The authors acknowledge funding from NINDS/NIH, R00-NS064197 and NINDS/NIH, R01NS083897 (both to D.L.S.).

1. Preparation of Media (Figure 1, Step 1)
<ol>
	<li>Slice Culture Medium
	<ol>
		<li>25 ml of slice culture medium is sufficient to prepare 5 glass bottom dishes with 2 slices per well.</li>
		<li>In a 50 ml conical tube, add 250 &#181;l of a 100x N2 solution and 500 &#181;l of a 50x B27 solution without vitamin A. Add DMEM/F12 to a volume of 22.5 ml.</li>
		<li>Filter sterilize solution and subsequently add 1.25 ml of heat inactivated horse serum and 1.25 ml of fetal bovine serum.</li>
		<li>Incubate solution in a water bath at 37&#160;&#176;C&#160;for the duration of the preparation of the slices.</li>
		<li>Add growth factors (FGF and EGF, final concentration of 10 ng/ml and 20 ng/ml, respectively) immediately prior to the beginning of culture. The composition of this medium has been optimized to promote the survival of cells in culture and to increase the proliferation rate of neural progenitors. This will thus maximize the probability to observe mitotic cells in the slice.</li>
	</ol>
	</li>
	<li>Full HBSS Dissection Solution
	<ol>
		<li>Prepare 500 ml of full HBSS by adding 50 ml of 10x HBSS, 1.25 ml of 1 M Hepes (pH 7.4, FC 2.5 mM), 6 ml of 2.5 M D-Glucose (FC 30 mM), 2.2 ml of 0.9 M NaHCO3 (FC 4 mM) and autoclaved diH2O to final volume of 500 ml.</li>
		<li>Filter sterilize solution and store at 4 &#176;C until the collection of uterine horns from the pregnant mouse. This solution can be kept at 4 &#176;C for weeks.</li>
		<li>Keep HBSS on ice during the whole dissection procedure.</li>
	</ol>
	</li>
	<li>Embedding solution
	<ol>
		<li>For the embedding of 4 embryonic brains, prepare 30 ml of 3% low melting agarose in the full HBSS solution. In a 50 ml conical tube, add 0.9 g of low melting agarose and full HBSS to 30 ml.</li>
		<li>Mix the solution with a vortex mixer, and melt in a microwave with the tube standing and the cap open.</li>
		<li>While in the microwave, monitor the solution to prevent overflow due to excessive boiling. When boiling starts, stop the microwave and vortex the solution.</li>
		<li>Repeat these steps until all the agarose has melted.</li>
		<li>Store the tube in a water bath at 42 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Dissection of the Embryos (Figure 1, Step 2)
<ol>
	<li>After careful euthanasia of the pregnant mouse, collect the uterine horns and transfer them into a 10 cm2&#160;Petri dish containing cold full 1x HBSS. The age of the embryo can vary depending upon the study. This protocol has been used successfully for culturing brain slices from embryonic days E12.5 to E17.5. Due to their size, younger brains tend to be more difficult to work with.</li>
	<li>Collect the embryos and dissect out the embryonic brains as described previously in the rat and mouse26,27, until a full embryonic brain including the hindbrain and the forebrain with the two&#160;cerebral hemispheres is obtained. These can be kept at RT until all brains have been dissected.</li>
	<li>Collect the embryos and dissect out the embryonic brains as described previously in the rat and mouse26,27, until a full embryonic brain including the hindbrain and the forebrain with the two&#160;cerebral hemispheres is obtained. These can be kept at RT until all brains have been dissected.</li>
</ol>
3. Embedding of the Embryonic Brains (Figure 1, Step 3)
<ol>
	<li>In a bucket containing ice, create a hole in ice to insert the embedding molds into.</li>
	<li>Pour the 3% agarose medium into a plastic mold and place that mold into the hole prepared in the ice.</li>
	<li>Stir the agarose medium with the tip of a digital thermometer until the temperature reaches 35 &#176;C.</li>
	<li>Promptly transfer the brain into the embedding medium.</li>
	<li>Critical Step: To remove the excess HBSS at the interface between the brain and the agarose, carefully and gently tumble/rotate the brain repeatedly in the embedding solution with the forceps.</li>
	<li>A cushion of gelled agarose will form at the bottom of the mold in contact with the ice. Once the cushion can be felt with the tip of the forceps while stirring the brain, position the brain with the dorsal side up. The brain should not sink to the very bottom of the mold.</li>
</ol>
4. Preparation of Agarose Block (Figure 1, Step 4)
<ol>
	<li>Let the agarose harden in the ice for at least 5 min.</li>
	<li>Using a razor blade, section the corners of the mold.</li>
	<li>Carefully carve an agarose block around the brain using a razor blade. Try to minimize the number of cuts to avoid perturbing the embedded brain.</li>
	<li>Make sure that the block has a larger surface area in the caudal region of the brain (versus the rostral region, see Figure 1, Step 4) to increase the stability of the block on the sectioning pedestal.</li>
	<li>The last cut should be the one on the caudal side of brain; this cut will define the correct sectioning plane with the vibratome.</li>
	<li>Do so by aligning the blade perpendicular to the rostro-caudal axis of the brain and the agarose block. Slide down the blade caudally along the rostro-caudal axis and make the final cut about 5 mm away from the most caudal region of the brain.</li>
	<li>Set the agarose/brain block aside and repeat embedding of other brains.</li>
</ol>
5. Transfer of the Embedded Brains into the Tray of the Vibratome (Figure 1, Step 5)
