This work was supported by the Institute for Research and Innovation in Biomedicine (IRIB), the Cell Imaging Platform of Normandy (PRIMACEN), Inserm, IBiSA, the University of Rouen, the European Regional Development Fund (ERDF &#8211; PeReNE, Interreg 4A), the LARC-Neurosciences Network and the R&#233;gion Haute-Normandie.

Animals (male or female Wistar rats) were born and bred in an accredited animal facility (approval B.76-451-04), according to the French guide for the care and use of laboratory animals. Experiments were conducted under the supervision of authorized investigators (M.B, D.V. and L.G.) in accordance with the European Community Council Directive (2010/63/UE of September 22, 2010) and the French Ministry of Agriculture.
1. Preparation of Media and Tools
<ol>
	<li>In a biological safety cabinet, prepare 1x Hank&#8217;s BSS (HBSS) in sterile water from 10x stock solution containing CaCl2 (1.85 g/l) /MgSO4 (0.9767 g/l) without MgCl2. Add NaHCO3 (350 &#181;g/ml) to 1x solution of HBSS.</li>
	<li>In a biological safety cabinet, add N2 supplement (from a 100x stock solution) and penicillin (100 units/ml) -streptomycin (0.1 mg/ml) solution to Dulbecco&#8217;s modified eagle medium (DMEM) nutrient mixture F-12 (1 : 1).</li>
	<li>In sterile conditions, prepare an aliquot (25 &#181;l, 2 mM) of the cytoplasmic fluorescent dye called Cell Tracker Green in DMSO (1.075 ml for 1 mg). Dilute one aliquot in 5 ml of DMEM in a 15 ml conical tube.</li>
	<li>Prepare filled ice-bucket to keep media at 4 &#176;C.</li>
	<li>Decontaminate laboratory bench tops and tools with 70% ethanol.</li>
</ol>
2. Dissection of Cerebellum from P10 Rats
<ol>
	<li>Rapidly decapitate rat pups (P10) with curved operating scissors behind ears in order to get the beginning of the spinal cord.</li>
	<li>At the back-side of the decapitated head, make midline incision of the skin from the neck to the nose with fine iris scissors and separate the skin from the skull with fine iris scissors and Dumont #3 forceps.</li>
	<li>Use fine iris scissors to delicately make two lateral incisions from the base to the rostral region of the skull. Remove the dissected skull with two #3 forceps. Detach the brain from any adherence with the skull using the same forceps.</li>
	<li>Transfer the brain with the spoon end of a spatula to Petri dish (&#216; 35 mm) containing 2 ml of ice-cold HBSS medium.</li>
	<li>Put the Petri dish containing the brain in a larger Petri dish (&#216; 100 mm) full of ice and transfer to the stage of the stereomicroscope.</li>
	<li>Under a stereomicroscope, isolate the cerebellum from the brain by dilaceration using two #3 forceps. Similarly, remove the residual spinal cord and the pial-membrane.</li>
	<li>Transfer the cerebellum in a new HBSS-filled Petri dish (&#216; 35 mm, 2 ml) with the spoon end of a spatula and keep on ice.</li>
</ol>
3. Preparation of Acute Cerebellar Slices
<ol>
	<li>Under the stereomicroscope, cut the cerebellum between the vermis and the right hemisphere as indicated by the two heads arrow in Figure 2A with a standard scalpel handle #3 solid and a #15 surgical blade.</li>
	<li>Put one drop of cyanoacrylate glue on the vibratome specimen disc and wait 15-25 sec to eliminate toxic solvent vapors.</li>
	<li>Collect the cut cerebellum with the spoon end of a spatula and remove excess HBSS with clean paper towels.</li>
	<li>Bring the cerebellum close to the specimen disc. Fix the cut edge to the specimen disc and wait 10 sec.</li>
	<li>Insert the specimen disc into the buffer tray with the manipulator, and rotate it so the transverse axis of the cerebellum is perpendicular to the knife holder. Fix the specimen disc with an Allen key and fill gently the buffer tray with HBSS medium until the cerebellum is covered.</li>
	<li>Load crushed ice into the cooling bath.</li>
	<li>Clean the blade three times with 70% ethanol to eliminate any oil.</li>
	<li>Insert the blade into the knife holder and secure with clamping screw.</li>
	<li>Place the blade edge right behind the rear edge (from user&#39;s view) of the specimen and&#160;define it as a starting point. Use forward command to define the ending point after the front edge of the specimen.</li>
	<li>Select sectioning speed at 2.5 and sectioning frequency at 8. Select trimming&#160;thickness at 180 &#181;m. Start tissue sectioning.</li>
