This work was supported by a Canadian Institutes of Health Research (CIHR) grant to A.S. J.K was partially supported by a Universit&eacute; Laval fellowship. A.S. is the recipient of a Canada Research Chair in postnatal neurogenesis.

1. Labeling Neuronal Precursors
Neuroblasts can be visualized using transgenic mice that selectively express fluorescent proteins in neuroblasts (i.e., Dcx-GFP, Gad67-GFP), by stereotaxically injecting viral particles encoding fluorescent proteins into the SVZ or RMS, or by grafting neuronal precursors from reporter mice (i.e., Dcx-GFP, Gad67-GFP) into the SVZ of wild-type mice. We describe the procedure for grafting and viral labeling of neuronal precursors.
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<ol>
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J., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+release+and+uptake+regulate+neuronal+precursor+migration+in+the+postnatal+subventricular+zone.">GABA release and uptake regulate neuronal precursor migration in the postnatal subventricular zone.</a> J. Neurosci. 24, 7623-7631 (2004).</li><li>Martini, F. J., Valdeolmillos, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actomyosin+contraction+at+the+cell+rear+drives+nuclear+translocation+in+migrating+cortical+interneurons.">Actomyosin contraction at the cell rear drives nuclear translocation in migrating cortical interneurons.</a> J. Neurosci. 30, 8660-8670 (2010).</li><li>Murase, S., Horwitz, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deleted+in+colorectal+carcinoma+and+differentially+expressed+integrins+mediate+the+directional+migration+of+neural+precursors+in+the+rostral+migratory+stream.">Deleted in colorectal carcinoma and differentially expressed integrins mediate the directional migration of neural precursors in the rostral migratory stream.</a> J. Neurosci. 22, 3568-3579 (2002).</li><li>Wilcock, A. C., Swedlow, J. R., Storey, K. 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C., Heintz, T., Young, S., Gordon, V., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tonic+activation+of+GLUK5+kainate+receptors+decreases+neuroblast+migration+in+whole-mounts+of+the+subventricular+zone.">Tonic activation of GLUK5 kainate receptors decreases neuroblast migration in whole-mounts of the subventricular zone.</a> J. Physiol. 586, 3783-3793 (2008).</li><li>Nam, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+features+of+postnatal+subventricular+zone+cell+motility:+a+two-photon+time-lapse+study.">Dynamic features of postnatal subventricular zone cell motility: a two-photon time-lapse study.</a> J. Comp. Neurol. 505, 190-208 (2007).</li><li>Kim, Y., Comte, I., Szabo, G., Hockberger, P., Szele, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+mouse+subventricular+zone+stem+and+progenitor+cells+are+sessile+and+epidermal+growth+factor+receptor+negatively+regulates+neuroblast+migration.">Adult mouse subventricular zone stem and progenitor cells are sessile and epidermal growth factor receptor negatively regulates neuroblast migration.</a> PLoS One. 4, e8122 (2009).</li><li>Bortone, D., Polleux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KCC2+expression+promotes+the+termination+of+cortical+interneuron+migration+in+a+voltage-sensitive+calcium-dependent+manner.">KCC2 expression promotes the termination of cortical interneuron migration in a voltage-sensitive calcium-dependent manner.</a> Neuron. 62, 53-71 (2009).</li><li>Comte, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galectin-3+maintains+cell+motility+from+the+subventricular+zone+to+the+olfactory+bulb.">Galectin-3 maintains cell motility from the subventricular zone to the olfactory bulb.</a> J. 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The correct targeting of neuronal precursors to the appropriate brain regions is a fundamental process underlying the proper assembly and function of neural circuitry. The vast majority of cells migrate during embryonic development and in the postnatal brain only in a few regions, such as the OB, dentate gyrus and cerebellum, neuronal displacement still takes place. The mechanisms orchestrating cell migration in the postnatal brain remain, however, poorly understood. Knowledge of the mechanisms and molecular pathways in...

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 <td>Name of the reagent</td>
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 <td>Sigma</td>
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 <td>Sigma</td>
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 <td>Sigma-Aldrich</td>
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 <td>Sigma</td>
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 <td>NaH2PO4xH2O</td>
 <td>EMD</td>
 <td>SX0710-1</td>
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 <td>Sigma-Aldrich</td>
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 <td>Neurobasal medium</td>
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 <td>EMD</td>
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 <td>Sutter Instrument </td>
 <td>P-97</td>
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 <td>Nanoliter injector</td>
 <td>WPI</td>
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 <td>Stereotaxic injection apparatus</td>
 <td>WPI</td>
 <td>502900</td>
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 <td>Micro drill system</td>
 <td>WPI</td>
 <td>501819</td>
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 <td>Vibratome</td>
 <td>Thermo Scientific</td>
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 <td>Wide-field fluorescent microscope</td>
 <td>Olympus</td>
 <td>BX61WIF</td>
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 <tr>
 <td>CCD camera</td>
 <td>Photometrics</td>
 <td>CS-HQ2-D</td>
 </tr>
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 <td>Ultra-quiet imaging chamber</td>
 <td>Harvard Apparatus </td>
 <td>64-1487</td>
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 <td>PH-1 Series 20 heater platform</td>
 <td>Harvard Apparatus </td>
 <td>64-0284</td>
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 <td>Heating system</td>
 <td>Warner Instruments </td>
 <td>TC-344B</td>
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 <td>40X water immersion objective</td>
 <td>Olympus</td>
 <td>1-UM587</td>
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 <tr>
 <td>10X water immersion objective</td>
 <td>Olympus</td>
 <td>1-UM583</td>
 </tr>
 <tr>
 <td>Lambda DG-4</td>
 <td>Sutter Instruments</td>
 <td>DG-4/OF</td>
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 <td>MetaMorph software</td>
 <td>Molecular Devices</td>
 <td>40000</td>
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 <td>Imaris software</td>
 <td>Bitplane</td>
 <td>BPI-IM70-F1</td>
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 </tbody>
</table>

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.

Watch this Scientific Journal Video about Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain at JoVE.com

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

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 ...

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14.8K Views.  Centre de Recherche Université Laval Robert-Giffard. 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.