Neuroblast migration is a crucial step in postnatal neurogenesis. The protocol described here can be used to investigate the role of candidate regulators of neuroblast migration by employing DNA/small hairpin RNA (shRNA) nucleofection and a 3D migration assay with neuroblasts isolated from the rodent postnatal rostral migratory stream.
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.
In the postnatal mammalian brain, generation of new neurons (neurogenesis) occurs throughout life and is restricted to two neurogenic niches: the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone of the dentate gyrus of the hippocampus1. Several recent studies have shown the important role of adult neurogenesis in facilitating learning and memory tasks2,3. Moreover, evidence of proliferation and recruitment of neural progenitors following brain injury4-7 raises the possibility of pharmacological activation of neurogenesis in neural repair.
Postnatal neurogenesis is strictly regulated in all its phases, which include neural progenitor proliferation, migration, differentiation, survival, and final synaptic integration of newly born neurons8. Neural progenitors (neuroblasts) derived from stem cells in the SVZ migrate over a great distance through the rostral migratory stream (RMS) towards the olfactory bulb (OB) where they mature into functional neurons9. Migratory neuroblasts are predominantly unipolar, with an elongated cell body extending a single leading process. These cells move in chains in a collective manner, sliding over one another10. Migration is a crucial step for the subsequent maturation of SVZ-derived progenitors into functional neurons11 and is controlled by multiple factors and guidance molecules including: polysialylated neural cell adhesion molecule (PSA-NCAM)12, Ephrins13, integrins14, Slits15, growth factors16 and neurotransmitters17, however the molecular mechanisms underlying this process are not fully understood. Investigating the intracellular signaling pathways regulating neuroblast migration will not only provide a better understanding of adult neurogenesis, but will also contribute to the development of the new therapeutic approaches to promote brain repair.
This manuscript describes a detailed protocol to study the role of candidate regulators of neuroblast migration in vitro using nucleofection and a 3D migration assay. Nucleofection is a cell transfection technique based on an improved method of electroporation. Cell-type specific electrical current and nucleofection solution allow the transfer of polyanionic macromolecules such as DNA and shRNA vectors and siRNA oligonucleotides directly into the cell nucleus and permit transfection of slowly dividing or mitotically inactive cells like embryonic and mammalian neurons18. This method is fast, relatively easy to perform and results in highly reproducible transfection of a broad range of cell types including primary neuroblasts and neurons19-21.
Dissociation of RMS tissue allows the isolation of migratory neuroblasts, which can be successfully nucleofected with DNA/shRNA vectors or siRNA oligos targeting genes of interest. Following nucleofection, neuroblasts are reaggregated in hanging drops and subsequently embedded in a three-dimensional Matrigel matrix. These conditions allow neuroblasts to migrate out of the cell aggregates recapitulating the migration mode observed in vivo, thus providing an excellent model system to investigate signaling pathways involved in neuroblast migration and to assess the influence of pharmacological treatments on the motility of these cells.
This procedure is in accordance with the UK Home Office Regulations (Animal Scientific Procedures Act, 1986). Scientists should follow the guidelines established and approved by their institutional and national animal regulatory organizations.
1. Dissection and Dissociation of Rat RMS Neuroblasts
Dissection medium (100 ml)
Hank's Balanced Salt Solution (HBSS) – 98.5 ml
5 M HEPES pH 7.4 – 0.5 ml
Penicillin-streptomycin (10,000 units/ml and 10,000 µg/ml) – 1 ml
Dissociation medium (2 ml)
HBSS – 1.760 ml
10x Trypsin (2.5%) – 200 µl
DNAse1 (1 mg/ml) – 40 µl
Dulbecco Modified Eagle's Medium (DMEM) + 10% Fetal Calf Serum (FCS) (40 ml)
DMEM – 36 ml
FCS – 4 ml
Complete medium (12 ml)
Neurobasal medium – 11.46 ml
B27 Supplement – 250 µl
L-Glutamine (200 mM) – 125 µl
Glucose (45%) – 165 µl
2. Filter-sterilize the DMEM + 10% FCS and the complete medium and preequilibrate them in a 37 °C /5% CO2 incubator.
2. Nucleofection
3. Embedding
4. 3D Migration Assay
Neuroblasts can be successfully isolated from dissected RMS tissue (Figure 1A) and embedded in a three-dimensional matrix. Cells isolated from either rat or mouse postnatal RMS are immunopositive for migratory neuroblast markers, such as doublecortin (DCX), βIII tubulin or PSA-NCAM (Figures 1B-C).
Dissociated neuroblasts can be efficiently nucleofected with DNA (e.g. a GFP-encoding plasmid, Figure 2) or shRNA plasmids (Figure 4) to achieve protein depletion, which can be assessed by western blot analysis (Figure 4B) or immunofluorescence (not shown).
Cells nucleofected with GFP-encoding plasmids migrate radially out reaggregated neuroblast clusters (Figure 3A). Quantification of relative migrated distance 24 hr post embedding (Figure 3B) shows no difference in migration between GFP-positive cells and GFP-negative, nonnucleofected cells (Figure 3C), indicating that nucleofection per se does not disrupt migration. There is also no significant difference in the extent of migration between nucleofected neuroblasts and neuroblasts directly migrating out of RMS explants (data not shown).
Figure 1. Dissection of RMS neuroblasts. (A) Schematic representation of RMS neuroblast dissection. For detailed description please refer to the text. (B) Isolated rat RMS cells are immunopositive for the migratory neuroblast makers DCX and βlll tubulin. Bar, 20 µm. (C) Cells migrating out of mouse RMS explants express the migratory neuroblast markers DCX and PSA-NCAM. Bar, 20 µm. Click here to view larger image.
Figure 2. Mouse neuroblast nucleofection. Dissociated mouse RMS neuroblasts were nucleofected with pMAX-GFP, reaggregated, embedded in a three-dimensional matrix and allowed to migrate for 6 hr. Neuroblasts migrating out of a reaggregated cell cluster (top, phase contrast pictures) show high transfection efficiency (bottom, GFP channel pictures). The right column panels show higher magnification pictures corresponding to the insets highlighted in the left column panels. Bars, 20 µm. Click here to view larger image.
Figure 3. 3D Migration assay. (A) Rat neuroblasts were nucleofected with pMAX-GFP (GFP) or pCAG-IRES-EGFP22 (EV), reaggregated, embedded in matrix and left to migrate for 24 hr. Cells were then fixed and immunostained for GFP (green) and βIII tubulin (red). Bar, 50 µm. (B) Measuring migration distance using ImageJ. The reaggregated cell cluster is divided into 6 equal sectors. The distance between the edge of the cluster (dotted line) and the furthest migrated cell is measured for each sector. (C) Quantification of the relative distance migrated by nucleofected cells (GFP-positive) and control, nonnucleofected cells (GFP-negative). Click here to view larger image.
Figure 4. Monitoring neuroblast migration after shRNA nucleofection. (A) Rat neuroblasts were nucleofected with a control shRNA vector (pCA-b-EGFPm5 Silencer 3, which also expresses EGFP23) or the same vector containing a shRNA targeting fascin, an actin-bundling protein24. Cells were reaggregated over 48 hr, embedded in matrix and left to migrate for 24 hr. Aggregates were then fixed and immunostained for GFP (green) and βIII tubulin (red). Bar, 50 µm. (B) Effective fascin depletion can be detected 50 hr after shRNA nucleofection by western blot analysis. Actin is shown here as a loading control. (C) Quantitative analysis of relative migration distance showing that fascin depletion significantly impairs neuroblast migration (mean ± SEM; **p<0.01; n=3 independent experiments). Click here to view larger image.
The migration of neuroblasts along the RMS to the final location in the OB is a fundamental step in postnatal neurogenesis. However, the molecular mechanisms controlling this complex process are far from being fully understood.
The experimental procedure described here allows the study of neuroblast migration in vitro. We have adapted a previously published protocol for isolating RMS neuroblasts from early postnatal mouse or rat25. To achieve optimal results it is important to master the dissection step, since it is crucial to keep the time interval between dissection and nucleofection to a minimum. After nucleofection, neuroblasts can be reaggregated, embedded in a three-dimensional matrix and left to migrate over a 24 hr period. Alternatively, for purposes other than migration (e.g. immunofluorescence or western blot analysis), cells can be immediately plated after nucleofection on polyornithine/laminin-coated coverslips, where they survive up to 4-5 days. Mouse and rat neuroblasts migrate in Matrigel to a similar extent, however mouse cells appear to have a stronger tendency to migrate in chains than rat cells.
Depending on the aim of the study, neuroblasts can be nucleofected with different plasmids encoding fluorescent proteins or wild type/mutant proteins of interest. For optimal protein expression plasmids with a CAG promoter (β-actin promoter with CMV enhancer and β-globin poly-A tail)26 are highly recommended. Moreover, siRNA oligos or shRNA plasmids can be nucleofected to knockdown targets of interest. Effective protein depletion can be visualized by immunofluorescence or by western blot (usually lysing embedded aggregates from 1 rat pup with 50 µl of standard lysis buffer).
Nucleofection is a relatively simple method to transfect primary neuroblasts, offers an easier and faster alternative to viral vector-mediated transfection, and can achieve high (~70-80%) transfection efficiency. It is critical to work quickly during the nucleofection procedure, since leaving neuroblasts in the nucleofection solution for a prolonged time drastically reduces cell viability.
The average cell yield from RMS dissection is relatively low for P7 mice (~5 x 105 cells/brain) in comparison to P7 rats (~1 x 106 cells/brain) and at least 3 x 106 cells per nucleofection are required to achieve transfection with ~50% efficiency. Moreover, rat neuroblasts appear to resist better to nucleofection compared to mouse neuroblasts. Therefore, early postnatal (P6-P7) rat pups might represent a convenient neuroblast source, also considering that the organization of rat and mouse RMS are remarkably similar27 and that the extent of rat and mouse neuroblast migration in vitro is also comparable. It is advisable not to keep the reaggregated clusters of nucleofected neuroblasts in suspension for longer than 48 hr to avoid abnormal effects on cell morphology and migration (our unpublished observations).
The 3D assay described here can be used to quantify neuroblast migration at a fixed time point after embedding in matrix (e.g. 24 hr). Aggregates of different sizes can be used in the analysis, since there is no significant correlation between the size of aggregates and migration distance (our unpublished observations). To visualize and further investigate the dynamics of neuroblast migration, time-lapse imaging can be used. It is recommended to carry out the migration analysis within a 24 hr interval after embedding, since the speed of neuroblasts appears to drastically decrease at longer time points (our unpublished observations).
There are some limitations to this protocol. 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 neuroblasts28. Second, the in vitro migration assay does not fully reproduce the complex architecture of the RMS observed in vivo. Indeed, although neuroblasts maintain the ability to migrate in a similar way to their in vivo counterparts, in the experimental setup described here they lack interactions with other RMS components such as astrocytes and blood vessels, which also contribute to regulate their motility9,29,30. This issue may be addressed in the future by optimization of three-dimensional coculture model systems.
In conclusion, combining nucleofection with a 3D migration assay represents a valuable tool to better understand the molecular mechanisms underlying neuroblast migration. 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 time-lapse imaging of brain slice cultures28,31,32.
The authors have nothing to disclose.
This work was funded by a Wellcome Trust Project Grant awarded to P.D. and G.L. (089236/Z/09/Z). S.G. was supported by a Biotechnology and Biological Sciences Research Council PhD studentship. We thank Matthieu Vermeren for the kind gift of the shRNA vector and Jennifer Shieh for valuable advice on neuroblast nucleofection.
Hank’s Balanced Salt Solution (HBSS) | Invitrogen Life Technologies | 14175129 | |
HEPES | Sigma-Aldrich | H3375-25G | |
Penicillin-Streptomycin | Invitrogen Life Technologies | 15140-122 | |
2.5% Trypsin-EDTA (10x) | Gibco | 15090-046 | store 200 µl aliquots at -20 °C |
DNAse I Vial (D2) | Worthington | LK003170 | ≥1,000 units per vial; store 50 µl aliquots at -20 °C |
Dulbecco Modified Eagle's Medium (DMEM) | Gibco | 11960-044 | |
Fetal Calf Serum (FCS) | Hyclone | SH3007902 | |
Neurobasal medium | Gibco | 21103-049 | |
B27 supplement | Invitrogen Life Technologies | 17504044 | |
L-Glutamine (200 mM) | Invitrogen Life Technologies | 25030-081 | |
D-(+)-Glucose solution (45%) | Sigma-Aldrich | G8769 | |
Matrigel Basement Membrane Matrix, Growth Factor Reduced (GFR), Phenol Red-free, 10 ml, LDEV-Free | BD Biosciences | 356231 | prepare 120 µl aliquots at 4 °C, then store at -80 °C |
PFA | Sigma-Aldrich | 441242 | |
Sucrose | BDH | 102745C | |
Goat serum | Sigma-Aldrich | 69023 | |
Triton X-100 | VWR International Ltd. | 306324N | |
BSA | Fisher Chemical | BPE9701-100 | |
Dako fluorescence mounting medium | Dako | S3023 | |
Rat neuron nucleofection kit | Lonza | VPG-1003 | |
Mouse neuron nucleofection kit | Lonza | VPG-1001 | |
Microsurgical knife | Angiotech | 7516 | |
McIlwain tissue chopper | The Mickle Laboratory Engineering Company | ||
Nucleofector II | Lonza |