	<li>Pick up each section using a wide-bore glass truncated Pasteur pipette and transfer to HBSS-containing Petri dish (&#216; 35 mm) kept on ice.</li>
	<li>Using two #5 forceps, remove the meninges carefully from the cerebellum when interfering with the blade. Collect a maximum of 5 slices per cerebellum (Figure 2B, C).</li>
	<li>Remove the meninges from the cerebellar slices carefully with two #5 forceps under the stereomicroscope and separate the lobules gently for better probe loading.</li>
</ol>
4. Fluorescent Staining of Living Interneurons
<ol>
	<li>Transfer cerebellar slices with a wide-bore glass truncated Pasteur pipette to a 6-well plate (max 3 slices/per well). Aspirate the HBSS medium.</li>
	<li>Incubate slices (3 max) in 5 ml of loading solution of the fluorescent dye (10 &#181;M).</li>
	<li>To protect from light, cover the microplate with aluminum foil. Put it on a gyro-moving table at 35 rpm for 10 min at RT to facilitate cell labeling.</li>
	<li>Transfer slices on the membrane of a Transwell insert (3.0 &#181;m pore size; Figure 2D) with a wide-bore glass truncated Pasteur pipette. Aspirate the loading medium with pipette.</li>
	<li>Remove the insert and fill the well with 1.9 ml of DMEM. Replace the insert and add 100 &#181;l of DMEM on top of the slice to cover the tissue.</li>
	<li>Place the plate containing the culture inserts in the incubator chamber (37 &#176;C, 5% CO2) for 2 hrs which is sufficient to observe GCs in the ML. Lie tissues flat to allow attachment on the insert membrane (Figure 2E). Ensure that slices are not drying.</li>
</ol>
5. Ex vivo Imaging Through Confocal Macroscopy
<ol>
	<li>Transfer the plate without the plastic lid into an incubator attached to the stand of a confocal macroscope. Place a glass cover on the plate insert of the macroscope. Keep the temperature of the chamber at 37.0 &#176;C &#177; 0.5 &#176;C, and supply the slices with constant gas flow (95% O2, 5% CO2) through the plate insert to maintain the pH constant. Wait for 2 additional hr before time-lapse experiment.</li>
	<li>To visualize GC migration in the tissue slices, illuminate the preparation with a 488 nm wavelength light by means of a laser diode through a confocal laser scanning macroscope equipped with a X2 dry objective (working distance: 39 mm, diameter: 58 mm, NA = 0.234), and detect fluorescence emission from 500 to 530 nm.
	<ol>
		<li>To finely resolve the movement of GCs, acquire images with an additional optical zoom factor of 1.5 to 2.0. Collect images of GNs in a single focal plane or up to 10 different focal planes along the z-axis every 30 min for up to 12 hr.</li>
	</ol>
	</li>
	<li>When necessary, remove the glass cover and add small volumes (1-10 &#181;l) of biological activators or inhibitors in DMEM with a 10 &#181;l pipette to study their effect on GC migration.</li>
</ol>
6. Cell Tracking
<ol>
	<li>For each time of the movie, perform z-stack projection through the ecart-type mode in ImageJ. Modulate the contrast and the brightness levels of the successive images to facilitate the identification and the tracking of labeled GCs. Map manually each position on the reference snapshot (at t = 0).
	<ol>
		<li>Use the &#8220;Manual tracking&#8221; plugin in the Analyse Particle Menu and determine by clicking the gravity point of each cell body during time-lapse. Export the raw tracking data in a spreadsheet.</li>
	</ol>
	</li>
	<li>Reorganize the exported raw tracking data from ImageJ with a smart home-made program (http://primacen.fr, written in PHP code) that identify each cell and associated positions. Using the program, calculate the total traveled distance and the average speed of migration for each cell. Classify and compare characteristics of cell migration in control and treatment conditions under appropriate filters using the same program.</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Development of the cerebellar system in relation to its evolution, structure, and functions. , (1997).</li><li>Komuro, H., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+modes+of+neuronal+migration+in+different+domains+of+developing+cerebellar+cortex.">Distinct modes of neuronal migration in different domains of developing cerebellar cortex.</a> Journal of Neuroscience. 18 (4), 1478-1490 (1998).