<ol>
	<li>Add a drop of glue onto the bottom of the vibratome tray. Place glue so that the agarose blocks will be positioned within reach of the vibrating blade.</li>
	<li>Place the agarose/brain block caudal side down on the edge of the spatula, with about 50% of the block tipping over the edge of the spatula.</li>
	<li>Apply the caudal face of the agarose block to the glue and slide the spatula away, maintaining the agarose block to the bottom of the tray with the shoulder of a pair of tweezers. Do not let the spatula directly contact the glue.</li>
	<li>Let the glue solidify at room temperature for 5 min.</li>
</ol>
6. Sectioning of the Embedded Brains (Figure 1, Step 6)
<ol>
	<li>Fill the vibratome tray with HBSS.</li>
	<li>Define the start and finish positions of the vibratome blade.</li>
	<li>Generate 200 - 250 &#181;m slices. With the VT1000s vibratome, use the following parameters: speed 2 - 4, frequency 8.</li>
	<li>Carefully remove the slices from the vibratome as they are cut. Transfer the slices with a spatula and the back end of tweezers into 12 well dishes filled with full HBSS. Alternatively some researchers have used a paintbrush in lieu of tweezers. Key point: During transfer, take care that the brain slices remain attached to the surrounding agarose.</li>
</ol>
7. Syto11 Staining of the Slices (Figure 1, Step 7)
<ol>
	<li>Dilute Syto11 to a final concentration of 0.5 - 1 &#181;M in the slice culture medium supplemented with growth factors.</li>
	<li>In the well of a 12-well plate, incubate slices from one brain in 2.5 ml of the staining solution for 1 hr at 37 &#176;C.</li>
	<li>Wash the slices in 2.5 ml of slice culture medium without staining solution for 20 min.</li>
</ol>
8. Mounting the Slices in a Glass Bottom Dish (Figure 1, Steps 8, 9)
<ol>
	<li>Prepare 15 &#181;l of embedding collagen solution per slice. Prepare a 1.5 mg/ml collagen solution by mixing 375 &#181;l of 3 mg/ml collagen type I solution with 75 &#181;l of 10x DMEM, 9.4 &#181;l of 1 M NaOH, and 290 &#181;l of H2O. Store on ice.</li>
	<li>Place a 15 &#181;l drop of the collagen solution at the bottom of a 35 mm Glass bottom Microwell Dish. Spread it with a pipet tip to match the size of the slice.</li>
	<li>Transfer the slice into the drop of collagen with a spatula and pair of tweezers. Key point: Mounting multiple slices in different dishes increases the probability to acquire slices suitable for imaging. Screen through different slices and select the ones that best meet the criteria described below in the &#34;Representative Results&#34; section.</li>
	<li>Let the slices incubate at RT for 10 min before transferring them into a 37 &#176;C incubator with 5% CO2.</li>
	<li>After 20 min, add 1.2 ml of slice culture medium into the Glass Bottom Microwell Dish. Add 600 &#181;l of medium at a time and spread it with a pipette tip.</li>
	<li>Capture a low magnification image of the brain slice to record the anatomical level and integrity of the slice.</li>
</ol>
9. Live Imaging of the Slices (Figure 1, Steps 10, 11)
<ol>
	<li>Let the slices recover in the incubator for at least 1 hr and 30 min at 37 &#176;C with 5% CO2 before live imaging.</li>
	<li>Image the slices with the microscope of choice, keeping in mind that Syto11 is very prone to photobleaching. Here an inverted spinning disk confocal microscope is used (Andor XD revolution spinning disk confocal microscope). Alternatively a laser scanning confocal microscope can be used. The following parameters are for the spinning disk microscope set-up.</li>
	<li>During the entire live imaging session, maintain slices at 37 &#176;C, 5% CO2 in a humidified incubation chamber attached to the microscope.</li>
	<li>Image cells with a 60X silicon oil objective with a working distance of 300 &#181;m, and a numerical aperture of 1.3 (for examples see Figures 3B, 3C, 3E, 3F, 4A, 4B). A 100X oil objective with a working distance of 130 &#181;m and a numerical aperture of 1.4 (for example see Figure 4C) can also be used. The resolution of the camera is 512 x 512.</li>
	<li>Image the cells in a 30 &#181;m z-stack, with the center of the z-stack located about 40 &#181;m below the surface of the slice. Trying to image deeper into the tissue can cause the objective to add mechanical pressure on the slice. This can affect the integrity of the brain slice. If planning to make 3D reconstructions of the cells, use a z-interval of no more than 2 &#181;m.</li>
	<li>Adjust the laser power and exposure times to limit photobleaching. Use exposure times ranging from 30 to 200 msec, depending on the intensity of the Syto11 signal.</li>
	<li>With a motorized stage, image multiple positions across multiple slices mounted in 1 dish. For example, image up to 20 positions scattered across 4 different slices in 1 single dish.</li>
	<li>For live imaging, use a temporal resolution of less than 5 min to enable the identification of the different phases of mitosis. Using Syto11 and/or histone H2B-EGFP, 4 - 5 hr of live imaging are sufficient to observe a significant number of mitotic cells. However, if imaging sparsely labeled mitotic cells (such as those achieved by in utero electroporation), then a longer imaging parameter (overnight) is appropriate and works well. 
