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.

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
<ol>
	<li>Prepare the solutions required for RMS dissection and dissociation:</li>
</ol>
Dissection medium (100 ml) 
Hank&#39;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 &#181;g/ml) - 1 ml
Dissociation medium (2 ml) 
HBSS - 1.760 ml 
10x Trypsin (2.5%) - 200 &#181;l 
DNAse1 (1 mg/ml) - 40 &#181;l
Dulbecco Modified Eagle&#39;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 &#181;l 
L-Glutamine (200 mM) - 125 &#181;l 
Glucose (45%) - 165 &#181;l
2. Filter-sterilize the DMEM + 10% FCS and the complete medium and preequilibrate them in a 37 &#176;C /5% CO2 incubator.
<ol>
	<li>Dissection
	<ol>
		<li>Sacrifice a P6-P7 rat litter (about 12 pups) by cervical dislocation and decapitate with scissors.</li>
		<li>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.</li>
		<li>Gently remove the cranial flaps with forceps and carefully remove the brain with a spatula, taking care to include the olfactory bulbs.</li>
		<li>Cut the most caudal third of the brain and discard it.</li>
		<li>Chop the brain tissue into 1.4 mm&#160;thick coronal slices using a tissue chopper.</li>
		<li>Place slices in dishes containing cold dissection medium and carefully separate them using a needle.</li>
		<li>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.</li>
		<li>Collect the RMS fragments with a plastic Pasteur pipette and place them in a small dish containing cold dissection medium on ice.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Dissociation
	<ol>
		<li>Replace the dissection medium with 2 ml of dissociation medium.</li>
		<li>Triturate the RMS fragments by gently pipetting the fragment suspension up and down about 10x using a P1000 pipette.</li>
		<li>Leave the tube with the tissue fragments in a 37 &#176;C water bath for 2 min.</li>
		<li>Pipette the solution again 10x and ensure that fragments have dissociated (the suspension should become cloudy).</li>
		<li>Inactivate the trypsin by adding 5 ml of prewarmed DMEM + 10% FCS.</li>
		<li>Centrifuge the cell suspension at 433 x g for 5 min.</li>
		<li>In the meantime aliquot the required amount of siRNA/DNA into Eppendorf tubes (usually 3-5 &#181;g DNA/shRNA or 5-9 &#181;g siRNA oligo per nucleofection, however the amount of DNA/siRNA may require optimization).</li>
		<li>Remove excess medium and resuspend the cell pellet by gentle pipetting in 5 ml of prewarmed DMEM + 10% FCS.</li>
		<li>Perform a cell count. Expect ~1 x 106 cells per rat pup brain. A minimum of 2.5 x 106 cells are required for each nucleofection, while optimal results are achieved using 3-4 x 106 cells per nucleofection.</li>
		<li>Centrifuge the cell suspension at 433 x g for 5 min. Make sure to remove as much medium as possible.</li>
	</ol>
	</li>
</ol>
2. Nucleofection
<ol>
	<li>Immediately resuspend the cell pellet in rat (or mouse if using mouse cells) neuron nucleofection solution previously incubated at room temperature. Use 100 &#181;l per nucleofection. Note: typically a rat litter of 12 pups is sufficient to perform 4 nucleofections and a mouse litter (12 pups) is sufficient to perform 2 nucleofections.</li>
	<li>Transfer 100 &#181;l of the cell suspension to each Eppendorf tube containing siRNA/DNA and mix gently 2-3x by pipetting with a P200 pipette.</li>
	<li>Add the sample (cell-DNA/siRNA suspension) to the bottom of the nucleofectioncuvette, taking care to avoid bubbles.</li>
	<li>Nucleofect using program G-013 (for rat cells) or O-005 (for mouse cells). One nucleofection takes approximately 5 sec.</li>
	<li>Quickly add 1 ml of prewarmed DMEM + 10% FCS to the nucleofected sample.</li>
	<li>Repeat steps 2.4 and 2.5 for all other samples. Note: for optimal results, the entire nucleofection procedure should last no longer than 5 min.</li>
	<li>Transfer each sample to a 15 ml tube containing 5 ml of prewarmed DMEM + 10% FCS using the plastic pipette provided by the nucleofection kit. Avoid transferring any cellular debris to the tube.</li>
	<li>Centrifuge samples at 433 x g for 5 min.</li>
	<li>Carefully remove all excess medium and resuspend the pellet in 25-30 &#181;l of prewarmed DMEM + 10% FCS using a P20 pipette. Do not use more than 30 &#181;l of medium.</li>
	<li>Pipette the suspension as a drop onto the inner side of a p35 dish lid.</li>
	<li>Invert the lid over the p35 dish containing 2 ml of complete medium (see also Figure 1).</li>
	<li>Leave in the incubator (37 &#176;C/5% CO2) for at least 5 hr and up to 7 hr. Longer incubation time allows better reaggregation of cell clusters.</li>
	<li>Transfer the hanging drops from the lid into the complete medium in the dish using a P1000 pipette with a cut tip.</li>