</li><li>Komuro, H., Yacubova, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+cerebellar+granule+cell+migration.">Recent advances in cerebellar granule cell migration.</a> Cellular and Molecular Life Sciences. 60 (6), 1084-1098 (2003).</li><li>Fahrion, J. 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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+cortical-layer-specific+control+of+neuronal+migration+by+pituitary+adenylate+cyclase-activating+polypeptide.">Cerebellar cortical-layer-specific control of neuronal migration by pituitary adenylate cyclase-activating polypeptide.</a> Neuroscience. 146 (2), 697-712 (2007).</li><li>Renaud, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plexin-A2+and+its+ligand,+Sema6A,+control+nucleus-centrosome+coupling+in+migrating+granule+cells.">Plexin-A2 and its ligand, Sema6A, control nucleus-centrosome coupling in migrating granule cells.</a> Nature Neuroscience. 4, 440-449 (2008).</li><li>Komuro, H., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+granule+cell+migration:+a+confocal+microscopic+study+in+acute+cerebellar+slice+preparations.">Dynamics of granule cell migration: a confocal microscopic study in acute cerebellar slice preparations.</a> Journal of Neuroscience. 15 (2), 1110-1120 (1995).</li><li>Nielsen, H. S., Hannibal, J., Fahrenkrug, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+pituitary+adenylate+cyclase+activating+polypeptide+(PACAP)+in+the+postnatal+and+adult+rat+cerebellar+cortex.">Expression of pituitary adenylate cyclase activating polypeptide (PACAP) in the postnatal and adult rat cerebellar cortex.</a> Neuroreport. 9 (11), 2639-2642 (1998).</li><li>Hannibal, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pituitary+adenylate+cyclase-activating+peptide+in+the+rat+central+nervous+system:+an+immunohistochemical+and+in+situ+hybridization+study.">Pituitary adenylate cyclase-activating peptide in the rat central nervous system: an immunohistochemical and in situ hybridization study.</a> Journal of Comparative Neurology. 453 (4), 389-417 (2002).</li><li>Garcia-Rocha, M., Avila, J., Armas-Portela, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-type+plasminogen+activator+(tPA)+is+the+main+plasminogen+activator+associated+with+isolated+rat+nerve+growth+cones.">Tissue-type plasminogen activator (tPA) is the main plasminogen activator associated with isolated rat nerve growth cones.</a> Neuroscience Letters. 180 (2), 123-126 (1994).</li><li>Ware, J. H., DiBenedetto, A. J., Pittman, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+tissue+plasminogen+activator+mRNA+in+the+developing+rat+cerebellum+and+effects+of+inhibiting+tissue+plasminogen+activator+on+granule+cell+migration.">Localization of tissue plasminogen activator mRNA in the developing rat cerebellum and effects of inhibiting tissue plasminogen activator on granule cell migration.</a> Journal of Neurobiology. 28 (1), 9-22 (1995).</li><li>Friedman, G. C., Seeds, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+plasminogen+activator+mRNA+expression+in+granule+neurons+coincides+with+their+migration+in+the+developing+cerebellum.">Tissue plasminogen activator mRNA expression in granule neurons coincides with their migration in the developing cerebellum.</a> Journal of Comparative Neurology. 360 (4), 658-670 (1995).</li><li>Seeds, N. W., Siconolfi, L. B., Haffke, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+extracellular+proteases+facilitate+cell+migration,+axonal+growth,+and+pathfinding.">Neuronal extracellular proteases facilitate cell migration, axonal growth, and pathfinding.</a> Cell and Tissue Research. 290 (2), 367-370 (1997).</li><li>Basham, M. E., Seeds, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasminogen+expression+in+the+neonatal+and+adult+mouse+brain.">Plasminogen expression in the neonatal and adult mouse brain.</a> Journal of Neurochemistry. 77 (1), 318-325 (2001).</li><li>Bourgault, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+conformational+determinants+of+pituitary+adenylate+cyclase-activating+polypeptide+(PACAP)+for+activation+of+the+PAC1+receptor.">Molecular and conformational determinants of pituitary adenylate cyclase-activating polypeptide (PACAP) for activation of the PAC1 receptor.</a> Journal of Medical Chemistry. 52 (10), 3308-3316 (2009).</li></ol>

