Name: live imaging of mitosis in the developing mouse embryonic cortex
Uploaded: 2014-06-04T13:45:00
Description: Watch this Scientific Journal Video about Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex at JoVE.com

Die Autoren danken Mittel aus NINDS / NIH, R00-NS064197 und NINDS / NIH, R01NS083897 (sowohl auf DLS).

1. Vorbereitung der Medien (Abb. 1, Schritt 1) <ol><li> Slice-Kultur-Medium <ol><li> 25 ml Kulturmedium Scheibe ist ausreichend, um 5 Glasbodenschalen mit 2 Scheiben pro gut vorbereiten. </li><li> In einem 50 ml konischen Röhrchen, fügen 250 ul einer 100fach N2-Lösung und 500 ul einer 50x-Lösung B27 ohne Vitamin A. In DMEM/F12 auf ein Volumen von 22,5 ml. </li><li> Filter-Lösung zu sterilisieren und anschließend hinzufügen, 1,25 ml hitzeinaktiviertem Pferdeserum und 1,25 ml fetales Rinderserum. </li><li> Inkubi...

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Der Hauptvorteil des Protokolls, die wir beschrieben haben, ist, dass sie eine dynamische zeitliche Auflösung der Mitose von neuronalen Vorläuferzellen. Typischerweise Assays zur Mitose im sich entwickelnden Gehirn visualisiert werden unter Verwendung von Immunfluoreszenz von fixierten Gewebeschnitten. Aber dieser Ansatz stellt nur eine Momentaufnahme der Mitose zu einem Zeitpunkt. Es gibt mehrere Schritte, die am kritischsten für die Abbildung der Mitose in Hirnschnitten sind: 1) Die Geh...

