Name: ex vivo imaging of postnatal cerebellar granule cell migration using confocal macroscopy
Uploaded: 2015-05-12T14:00:00
Duration: 9 min 10 s
Description: Watch this Scientific Journal Video about Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy at JoVE.com

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>

<ol>
	<li>Komuro, Y., Kumada, T., Ohno, N., Foote, K. D., Komuro, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+in+the+Cerebellum.">Migration in the Cerebellum.</a> Cellular Migration and Formation of Neuronal Connections: Comprehensive Developmental Neuroscience. 2, 281-297 (2013).</li><li>Altman, J., Bayer, S. 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. K., 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=Rescue+of+neuronal+migration+deficits+in+a+mouse+model+of+fetal+Minamata+disease+by+increasing+neuronal+Ca2++spike+frequency.">Rescue of neuronal migration deficits in a mouse model of fetal Minamata disease by increasing neuronal Ca2+ spike frequency.</a> Proceedings of National Academy of Sciences USA. 109 (13), 5057-5062 (2012).</li><li>Raoult, E., 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=Pituitary+adenylate+cyclase-activating+polypeptide+(PACAP)+stimulates+the+expression+and+the+release+of+tissue+plasminogen+activator+(tPA)+in+neuronal+cells:+involvement+of+tPA+in+the+neuroprotective+effect+of+PACAP.">Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates the expression and the release of tissue plasminogen activator (tPA) in neuronal cells: involvement of tPA in the neuroprotective effect of PACAP.</a> Journal of Neurochemistry. 119 (5), 10-1111 (2011).</li><li>Cameron, D. 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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s modified eagle medium (DMEM) nutrient mixture F-12</td>
			<td>Sigma-Aldrich</td>
			<td>D8437</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;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 et al., 200919</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></td>
			<td></td>
			<td></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 &#181;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>CO2 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>CO2-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></td>
			<td></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).

Watch this Scientific Journal Video about Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy at JoVE.com

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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Research

JoVE Journal

Neuroscience

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 ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

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 ...

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering the molecular signals that cooperatively guide neuronal migration in the embryonic brain is therefore important to understand how the complex neural networks form which later support postnatal life. Facial branchiomotor (FBM) neurons in the mouse embryo hindbrain migrate from rhombomere (r) 4 caudally to form the paired facial nuclei in the r6-derived region of the hindbrain. Here we provide a detailed protocol for wholemount ex vivo culture of mouse embryo hindbrains suitable to investigate the signaling pathways that regulate FBM migration. In this method, hindbrains of E11.5 mouse embryos are dissected and cultured in an open book preparation on cell culture inserts for 24 hr. During this time, FBM neurons migrate caudally towards r6 and can be exposed to function-blocking antibodies and small molecules in the culture media or heparin beads loaded with recombinant proteins to examine roles for signaling pathways implicated in guiding neuronal migration.

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Embryonic neurons are born in the ventricular zone of the neural tube, but migrate to reach appropriate targets. Facial branchiomotor (FBM) neurons are a useful model to study neuronal migration. This protocol describes the wholemount ex vivo culture of mouse embryo hindbrains to investigate mechanisms that regulate FBM migration.

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering ...

Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique &#8211; described here &#8211; is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis13,14. Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer&#8217;s or Parkinson&#8217;s disease.

Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature.

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating ...

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

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 ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.