The authors have nothing to disclose.

This protocol describes the culture of acute P10 rat cerebellar slices in the Transwell system and the fluorescent labeling of GC with a green fluorescent dye to study cell migration during postnatal development through confocal macroscopy. This protocol allows observations of cell migration for a period up to 12 hr and testing of the possible roles of regulating factors in migration including agonists or antagonists of neuropeptides, enzyme inhibitors, cell signaling modulators or toxic substances during the experiment. A small hole in the membrane insert with a pipette tip is necessary to facilitate administration of compounds in the incubation medium. A home-made curved tip pipette can be used to facilitate the delivery of solution.
One issue of cell migration studies on living tissue slices is that the movements of the tissue itself can make cell tracking difficult. Whereas previous approaches have proposed to gently stabilize slices with a nylon mesh or a thin layer of rat tail collagen7,17, a major advantage of this technology is the simple and direct transfer of a 6-well culture plate containing cerebellar slices on membrane inserts from the CO2 incubator under the objective of a confocal macroscope. Integrated temperature and CO2 controllers also provide appropriate and constant environmental parameters essential for cell migration9. Therefore, culture conditions are kept during observations and tissue movements are minimized since slices are well attached to the membrane insert. Stabilization of the tissue is verified by following the position of slice edges or Purkinje cells that should be fixed references during acquisition. In addition, cerebellar slices (between 12 and 18) distributed in the 6 wells of the plate can be quickly observed in detail with a motorized stage and an optical zoom. Due to the large working distance (X2, 39 mm) of the dry objective, epi-observation is free of immersion and administration of compounds in the culture medium is much easier. Therefore, environmental parameters and culture support similarities in CO2 incubator and confocal macroscopy lead to maximum conservation of the biological sample.
Another advantage of the protocol is the large field of view and consequently the large number of cells that can be observed simultaneously. For example, we have previously determined that the density of fluorescent GCs with radial migration in the ML was 1124 &#177; 138 cell/mm2 18. Confocal macroscopy (X2, NA = 0.234) has a lower lateral resolution compared to confocal microscopy (40X, NA = 1.25) but cell bodies of GCs can be easily tracked and the average speed of migration is comparable between the two technological approaches7,18.
Besides technical improvement for image acquisitions, the quality of tissue slices and the quality of labeling are key points for successful experiments. Always keep media and tissues on ice during dissection processes, eliminate oil on vibratome blades and do not utilize slices of tissue in contact with glue. Sagittal and transverse sections are adapted to radial and tangential migration respectively. Use different lengths of incubation for appropriate detection in the different cortical layers of the cerebellum. Long incubation times (up to 8 hr) are necessary to detect the migration of numerous GCs in the PCL and the IGL. Since GC migration is a physiological process during specific spatio-temporal windows, the positive control is that the cells have to migrate properly. In particular, numerous spindle GC in the ML is one of the main health indicator for sagittal cerebellar slices. For starting experiments, observation of GC movements in the ML is suggested. Indeed, the shape of GC with vertically elongated cell body should be considered as a reference point to start acquisition with successive control (2 hr) and treatment (2 hr) periods that can be easily performed in the ML.
Fluorescent dyes like the Cell Tracker family or fluorescent proteins expressed through genetic constructs can be used as tracers for cell migration studies. Due to the slow kinetics of GC migration (1 stack every 30 min), multicolor experiments can also be performed in sequential mode since 4 lasers beams (405, 488, 532 and 633 nm) are available on the system. Considering centripetal and centrifugal radial migration, tracking of other interneurons can also be realized18. In particular, less numerous cell types can be more easily localized with a large field of view. Finally, this protocol can be used to study cell migration at other stages of cerebellar development but also other brain areas.