Das übergeordnete Ziel dieses Protokolls ist zu beschreiben, wie die Live-Darstellung von neuronalen Vorläufer Mitose in der embryonalen Hirnschnitten durchzuführen. Mit Live-Bildgebung von Hirnschnitten in Kultur, bietet das Protokoll eine einfache Methode, um mehrere Aspekte der Mitose in neuralen Vorläuferzellen in einem Umfeld sehr ähnlich zu einem in-vivo-Einstellung testen. Es kann an Gehirnen von mutierten Tieren und / oder Gehirn, die mit in utero Elektroporation 1-5 manipuliert wurden) angewendet werden. Diese Technik eignet sich auch hervorragend, um die Wirkung von pharmakologischen Mitteln auf neuralen Vorläufer &#39;Mitose zu testen, durch einfaches Hinzufügen eines Mittels zu dem Kulturmedium. In der Summe wird dieser Artikel machen eine technisch anspruchsvolle Protokoll zugänglich, die das Studium der Neurogenese. Während der Neurogenese, unterschiedliche neuronale Vorläuferpopulationen unterziehen präzise Divisionen zu Neuronen, die schließlich zu den sechs kortikalen Schichten der erwachsenen Neocortex 08.06 beitragen erzeugen. Früh in kortikalen Entwicklung, dehnt sich die neuralen Vorläuferpool neuroepithelialen (NE) Zellen teilen sich symmetrisch zur Selbsterneuerung. NE Zellen wandeln dann in radiale Gliazellen (RGZ). Zunächst RGZ symmetrisch teilen, um zwei neue RGZ produzieren, jedoch während der Großteil der Neurogenese, ist Haupt-Modus der Aufteilung RGZ &#39;asymmetrisch. Bei der asymmetrischen Teilung, gibt ein RGC zu einer neuen RGC und entweder mit einem post-mitotischen Neuronen oder eine spezialisierte Vorläufer (entweder eine kurze neurale Vorläufer (SNP), einem äußeren radialen Gliazellen (ORG) oder einem Zwischenvorläufer (INP) 2,3,7,9. INPS, SNPs und ORGs können dann erzeugen Neuronen an der Unter ventrikuläre, ventrikuläre und basalen Regionen des Kortex. Daher ist jeweils Zellteilung der Vorläuferzellen eine grundlegende Verfahren zur Erzeugung von Neuronen des Neocortex. Zahlreiche Studien weisen auf eine Korrelation zwischen der spezifischen Merkmale der mitotischen RGZ und das Schicksal der Tochterzellen. Haydar et al. Und Takahashi et al.haben gezeigt, dass die Dauer und RGC mitotischen Zellzyklus Anstieg der Neurogenese als Erlös, hallte eine Feststellung, in Folgestudien 10-13. Eine Reihe von Studien haben vorgeschlagen, dass mitotischen Spindel-Orientierung bezüglich des Ventrikels beeinflusst Aspekte der Neurogenese und Kortikogenese, einschließlich Arten von Neuronen erzeugt und Lage der Nachkommen im Gehirn, jeweils 3,10,14-16. Ob Spaltebene Orientierung Zelle Schicksal direkt beeinflusst, ist umstritten, aber die Schlussfolgerung bleibt, dass diese Parameter mitotische Auswirkungen der Neurogenese. Weitere unterstreicht die Bedeutung der Mitose ist die Beobachtung, dass viele Gene, die in der Mechanik der Mitose beteiligt sind entscheidend für die Neurogenese und für die richtige Entwicklung des Gehirns 17-20. Mitose ist ein dynamischer Prozess, aber bis heute die meisten Studien Detaillierung neuralen Vorläufer Mitose nutzen Analyse der Gewebeschnitten oder Bildgebung von neuralen Vorläuferzellen durch in vitro-Zellkultur. So sind die Mainstream-Methoden, um beurteilen zu können Mitose nur eine Momentaufnahme dieses Prozesses und nicht zu entdecken, wie Zellen verhalten sich in einem Gewebe. Live-Darstellung von neuralen Vorläufer Mitose ist zunehmend ein wichtiges Werkzeug für das Verständnis der neuralen Vorläuferfunktion geworden. Beispiele finden Sie diese Verweise 4,8,10,21-25. Mehrere hervorragende Protokolle für die Vorbereitung und Bildgebung von Hirnschnitten 26,27 erschienen. Doch bis heute hat ein umfassendes Protokoll für Abbildung und Analyse der Mitose nicht beschrieben worden, noch im Video demonstriert. Diese Technik bietet einige wesentliche Vorteile gegenüber Fest Analyse von Hirnschnitten. Zeitraffer-Analyse von Hirnschnitten ermöglicht die Erzeugung von deutlich mehr Datenpunkte, die in einer flexiblen Weise analysiert werden kann. Zuerst werden die Daten an den einzelnen Zeitpunkten im Verlauf von mehreren Minuten oder mehreren Stunden gesammelt. Man kann einzelne Zeitpunkte (eine statische Montage erstellen) oder analysierenkann zu verschiedenen Zeitpunkten in Filmen zu kombinieren. Zweite konfokale Bildgebung von Scheiben ermöglicht die Erzeugung von Daten mit unterschiedlichen Abschnitten in Z Hirnschnitten. Als Ergebnis können die einzelnen Abschnitte analysiert werden. Alternativ können Stapel von einzelnen Abschnitte in eine maximale Intensitätsprojektion kombiniert werden. Drittens wird die Analyse im Rahmen eines Gewebes durchgeführt, offenbart, wie Zellteilung relativ zu benachbarten Zellen und Strukturen. Viertens ist es ideal um die Analyse von Mutanten, die einige Hinweise von mitotischen Defekte zeigen geeignet. Gemeinsam dieses Protokoll wird zur Klärung kritischen Schritte, um Ermittler, die für die Durchführung von Live-Bildgebung neuronaler Vorläufer Mitose in den eigenen Labors wollen helfen.

