ( Falenta et al, 2013 ). The presented data are obtained from at least 3 independent experiments, with at least 10 explants examined per condition in each experiment.…”