In the developing cerebellum, eight different types of neurons are produced sequentially between the second embryonic week and the second postnatal week in rodents1. Originating initially from, a primary germinal zone, immature granule cells (GC) are the last neurons to be produced from the external granular layer (EGL), a secondary germinal zone2. During the first three postnatal weeks, the cerebellar cortex is a foliated structure organized in four layers including the EGL, the molecular layer (ML), the Purkinje cell layer (PCL) and the internal granular layer (IGL) (Figure 1). Through centripetal migration, immature GCs, glutamatergic interneurons, reach the IGL within approximately 2 days. By the third postnatal week, the EGL disappears and the IGL constitutes what is called the granular layer (GL) in the adult cerebellum. In the GL, GCs receive excitatory synaptic inputs from mossy fibers and unipolar brush cells, and inhibitory synaptic inputs from Golgi cell axons. In the ML, GC axons make excitatory synapses with GABAergic neurons including Purkinje cells, basket cells, stellate cells, and Golgi cells2.
Real-time observation of cell movement in acute cerebellar slices obtained from early postnatal rodents demonstrates that GCs modify their shape concomitantly with changes in the modality and speed of migration during their route in the cerebellar cortex3. During first two postnatal weeks, GC precursors actively proliferate at the top of the EGL. In the middle part of the EGL, postmitotic GCs migrate tangentially in the direction of their larger process. At the EGL-ML border, GCs slow their movement, the cells begin to enter a short vertical descending process into the ML. In the ML, GCs have a vertically elongated cell body, a thin trailing process and a more voluminous leading process, and migrate radially along the Bergmann glial fibers. In the PCL, GCs stop their movement but after a prolonged stationary phase (2 hr), they cross the PCL-IGL border. In the IGL, GCs migrate towards the bottom of the layer in the absence of glial fiber support. Once the tips of the leading process approach the IGL-white matter (WM) border, GCs slow and stop their movement. Transverse sections of the cerebellum are preferred for tangential migration studies in the EGL while sagittal slices are dedicated to radial migration in the ML, PCL and IGL. Some regulatory factors of GC movements including neuropeptides (e.g., somatostatin, PACAP) have been identified so far but the complete mechanisms involved in the spatio-temporal control of GC migration in each cortical layer are still largely unknown1,4,5,6.
GC migration has been studied during the last 20 years through video- and confocal microscopy using either transmitted light illumination for isolated cultured cells or fluorescence detection for acute cerebellar slices. Initially lipophilic DiI, and more recently &#8220;Cell Tracker&#8221; dyes and cell-expressed fluorescent proteins were used for confocal or two-photon microscopy7,8. Successful experiments depend on a number of specific procedures that make the protocol simple but not easy. In particular, acute slices have to be stabilized during observations generally with a home-made nylon mesh network9. The intensity of light illumination has to be as low as possible to avoid phototoxicity and photobleaching as proposed by multipoint scanning confocal microscope approach. In addition, temperature and CO2 are key environmental parameters since instability may affect neuronal migration. To facilitate and refine the experimental procedures, we have developed a confocal macroscopy protocol that limits slice movements, ensures constant environmental parameters, reduces photobleaching, increases field of view (on the order of milimeters) and consequently the number of cells (dozens) that can be tracked through image analysis. Thus, 180 &#181;m thick slices are cultured on membrane inserts, and 6-well plates are directly transferred under a 2X motorized objective of a commercial confocal macroscope equipped with a large incubation chamber, temperature and CO2 controllers and a vibration control system. Time-lapses and z-stacks are then performed over several hours and pharmacological tools or bioactive molecules can be added or delivered in the incubation medium. This method could also be adapted to study the migration of the other types of neurons in the cerebellum or cerebrum at different developmental stages.