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		<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

Obwohl von kurzer Dauer ist Mitose ein komplexes und dynamisches mehrstufiges Verfahren grundlegend für die Entwicklung der Organe, einschließlich des Gehirns. In der sich entwickelnden Großhirnrinde kann abnorme Mitose von neuronalen Vorläuferzellen Defekte in der Gehirngröße und Funktion führen. Daher besteht ein kritischer Bedarf an Werkzeugen, die Mechanismen der neuralen Vorläufer Mitose verstehen. Kortikale Entwicklung in Nagetieren ist eine herausragende Modell für die Untersuchung dieses Prozesses. Neurale Vorläufer Mitose wird häufig in festen Hirnschnitten untersucht. Dieses Protokoll wird im Detail beschreiben einen Ansatz für die Live-Darstellung der Mitose in ex vivo embryonalen Hirnschnitten. Wir werden die wichtigsten Schritte für dieses Verfahren zu beschreiben, welche sind: Gehirn-Extraktion, Gehirn Einbettung Vibratom Schnitte von Hirnschnitten, Färbung und Kultivierung von Scheiben und Zeitraffer-Bildgebung. Wir werden dann zeigen und beschreiben im Detail, wie der nach dem Erwerb der Mitose Analyse durchzuführen. Wir sind repräsentative Ergebnisse from dieser Test mit dem Vitalfarbstoff Syto11 transgene Mäuse (Histon H2B-EGFP und Centrin-EGFP) und in utero Elektroporation (mCherry-α-Tubulin). Wir werden diskutieren, wie dieses Verfahren am besten optimiert werden kann und wie sie für die Untersuchung der genetischen Regulation der Mitose geändert werden. Live-Imaging der Mitose in Hirnschnitten ist ein flexibler Ansatz zur Bewertung der Auswirkungen von Alter, Anatomie, und genetische Störung in einer kontrollierten Umgebung, und eine große Menge von Daten mit hoher zeitlicher und räumlicher Auflösung zu erzeugen. Daher dieses Protokoll wird die bestehenden Werkzeuge für die Analyse von neuronalen Vorläufer Mitose ergänzen.

Der Erfolg dieses Tests und die Beobachtung von mehreren mitotischen Zellen während einer Live-Imaging-Sitzung weitgehend abhängig sowohl von der Integrität und der anatomischen Ebene der Scheibe, wo Akquisitionen vorgenommen werden. Wie nachstehend erörtert, ist der anatomischen Ebene einer Scheibe ein wichtiger Faktor. 3A zeigt rostro-kaudal-und medial-lateral-Standorten, wo wir am erfolgreichsten. Weitere Diskussionen zu diesem Thema finden Sie in Noctor 26. Die Integrität der Scheibe...

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

Neurale Vorläufer Mitose ist ein kritischer Parameter der Neurogenese. Viel von unserem Verständnis der neuralen Vorläufer Mitose basiert auf der Analyse von festen Gewebe basiert. Live-Bildgebung in der embryonalen Hirnschnitten ist eine vielseitige Technik zur Mitose mit hoher zeitlicher und räumlicher Auflösung in einer kontrollierten Umgebung zu beurteilen.

Live-Imaging der Mitose in der Entwicklung von embryonalen Maus-Cortex

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

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.

Chronische Imaging of Mouse Visual Cortex Mit einem ausgedünnten Schädel Vorbereitung

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

Forschung

Neurowissenschaften

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.

Multiple-Maus Neuroanatomische 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 ...

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.

Zeitraffer-Live-Imaging von klonal verbundenen neuronalen Vorläuferzellen in der Entwicklung von Zebrafisch Vorderhirnneuronen

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.

Hochauflösende Live-Bildgebung von Zell-Verhalten in der Entwicklung von Neuroepithel

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.

Zwei-Photonen-<em&gt; In vivo</em&gt; Imaging von dendritischen Dornen in der Maus Cortex Mit einem ausgedünnten Schädel Vorbereitung

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.

Live-Imaging des Ependymale Cilia in der Seitenventrikel des Maus-Gehirn-

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.

Die gleichzeitige Zwei-Photonen<em&gt; In Vivo</em&gt; Imaging der synaptischen Eingänge und Postsynaptische Targets in der Maus retrosplenialen Cortex

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.

Bildgebung des primären Hirnrinde Zellen unter Verwendung einer 2D Kultursystem zu leben

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.

Ex Utero Elektroporation und organotypischen Slice Kulturen der embryonalen Mäusegehirnen für Live-Aufnahmen von Migration Gabaergen Interneuronen

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.

Nicht-invasive Modulation und Robotik-Mapping von Motor Cortex im sich entwickelnden Gehirn

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