	Note: Using this protocol, we typically observe on average 10 mitotic cells per position. As stated above, imaging multiple mounted slices increases the probability of having a successful experiment. With this approach we typically identify mitotic cells in virtually all experiments performed with Syto11 or histone H2B-EGFP.</li>
</ol>
10. Post-acquisition Analysis of Mitosis (Figure 2)
All the procedures in this section have been optimized in Fiji (ImageJ), which is advantageous because it is a free open source software. Other software solutions are available to perform the same tasks, such as Metamorph, Imaris, and Amira.
<ol>
	<li>Identification of Mitotic Figures in Fiji
	<ol>
		<li>After opening the dataset for one position as a &#34;hyperstack&#34;, select one z-plane (see Figure 2A for the location of the scroll bar) where the tissue and cells look healthy (see criteria in the discussion section). Scroll across the temporal dimension to identify cells going through anaphase, as it is the easiest mitotic phase to identify.</li>
		<li>Scroll back in time to identify the time point where the cell enters mitosis (DNA condensation). The cell may move across z-planes, but the temporal resolution and the z-stack parameters described earlier have been optimized to enable this process.</li>
		<li>In a spreadsheet, record the 4D coordinates of the cell in the hyperstack (X,Y,Z and time, see Figure 2A to visualize the location of these figures in the Fiji interface) as it enters mitosis. Knowing these coordinates will prevent counting the same cell several times.</li>
		<li>Scroll forward in time and record the time when the cell progresses from one phase to another. Document the duration of each mitotic phase for one given cell.</li>
		<li>Proceed with other cells, across the full dataset (multiple cells within and across positions).</li>
	</ol>
	</li>
	<li>3D Reconstruction of the Cells and Quantitation of Rotation During Metaphase (Adapted from Haydar et al, 2003) (Figure 2B).
	<ol>
		<li>After having identified multiple mitotic cells, use the coordinates of a cell and scroll forward in time until the beginning of metaphase.</li>
		<li>Rotate the entire &#34;hyperstack&#34; so that the ventricular border is planar (using the &#34;Image/Transform/Rotate&#8230;&#34; command). Use the &#34;angle&#34; tool to determine the angle to apply for this rotation.</li>
		<li>Identify the z-plane where the cell of interest is the most visible. In that plane, use the &#34;rectangle selection&#34; tool to draw a selection that includes the whole cell.</li>
		<li>Identify the z-planes that include the cell of interest. Use the &#34;Image/Duplicate&#34; command to create a &#34;sub-stack&#34;. In the dialog box, leave &#34;Duplicate hyperstack&#34; checked. The &#34;Slices (z)&#34; parameter corresponds to the z-planes including the whole cell, and the &#34;Frames (t)&#34; parameter corresponds to the current time point.</li>
		<li>If the resolution is poor, increase the size of the &#34;sub-stack&#34; using the &#34;Image/Adjust/Size&#8230;&#34; command.</li>
		<li>Generate a 3D reconstruction of the cell at the current time point using the &#34;Image/Stacks/3D project&#8230;&#34; command. The parameters for this command are displayed in Figure 2B. The &#34;Slice spacing&#34; corresponds to the interval between each of the z-plane images (&#181;m). Important: make sure that the physical dimension (&#181;m) of the sub-stack has been conserved. If not, the interpolation of the pixels in the z dimension will be incorrect, and the 3D reconstruction will be inaccurate.</li>
		<li>Using the scrollbar, rotate the resulting 3D reconstruction so that the edge of the metaphase plate is visible. The number of the frame corresponds to the beta angle described in Figure 3B, record this number. Using the &#34;angle&#34; tool and the &#34;Analyze/Measure&#8230;&#34; command, measure the angle between the horizontal and a line bisecting the metaphase plate perpendicularly (Figure 2B). This is the angle alpha.</li>
		<li>Record these angles for all the metaphase time points of the cell of interest. The angle of rotation between time points (t) and (t-1) equals to the absolute value of the difference between an angle at (t) and this angle at (t-1).</li>
	</ol>
	</li>
	<li>Measuring the Orientation of the Cleavage Plane
	<ol>
		<li>3D reconstruct a cell of interest at one time point during anaphase following the instructions described for the 3D reconstruction of metaphase plates.</li>
		<li>Rotate the reconstruction until the edges of the two plates formed by the segregating chromosomes are visible.</li>
		<li>Use the angle tool to measure the angle between the horizontal (the ventricular edge) and a line parallel to the two plates formed by the segregating chromosomes. This is the angle of the cleavage plane (Figure 2C).</li>
	</ol>
	</li>
	<li>Generation of Movies
	<ol>
		<li>As the cells often move across different z-planes, identify the z-planes occupied by the cell during the live imaging session. Generate a &#34;Maximum intensity projection&#34; of these z-planes using the &#34;Image/Stacks/Z project&#8230;&#34; command. Save the resulting stack as a &#34;.TIF&#34; file.</li>
		<li>Open this file in the 1.45 version of ImageJ as Fiji does not include a stable plugin for the generation of &#34;.mov&#34; or &#34;.avi&#34; files.</li>
		<li>Use the &#34;File/Save as/&#8230;&#34; command of ImageJ to generate a movie in a format compatible with your down-stream applications.</li>
	</ol>
	</li>
</ol>