	<li>Incubate at 37 &#176;C/5% CO2 for 24 hr for DNA nucleofections and 48 hr for siRNA/ shRNA nucleofections.</li>
</ol>
3. Embedding
<ol>
	<li>Prepare complete medium (25 ml) and preequilibrate it at 37 &#176;C/5% CO2 for a few hours.</li>
	<li>Take out the frozen aliquots of basement membrane matrix from -80 &#176;C freezer and thaw on ice in the cold room.</li>
	<li>For each nucleofection prepare a 6 cm dish containing up to eight 13 mm sterile coverlips.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Add complete medium to the thawed matrix in a 1:3 ratio. For example, mix 40 &#181;l of complete medium with 120 &#181;l of matrix by pipetting. This amount of matrix is sufficient for embedding aggregates on eight 12 mm coverslips.</li>
	<li>Transfer the reaggregated cell clusters to a 15 ml tube and centrifuge at 433 x g for 5 min.</li>
	<li>Remove excess medium and resuspend the pellet in 10 &#181;l of complete medium.</li>
	<li>Place 2 &#181;l of cell aggregate suspension onto each sterile coverslip and add 18 &#181;l of matrix/complete medium mixture. Use the pipette tip to spread the matrix over the entire coverslip.</li>
	<li>Immediately place the 6 cm dish containing the coverslips in the 15 cm dish and leave in the incubator (37&#176;C /5% CO2) 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.</li>
	<li>Incubate for 24 hr at 37 &#176;C /5% CO2 to let neuroblasts migrate out of the cell aggregates.</li>
</ol>
4. 3D Migration Assay
<ol>
	<li>Prepare the solutions required for immunostaining.
	<ol>
		<li>Prepare the block solution: 
		Goat block solution (50 ml) 
		Phosphate Buffered Saline (PBS) - 5 ml 
		Goat serum - 7.5 ml 
		10% Triton X-100 - 1.5 ml 
		BSA - 50 mg 
		H2O - 36 ml</li>
		<li>Filter and store at 4 &#176;C.</li>
		<li>Prepare the fixing solution: 
		Fixing solution (100 ml) 
		Paraformaldehyde (PFA) - 4 g CAUTION: always handle PFA under a hood 
		Sucrose - 20 g (optional) 
		PBS up to 100 ml</li>
		<li>On a hot plate and under constant stirring dissolve the PFA in 80 ml PBS maintained at 65 &#176;C.</li>
		<li>Once the PFA has dissolved, add 20 g of sucrose.</li>
		<li>Adjust pH to 7.4 (usually by adding ~60 &#181;l 1 M NaOH per 100 ml of solution).</li>
		<li>Bring up to a total volume of 100 ml with PBS.</li>
	</ol>
	</li>
	<li>Immunostaining
	<ol>
		<li>Place coverslips in a 24-well plate.</li>
		<li>Rinse coverslips with PBS 2x.</li>
		<li>Fix the RMS neuroblast aggregates with fixing solution for 45 min at room temperature.</li>
		<li>Rinse coverslips with PBS 3x (5 min/wash - on a rocking platform).</li>
		<li>Block for 30-60 min&#160;with goat block solution.</li>
		<li>Dilute primary antibodies in goat block solution and incubate overnight at 4 &#176;C. (If desired, fluorescent phalloidin (1:400) and Hoechst dye (1:10,000) can also be added to the primary antibody solution to visualize filamentous actin and nuclei).</li>
		<li>Rinse coverslips with PBS 3x&#160;(5 min/wash).</li>
		<li>Dilute secondary antibodies in goat block solution and incubate for 2 hr at room temperature.</li>
		<li>Rinse coverslips with PBS 3x&#160;(5 min/wash)</li>
		<li>Mount coverslips with fluorescent mounting medium and leave to dry overnight at room temperature.</li>
	</ol>
	</li>
	<li>Migration Analysis
	<ol>
		<li>Capture images of fixed RMS neuroblast aggregates with a fluorescent microscope using a 10X&#160;objective. Include a scale bar in a sample image.</li>
		<li>To set up the scale for quantification, measure the scale bar in the image by selecting the &#39;Straight line&#39; tool on the ImageJ tool bar.</li>
		<li>Choose the &#39;Analyze&#39; option and click on &#39;set the scale&#39;.</li>
		<li>In the scale window set the &#39;known distance&#39; and tick the &#39;global&#39; box to keep the same settings for all measurements.</li>
		<li>Use the &#39;Segmented line&#39; tool on the ImageJ tool bar to measure the distance from the edge of the aggregate to the furthest migrated neuroblast in 6 different sectors around the entire aggregate (Figure 3B). Consider only isolated aggregates for analysis.</li>
		<li>Calculate an average migration distance from the 6 values obtained for each aggregate.</li>
		<li>Measure 10-20 aggregates for each condition in every independent experiment and pool results from a minimum of three independent experiments. Always include a nucleofection control (e.g.&#160;GFP or a control sh/siRNA).</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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 (&#946;-actin promoter with CMV enhancer and &#946;-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 &#181;l 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.

