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 wa...

<ol>
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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+ventricular+zone+neural+precursors+contributes+to+neuronal+fate+diversity+in+the+postnatal+neocortex.">Heterogeneity in ventricular zone neural precursors contributes to neuronal fate diversity in the postnatal neocortex.</a> Journal of Neuroscience. 30, 7028-7036 (2010).</li><li>Konno, D., 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=Neuroepithelial+progenitors+undergo+LGN-dependent+planar+divisions+to+maintain+self-renewability+during+mammalian+neurogenesis.">Neuroepithelial progenitors undergo LGN-dependent planar divisions to maintain self-renewability during mammalian neurogenesis.</a> Nat Cell Biol. 10, 93-101 (2008).</li><li>LoTurco, J. J., Manent, J. -. 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F., Ang, E., 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=Mitotic+spindle+rotation+and+mode+of+cell+division+in+the+developing+telencephalon.">Mitotic spindle rotation and mode of cell division in the developing telencephalon.</a> Proc Natl Acad Sci USA. 100, 2890-2895 (2003).</li><li>Takahashi, T., Nowakowski, R. S., Caviness, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+cycle+of+the+pseudostratified+ventricular+epithelium+of+the+embryonic+murine+cerebral+wall.">The cell cycle of the pseudostratified ventricular epithelium of the embryonic murine cerebral wall.</a> J Neurosci. 15, 6046-6057 (1995).</li><li>Pilaz, L. -. 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V., 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=Mitotic+spindle+orientation+predicts+outer+radial+glial+cell+generation+in+human+neocortex.">Mitotic spindle orientation predicts outer radial glial cell generation in human neocortex.</a> Nature Communications. 4, 1665 (2013).</li><li>Postiglione, M. P., 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=Mouse+Inscuteable+Induces+Apical-Basal+Spindle+Orientation+to+Facilitate+Intermediate+Progenitor+Generation+in+the+Developing+Neocortex.">Mouse Inscuteable Induces Apical-Basal Spindle Orientation to Facilitate Intermediate Progenitor Generation in the Developing Neocortex.</a> Neuron. 72, 269-284 (2011).</li><li>Silver, D. L., 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=The+exon+junction+complex+component+Magoh+controls+brain+size+by+regulating+neural+stem+cell+division.">The exon junction complex component Magoh controls brain size by regulating neural stem cell division.</a> Nat Neurosci. 13, 551-558 (2010).</li><li>Pulvers, J. N., 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=Mutations+in+mouse+Aspm+(abnormal+spindle-like+microcephaly+associated)+cause+not+only+microcephaly+but+also+major+defects+in+the+germline.">Mutations in mouse Aspm (abnormal spindle-like microcephaly associated) cause not only microcephaly but also major defects in the germline.</a> Proc Natl Acad Sci USA. , 107-16595 (2010).</li><li>Feng, Y., Walsh, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+spindle+regulation+by+Nde1+controls+cerebral+cortical+size.">Mitotic spindle regulation by Nde1 controls cerebral cortical size.</a> Neuron. 44, 279-293 (2004).</li><li>Megraw, T. L., Sharkey, J. T., Nowakowski, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdk5rap2+exposes+the+centrosomal+root+of+microcephaly+syndromes.">Cdk5rap2 exposes the centrosomal root of microcephaly syndromes.</a> Trends Cell Biol. 21, 1-11 (2011).</li><li>Bond, 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=A+centrosomal+mechanism+involving+CDK5RAP2+and+CENPJ+controls+brain+size.">A centrosomal mechanism involving CDK5RAP2 and CENPJ controls brain size.</a> Nat Genet. 37, 353-355 (2005).</li><li>Shu, T., 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=Doublecortin-like+kinase+controls+neurogenesis+by+regulating+mitotic+spindles+and+M+phase+progression.">Doublecortin-like kinase controls neurogenesis by regulating mitotic spindles and M phase progression.</a> Neuron. 49, 25-39 (2006).</li><li>Nelson, B. R., Hodge, R. D., Bedogni, F., Hevner, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Interactions+between+Intermediate+Neurogenic+Progenitors+and+Radial+Glia+in+Embryonic+Mouse+Neocortex:+Potential+Role+in+Dll1-Notch+Signaling.">Dynamic Interactions between Intermediate Neurogenic Progenitors and Radial Glia in Embryonic Mouse Neocortex: Potential Role in Dll1-Notch Signaling.</a> Journal of Neuroscience. 33, 9122-9139 (2013).</li><li>Hu, D. 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=Dynein+recruitment+to+nuclear+pores+activates+apical+nuclear+migration+and+mitotic+entry+in+brain+progenitor+cells.">Dynein recruitment to nuclear pores activates apical nuclear migration and mitotic entry in brain progenitor cells.</a> Cell. 154, 1300-1313 (2013).</li><li>Noctor, S. 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. 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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

15.1K ビュー.  Duke University Medical Center. 神経前駆有糸分裂は、神経新生の重要なパラメータである。神経前駆糸分裂の我々の理解の多くは、固定された組織の分析に基づいている。胚性脳スライス内のライブイメージングは​​、制御された環境で、高い時間·空間分解能で有糸分裂を評価するための汎用的な手法である。 

ビデオ: 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 ...