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C., Mart&#237;nez-Cerde&#241;o, V., Ivic, L., Kriegstein, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+neurons+arise+in+symmetric+and+asymmetric+division+zones+and+migrate+through+specific+phases.">Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases.</a> Nature Publishing Group. 7, 136-144 (2004).</li><li>Tyler, W. A., Haydar, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+Genetic+Fate+Mapping+Reveals+a+Novel+Route+of+Neocortical+Neurogenesis,+Which+Is+Altered+in+the+Ts65Dn+Mouse+Model+of+Down+Syndrome.">Multiplex Genetic Fate Mapping Reveals a Novel Route of Neocortical Neurogenesis, Which Is Altered in the Ts65Dn Mouse Model of Down Syndrome.</a> Journal of Neuroscience. 33, 5106-5119 (2013).</li><li>Noctor, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-Lapse+Imaging+of+Fluorescently+Labeled+Live+Cells+in+the+Embryonic+Mammalian+Forebrain.">Time-Lapse Imaging of Fluorescently Labeled Live Cells in the Embryonic Mammalian Forebrain.</a> CSH Protoc. , (2011).</li><li>Elias, L., Kriegstein, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+slice+culture+of+E18+rat+brains.">Organotypic slice culture of E18 rat brains.</a> Journal of visualized experiments : JoVE. , (2007).</li><li>Shitamukai, A., Konno, D., Matsuzaki, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oblique+radial+glial+divisions+in+the+developing+mouse+neocortex+induce+self-renewing+progenitors+outside+the+germinal+zone+that+resemble+primate+outer+subventricular+zone+progenitors.">Oblique radial glial divisions in the developing mouse neocortex induce self-renewing progenitors outside the germinal zone that resemble primate outer subventricular zone progenitors.</a> Journal of Neuroscience. 31, 3683-3695 (2011).</li><li>Hadjantonakis, A. -. K., Papaioannou, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+in+vivo+imaging+and+cell+tracking+using+a+histone+fluorescent+protein+fusion+in+mice.">Dynamic in vivo imaging and cell tracking using a histone fluorescent protein fusion in mice.</a> BMC Biotechnol. 4, 33 (2004).</li><li>Higginbotham, H., Bielas, S., Tanaka, T., Gleeson, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mouse+line+with+green-fluorescent+protein-labeled+Centrin+2+allows+visualization+of+the+centrosome+in+living+cells.">Transgenic mouse line with green-fluorescent protein-labeled Centrin 2 allows visualization of the centrosome in living cells.</a> Transgenic Res. 13, 155-164 (2004).</li><li>Walantus, W., Castaneda, D., Elias, L., Kriegstein, A. 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The authors declare they have no competing financial interests.

The major advantage of the protocol we have described is that it provides a dynamic temporal resolution of mitosis of neural progenitors. Typically, assays to visualize mitosis in the developing brain are performed using immunofluorescence of fixed tissue sections. But this approach only provides a snapshot of mitosis at one time point.
There are several steps that are most critical for imaging mitosis in brain slices: 1) The brain slices should remain attached to the agarose, to preserve the ...

The overall goal of this protocol is to describe how to perform live imaging of neural progenitor mitosis in embryonic brain slices. Using live imaging of brain slices in culture, this protocol provides a simple method to assay multiple aspects of mitosis in neural progenitors in an environment highly similar to an in vivo setting. It can be applied to brains of mutant animals and/or brains that have been manipulated with in utero electroporation)1-5. This technique is also ideal to test the effect of pharmacological agents on neural precursors&#39; mitosis, by simply adding an agent to the culture medium. In sum, this article will make a technically challenging protocol accessible to those studying neurogenesis.
During neurogenesis, distinct neural progenitor populations undergo precise divisions to generate neurons that eventually contribute to the six cortical layers of the adult neocortex6-8. Early in cortical development, the neural precursor pool expands as neuroepithelial (NE) cells divide symmetrically to self-renew. NE cells then convert into radial glial cells (RGCs). Initially RGCs divide symmetrically to produce two new RGCs, however during the bulk of neurogenesis, RGCs&#39; main mode of division is asymmetric. In asymmetric division, 1 RGC gives rise to a new RGC and either a post-mitotic neuron, or a more specialized progenitor (either a short neural precursor (SNP), an outer radial glia (ORG), or an intermediate progenitor (INP)2,3,7,9. INPs, SNPs, and ORGs can then generate neurons at the sub-ventricular, ventricular, and basal regions of the cortex, respectively. Hence, cell division of progenitors is a fundamental process for generating neurons of the neocortex.
Numerous studies point to a correlation between specific mitotic traits of RGCs and the fate of daughter cells. Haydar et al. and Takahashi et al. have shown that RGC mitotic duration and cell cycle length increase as neurogenesis proceeds, a finding echoed in follow up studies10-13. A number of studies have suggested that mitotic spindle orientation relative to the ventricle influences aspects of neurogenesis and corticogenesis, including types of neurons generated and location of progeny in the brain, respectively3,10,14-16. Whether cleavage plane orientation directly influences cell fate is controversial, but the conclusion remains that this mitotic parameter impacts neurogenesis. Further underscoring the importance of mitosis is the observation that many genes involved in the mechanics of mitosis are crucial for neurogenesis and for proper brain development17-20.
Mitosis is a dynamic process, yet to date most studies detailing neural progenitor mitosis utilize analysis of fixed tissue sections or imaging of neural progenitors via in vitro cell culture. Thus, the mainstream methods to evaluate mitosis only provide a snapshot of this process and fail to uncover how cells behave in a tissue. Live imaging of neural progenitor mitosis has increasingly become a critical tool for understanding neural progenitor function. For examples please see these references4,8,10,21-25. Several outstanding protocols have been published for preparation and imaging of brain slices26,27. However to date, a comprehensive protocol for imaging and analysis of mitosis has not been described nor demonstrated in video.
This technique offers several significant advantages over fixed analysis of brain sections. Time-lapse analysis of brain slices enables generation of significantly more data points that can be analyzed in a flexible fashion. First, data is gathered at individual time points over the course of several minutes or several hours. One can analyze individual time points (to create a static montage) or can combine different time points into movies. Second, confocal imaging of slices enables generation of data at different Z sections in brain slices. As a result, individual sections can be analyzed. Alternatively, stacks of individual sections can be combined into a maximum intensity projection. Third, analysis is done in the context of a tissue, revealing how cells divide relative to neighboring cells and structures. Fourth, it is ideally suited to analysis of mutants that show some evidence of mitotic defects. Together this protocol will help clarify critical steps to aid investigators who wish to carry out live imaging of neural progenitor mitosis in their own laboratories.