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.

<table><tbody><tr><td>Hank&amp;rsquo;s Balanced Salt Solution (HBSS)</td><td>Invitrogen Life Technologies</td><td>14175129</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375-25G</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Invitrogen Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>2.5% Trypsin-EDTA (10x)</td><td>Gibco</td><td>15090-046</td><td>store 200 &amp;micro;l aliquots at -20 &amp;deg;C</td></tr><tr><td>DNAse I Vial (D2)</td><td>Worthington</td><td>LK003170</td><td>&amp;ge;1,000 units per vial; store 50 &amp;micro;l aliquots at -20 &amp;deg;C</td></tr><tr><td>Dulbecco Modified Eagle&#039;s Medium (DMEM)</td><td>Gibco</td><td>11960-044</td><td></td></tr><tr><td>Fetal Calf Serum (FCS)</td><td>Hyclone</td><td>SH3007902</td><td></td></tr><tr><td>Neurobasal medium</td><td>Gibco</td><td>21103-049</td><td></td></tr><tr><td>B27 supplement</td><td>Invitrogen Life Technologies</td><td>17504044</td><td></td></tr><tr><td>L-Glutamine (200 mM)</td><td>Invitrogen Life Technologies</td><td>25030-081</td><td></td></tr><tr><td>D-(+)-Glucose solution (45%)</td><td>Sigma-Aldrich</td><td>G8769</td><td></td></tr><tr><td>Matrigel Basement Membrane Matrix, Growth Factor Reduced (GFR), Phenol Red-free, 10 ml, LDEV-Free</td><td>BD Biosciences</td><td>356231</td><td>prepare 120 &amp;micro;l aliquots at 4 &amp;deg;C, then store at -80 &amp;deg;C</td></tr><tr><td>PFA</td><td>Sigma-Aldrich</td><td>441242</td><td></td></tr><tr><td>Sucrose</td><td>BDH</td><td>102745C</td><td></td></tr><tr><td>Goat serum</td><td>Sigma-Aldrich</td><td>69023</td><td></td></tr><tr><td>Triton X-100</td><td>VWR International Ltd.</td><td>306324N</td><td></td></tr><tr><td>BSA</td><td>Fisher Chemical</td><td>BPE9701-100</td><td></td></tr><tr><td>Dako fluorescence mounting medium</td><td>Dako</td><td>S3023</td><td></td></tr><tr><td>Rat neuron nucleofection kit</td><td>Lonza</td><td>VPG-1003</td><td></td></tr><tr><td>Mouse neuron nucleofection kit</td><td>Lonza</td><td>VPG-1001</td><td></td></tr><tr><td>Microsurgical knife</td><td>Angiotech</td><td>7516</td><td></td></tr><tr><td>McIlwain tissue chopper</td><td>The Mickle Laboratory Engineering Company</td><td></td><td></td></tr><tr><td>Nucleofector II</td><td>Lonza</td><td></td><td></td></tr></tbody></table>