<table><tbody><tr><td>Dulbecco&amp;rsquo;s modified eagle medium (DMEM) nutrient mixture F-12</td><td>Sigma-Aldrich</td><td>D8437</td><td></td></tr><tr><td>Hank&#039;s balanced salt solution 10x</td><td>Sigma-Aldrich</td><td>H1641</td><td></td></tr><tr><td>PACAP38</td><td>INRS, Canada</td><td></td><td>Bourgault &lt;em&gt;et al.&lt;/em&gt;, 2009&lt;sup&gt;19&lt;/sup&gt;</td></tr><tr><td>PAI-1</td><td>Calbiochem</td><td>528208</td><td></td></tr><tr><td>N-2 supplement</td><td>Fisher Scientific / Gibco/ invitrogen</td><td>O973</td><td></td></tr><tr><td>Cyanoacrylate glue</td><td>Loctite</td><td></td><td></td></tr><tr><td>Cell Tracker Green CMFDA</td><td>Invitrogen</td><td>C2925</td><td></td></tr><tr><td>Polyester Transwell-Clear inserts</td><td>Corning</td><td>3452</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P0781</td><td></td></tr><tr><td>6-well cell culture cluster</td><td>Corning</td><td>3516</td><td></td></tr><tr><td>DMSO</td><td>Fisher Scientific</td><td>BP231-100</td><td></td></tr><tr><td>Tissue culture dish 35 mm diameter</td><td>BD Falcon</td><td>353004</td><td></td></tr><tr><td>Tissue culture dish 100 mm diameter</td><td>Thermo SCIENTIFIC</td><td>130182</td><td></td></tr><tr><td>Polypropylen tube (15 ml)</td><td>BD Falcon</td><td>352096</td><td></td></tr><tr><td>Ethanol 70%</td><td>Fisher Chemical</td><td>E/0800DF/21</td><td></td></tr><tr><td>Biological safety cabinet fume hood</td><td>Thermo Scientific</td><td>MSC9 Class II A2</td><td></td></tr><tr><td>Adjustable-volume pipette (0.5-10 &amp;micro;L)</td><td>Eppendorf</td><td>4910 000.018</td><td></td></tr><tr><td>Gyro-rocker, SSL3</td><td>Stuart</td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator, Hera Cell 150</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Vibrating blade microtome, VT1000S</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Confocal macroscope, TCS LSI</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Temperature controller</td><td>PeCon</td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt;-controller</td><td>PeCon</td><td></td><td></td></tr><tr><td>Stereomicroscope, M205 C</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Operating scissors, curved, blunt/blunt</td><td>Medicon</td><td>03.03.17</td><td></td></tr><tr><td>Hardened fine iris scissors, straight, sharp/sharp</td><td>FST</td><td>149090-11</td><td></td></tr><tr><td>Dumont #3 and #5 forceps</td><td>FST</td><td>11293-00 and 11252-20</td><td></td></tr><tr><td>Vibratome injector blades/single edge</td><td>Leica Microsystems</td><td>39053250</td><td></td></tr><tr><td>Standard scalpel handle #3 solid</td><td>FST</td><td>10003-12</td><td></td></tr><tr><td>Surgical blade #15</td><td>Swann-Morton</td><td>205</td><td></td></tr></tbody></table>

ex vivo imaging of postnatal cerebellar granule cell migration using confocal macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

In the early postnatal cerebellum, GCs exhibit significant changes in their mode and speed of migration as they cross different cortical layers1 (Figure 1). This section illustrates examples of results that can be obtained by studying GC migration in their natural cellular milieu. P10 rat cerebellar tissue slices labeled with a green fluorescent dye are examined under a confocal macroscope (Figure 3A) and we show that GCs migrate radially in the ML with an average speed of 18 &#181;m/hr (Figure 3B, C). To date, the role of interactions/communications between neuronal and glial cells including the regulatory factors and molecular mechanisms involved in the control of cell migration in each cortical layer are largely unknown. Consequently, the main issue is to identify neuropeptides, neurotransmitters, neurotrophins and extracellular matrix components that could play a role in these cortical layer-specific changes of the speed during their migration process. Pituitary adenylate cyclase-activating polypeptide (PACAP) is detected mainly in the PCL, but also in the ML and the IGL during the first two postnatal weeks in rodents7,10,11. Application of PACAP38 (10-6 M) to the culture medium resulted in a 79% speed decrease of the GC in the ML. For example, the migration velocity of GCs in the ML dropped from 11.9 &#181;m/hr in control conditions to 2.5 &#181;m/hr after administration of PACAP38 (Figure 4A). Tissue-type plasminogen activator (tPA) is a member of the proteolytic cascade that leads to the degradation of the extracellular matrix (EM) components such as cell adhesion molecules or laminin12,13. tPA and plasminogen, a substrate of tPA, are detected in cortical layers during development of the postnatal cerebellum14,15,16. Administration of PAI-1 (10-7 M), an inhibitor of endogenous tPA, reduced by 78% the GC migration in the ML. For example, GCs reduced migration speed in the ML from 19.2 &#181;m/hr in control conditions to 4.2 &#181;m/hr after addition of PAI-1 (Figure 4B). These results indicate that PACAP exerts a direct inhibitory effect on GC movements and that the serine protease tPA facilitates the migration of GCs in the ML of the developing rat cerebellum.
<img alt="Figure 1" src="/files/ftp_upload/52810/52810fig1.jpg" /> 
Figure 1: 3D representation of GC migration in the postnatal cerebellar cortex. 1-4, Extension of GC processes and tangential migration in the EGL. 5, Radial migration in the ML along Bergmann glial fibers. 6, Transient stationary phase in the PCL. 7, Glial-independent radial migration in the IGL. 8, Completion of GC migration in the IGL. GC, granule cell, in red; EGL, external granular layer; B, Bergmann glia, in dark purple; G, Golgi cell, in yellow; cf, climbing fibers, in blue; g, postmigratory granule cells, in light green; IGL, internal granular layer; mft, mossy fiber terminal, in dark green; ML, molecular layer; P, Purkinje cell, in light purple; PCL, Purkinje cell layer. This figure has been modified from 5.
<img alt="Figure 2" src="/files/ftp_upload/52810/52810fig2.jpg" /> 
Figure 2: Ex vivo culture of P10 cerebellar slices. (A) Dissected cerebellum from P10 rat. Scale bar = 6 mm. (B) Micrograph of living 180 &#181;m-thick cerebellar slice through stereomicroscopy. Scale bar = 3 mm. (C) At a higher magnification, the four cortical layers (EGL, ML, PCL, IGL) of the cerebellum are already distinguishable. Scale bar = 1 mm. (D) After fluorescent labeling, tissue slices are placed on culture inserts (24 mm diameter) into a 6-well plate. (E) Schematic representation of a culture insert with tissue culture treated polyester membrane. <a href="https://www.jove.com/files/ftp_upload/52810/52810fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52810/52810fig3.jpg" /> 
Figure 3: Dynamic migration of GCs in the cortical layers of rat P10 cerebellum. (A) Macroconfocal view (xyz, 2D projection) of a P10 rat cerebellar slice in which GCs are labeled with a green cytoplasmic fluorescent dye. Scale bar = 75 &#181;m. (B) Time-lapse imaging showing GC movements in the ML by confocal macroscopy for 4 hr in control conditions. Asterisk (*) symbol marks the GC soma. Elapsed time (in min) is indicated on the bottom of each photomicrograph. Scale bar = 10 &#181;m. (C) Sequential changes in the distance traveled by GC soma.
<img alt="Figure 4" src="/files/ftp_upload/52810/52810fig4.jpg" /> 
Figure 4: Effect of neuropeptide and protease inhibitor of GC migration. (A) GC was tracked in the ML by confocal macroscopy for 2 hr in control conditions and then for 2 hr in the presence of Pituitary adenylate cyclase-activating polypeptide (PACAP). (B) GC was tracked in the ML by confocal macroscopy for 2 hr in control conditions and then for 2 hr in the presence of plasminogen activator inhibitor-1 (PAI-1).

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Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

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Neuroscience

9.4K Views.  Inserm, IRIB, University of Rouen. During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

Video: Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

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 ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

Medicine

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

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 ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...

Immunology and Infection

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent cerebellum is an ideal system to track the development and morphogenesis of a single cell type, the cerebellar granule neuron (CGN), across time. Here, in vivo electroporation of granule neuron progenitors in the developing mouse cerebellum was employed to sparsely label cells for subsequent morphological analyses. The efficacy of this technique is demonstrated in its ability to showcase key developmental stages of CGN maturation, with a specific focus on the formation of dendritic claws, which are specialized structures where these cells receive the majority of their synaptic inputs. In addition to providing snapshots of CGN synaptic structures&#160;throughout cerebellar development, this technique can be adapted to genetically manipulate granule neurons in a cell-autonomous manner to study the role of any gene of interest and its effect on CGN morphology, claw development, and synaptogenesis.

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Here we describe a method to visualize synaptogenesis of granule neurons in the mouse cerebellum over the time course of postnatal brain development when these cells refine their synaptic structures and form synapses to integrate themselves into the overall brain circuit.

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent ...

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 ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

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