<table border="0" cellpadding="0" cellspacing="0" style="width:739px;" width="738">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:143px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:261px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100X N2</td>
			<td>Life technologies</td>
			<td>17502048</td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50X B27 without vitamin A</td>
			<td>Life technologies</td>
			<td>12587010</td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12</td>
			<td>Life technologies</td>
			<td>11320033</td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Heat-inactivated horse serum</td>
			<td>Sigma Aldrich</td>
			<td>H1138-500ml</td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heat-inactivated calf serum</td>
			<td>Sigma Aldrich</td>
			<td>F4135-500ML</td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FGF</td>
			<td>R&#38;D Sytems</td>
			<td>3139-FB-025</td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGF</td>
			<td></td>
			<td></td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10X HBSS</td>
			<td>Life technologies</td>
			<td>14065-056</td>
			<td style="width:261px;">for the dissection of the embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hepes Free Acid</td>
			<td>Sigma Aldrich</td>
			<td>&#160;H4034</td>
			<td style="width:261px;">dilute to 1M (pH7.4)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2.5M D-Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G8769</td>
			<td style="width:261px;">for the dissection of the embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.9M NaHC03</td>
			<td>Life technologies</td>
			<td>25080-094</td>
			<td style="width:261px;">for the dissection of the embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">low-melting agarose</td>
			<td>Fisher&#160;</td>
			<td>BP165-25</td>
			<td style="width:261px;">for generating slices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Loctite 404 glue</td>
			<td>Loctite 404</td>
			<td>46551</td>
			<td style="width:261px;">keep at 4&#730;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">syto11</td>
			<td>Life technologies</td>
			<td>S7573</td>
			<td style="width:261px;">Make 5&#181;l aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3 mg/ml collagen type I&#160;</td>
			<td>Life technologies</td>
			<td>A1048301</td>
			<td style="width:261px;">for culturing slices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glass bottom dish</td>
			<td>MatTek</td>
			<td>P35G-1.5-14-C</td>
			<td style="width:261px;">for culturing slices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">petri dishes</td>
			<td></td>
			<td></td>
			<td style="width:261px;">for dissection of the embryos</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">digital thermometer</td>
			<td></td>
			<td></td>
			<td style="width:261px;">to measure the temperature of the agarose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">spatula</td>
			<td></td>
			<td></td>
			<td style="width:261px;">to transfer brains</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paintbrush</td>
			<td></td>
			<td></td>
			<td style="width:261px;">alternative to transfer brains</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vibratome</td>
			<td>Leica</td>
			<td>VT1000s</td>
			<td style="width:261px;">for generating slices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dissecting microscope</td>
			<td></td>
			<td></td>
			<td style="width:261px;">dissecting out embryos</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">imaging microscope</td>
			<td></td>
			<td></td>
			<td style="width:261px;">A confocal microscope is required, it needs to be equipped with an incubation chamber</td>
		</tr>
	</tbody>
</table>