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 &#39;hanging drops&#39; 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.

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), &#946;III tubulin or PSA-NCAM (Figures 1B-C).&#160;
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).
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/50989/50989fig1.jpg" width="500px" /> 
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 &#946;lll tubulin. Bar, 20 &#181;m. (C) Cells migrating out of mouse RMS explants express the migratory neuroblast markers DCX and PSA-NCAM. Bar, 20 &#181;m. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50989/50989fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="6in" src="/files/ftp_upload/50989/50989fig2.jpg" width="500px" /> 
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 &#181;m. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50989/50989fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="6in" src="/files/ftp_upload/50989/50989fig3.jpg" width="500px" /> 
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 &#946;III tubulin (red). Bar, 50 &#181;m. (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). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50989/50989fig3highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 4" fo:content-width="6in" src="/files/ftp_upload/50989/50989fig4.jpg" width="500px" /> 
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 &#946;III tubulin (red). Bar, 50 &#181;m. (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 &#177; SEM; **p&#60;0.01; n=3 independent experiments). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50989/50989fig4highres.jpg" target="_blank">Click here to view larger image</a>.

Watch this Scientific Journal Video about Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro at JoVE.com

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

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.

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 ...

nucleofection-rodent-neuroblasts-to-study-neuroblast-migration

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

11.4K Views.  King's College London. 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.

Video: Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

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

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 processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. 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 network1, 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 migration7-10. 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.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

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

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.

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

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Murine Neural Plate Targeting by In Utero Nano-Injection (NEPTUNE) at Embryonic Day 7.5

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously largely been restricted to the manipulation of embryonic stages post-neurulation. A protocol was developed to inject the amniotic cavity at embryonic day (E)7.5 and deliver lentivirus, encoding cDNA or shRNA, targeting &gt;95% of the neural plate and neural crest cells, contributing to the future brain, spinal cord, and peripheral nervous system. This protocol describes the steps necessary to achieve successful transduction, including grinding of the glass capillary needles, pregnancy verification, developmental staging using ultrasound imaging, and optimal injection volumes matched to embryonic stages. 
Following this protocol, it is possible to achieve transduction of &gt;95% of the developing brain with high-titer lentivirus and thus perform whole-brain genetic manipulation. In contrast, it is possible to achieve mosaic transduction using lower viral titers, allowing for genetic screening or lineage tracing. Injection at E7.5 also targets ectoderm and neural crest contributing to distinct compartments of the eye, tongue, and peripheral nervous system. This technique thus offers the possibility to manipulate gene expression in mouse neural-plate- and ectoderm-derived tissues from preneurulation stages, with the benefit of reducing the number of mice used in experiments.

Murine Neural Plate Targeting by In Utero Nano-Injection NEPTUNE at Embryonic Day 7.5

In this protocol, we describe how to inject the mouse amniotic cavity at E7.5 with lentivirus, leading to uniform transduction of the entire neural plate, with minimal detrimental effects on survival or embryonic development.

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously ...

Developmental Biology

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Visualizing Neuronal and Glial Cell Dynamics in the Developing Mouse Brain

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

Author Spotlight: Exploring Cell Migration and Gene Roles in the Developing Brain

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain ...

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

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Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices