Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

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.


Introduction
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 hippocampus 1 . Several recent studies have shown the important role of adult neurogenesis in facilitating learning and memory tasks 2,3 . Moreover, evidence of proliferation and recruitment of neural progenitors following brain injury [4][5][6][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 neurons 8 . 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 neurons 9 . 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 another 10 . Migration is a crucial step for the subsequent maturation of SVZ-derived progenitors into functional neurons 11 and is controlled by multiple factors and guidance molecules including: polysialylated neural cell adhesion molecule (PSA-NCAM) 12 , Ephrins 13 , integrins 14 , Slits 15 , growth factors 16 and neurotransmitters 17 , 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 neurons 18 . 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 neurons [19][20][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 2. Make an anteroposterior incision in the skin along the mid-sagittal suture from the nose to the cerebellum with a scalpel blade. Peel the skin off and repeat the same incision along the skull. 3. Gently remove the cranial flaps with forceps and carefully remove the brain with a spatula, taking care to include the olfactory bulbs. 4. Cut the most caudal third of the brain and discard it. 5. Chop the brain tissue into 1.4 mm thick coronal slices using a tissue chopper. 6. Place slices in dishes containing cold dissection medium and carefully separate them using a needle. 7. The RMS appears as a triangular, translucent area in the centre of OB sections and as a small, circular area in more caudal brain slices. Cut the RMS out of each slice with a microsurgical knife, taking care to avoid including surrounding tissue. In P7 rat pups, usually the ~8 most rostral slices (including OB) contain the RMS. 8. Collect the RMS fragments with a plastic Pasteur pipette and place them in a small dish containing cold dissection medium on ice. 9. When the dissection is complete, transfer the RMS fragments into a 15 ml tube with a plastic pipette. Leave fragments to settle at the bottom of the tube.
2. Dissociation 1. Replace the dissection medium with 2 ml of dissociation medium. 2. Triturate the RMS fragments by gently pipetting the fragment suspension up and down about 10x using a P1000 pipette. 3. Leave the tube with the tissue fragments in a 37 °C water bath for 2 min. 4. Pipette the solution again 10x and ensure that fragments have dissociated (the suspension should become cloudy). 5. Inactivate the trypsin by adding 5 ml of prewarmed DMEM + 10% FCS. 6. Centrifuge the cell suspension at 433 x g for 5 min. 7. In the meantime aliquot the required amount of siRNA/DNA into Eppendorf tubes (usually 3-5 µg DNA/shRNA or 5-9 µg siRNA oligo per nucleofection, however the amount of DNA/siRNA may require optimization). 8. Remove excess medium and resuspend the cell pellet by gentle pipetting in 5 ml of prewarmed DMEM + 10% FCS. 9. Perform a cell count. Expect ~1 x 10 6 cells per rat pup brain. A minimum of 2.5 x 10 6 cells are required for each nucleofection, while optimal results are achieved using 3-4 x 10 6 cells per nucleofection.

Embedding
1. Prepare complete medium (25 ml) and preequilibrate it at 37 °C/5% CO 2 for a few hours. 2. Take out the frozen aliquots of basement membrane matrix from -80 °C freezer and thaw on ice in the cold room.
3. For each nucleofection prepare a 6 cm dish containing up to eight 13 mm sterile coverlips. 4. Place the dishes on an ice box covered with cling film. It is important to keep the coverslips cool to prevent matrix solidification during the embedding procedure. 5. To maintain humidity, place a strip of damp tissue inside a 15 cm dish that will be used to hold up to three 6 cm dishes containing the embedded neuroblasts. 6. Add complete medium to the thawed matrix in a 1:3 ratio. For example, mix 40 µl of complete medium with 120 µl of matrix by pipetting. This amount of matrix is sufficient for embedding aggregates on eight 12 mm coverslips. 7. Transfer the reaggregated cell clusters to a 15 ml tube and centrifuge at 433 x g for 5 min. 8. Remove excess medium and resuspend the pellet in 10 µl of complete medium. 9. Place 2 µl of cell aggregate suspension onto each sterile coverslip and add 18 µl of matrix/complete medium mixture. Use the pipette tip to spread the matrix over the entire coverslip. 10. Immediately place the 6 cm dish containing the coverslips in the 15 cm dish and leave in the incubator (37°C /5% CO 2 ) for 15-20 min. When the matrix has solidified, gently add 5 ml complete medium to each 6 cm dish taking care to push down any floating coverslip with a pipette tip. 11. Incubate for 24 hr at 37 °C /5% CO 2 to let neuroblasts migrate out of the cell aggregates. 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.

3D Migration Assay
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 10 5 cells/brain) in comparison to P7 rats (~1 x 10 6 cells/brain) and at least 3 x 10 6 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 similar 27 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 neuroblasts 28 . 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 motility 9,29,30 . 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 timelapse imaging of brain slice cultures 28,31,32 .

Disclosures
The authors have nothing to disclose.