live imaging of mitosis in the developing mouse embryonic cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

The success of this assay and the observation of multiple mitotic cells during one live imaging session largely depend upon both the integrity and anatomical level of the slice where acquisitions are made. As discussed below, the anatomical level of a slice is an important factor. Figure 3A shows rostro-caudal and medial-lateral locations where we have been most successful. For additional discussion of this subject please see Noctor26. The integrity of the slice can vary in different regions o...

Watch this Scientific Journal Video about Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex at JoVE.com

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

live-imaging-of-mitosis-in-the-developing-mouse-embryonic-cortex

Research

JoVE Journal

Neuroscience

15.2K Views.  Duke University Medical Center. Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Video: Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

Non-Invasive Modulation and Robotic Mapping of Motor Cortex in the Developing Brain

Mapping the motor cortex with transcranial magnetic stimulation (TMS) has potential to interrogate motor cortex physiology and plasticity but carries unique challenges in children. Similarly, transcranial direct current stimulation (tDCS) can improve motor learning in adults but has only recently been applied to children. The use of tDCS and emerging techniques like high&#8211;definition tDCS (HD-tDCS) require special methodological considerations in the developing brain. Robotic TMS motor mapping may confer unique advantages for mapping, particularly in the developing brain. Here, we aim to provide a practical, standardized approach for two integrated methods capable of simultaneously exploring motor cortex modulation and motor maps in children. First, we describe a protocol for robotic TMS motor mapping. Individualized, MRI-navigated 12x12 grids centered on the motor cortex guide a robot to administer single-pulse TMS. Mean motor evoked potential (MEP) amplitudes per grid point are used to generate 3D motor maps of individual hand muscles with outcomes including map area, volume, and center of gravity. Tools to measure safety and tolerability of both methods are also included. Second, we describe the application of both tDCS and HD-tDCS to modulate the motor cortex and motor learning. An experimental training paradigm and sample results are described. These methods will advance the application of non-invasive brain stimulation in children.

We demonstrate protocols for the modulation (tDCS, HD-tDCS) and mapping (robotic TMS) of the motor cortex in children.

Mapping the motor cortex with transcranial magnetic stimulation (TMS) has potential to interrogate motor cortex physiology and plasticity but carries ...