eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Dissection of Chicken Embryo Day 14 (e14) Cerebellum
<ol>
	<li>Incubate brown fertilized hens&#39; eggs at 38 &#176;C to embryonic Day 14.</li>
	<li>Using egg scissors, decapitate the chick embryo in ovo and remove the head to a Petri dish containing ice-cold Phosphate buffer saline (PBS) (Figure 1A).</li>
	<li>Using standard forceps, make incisions behind each eye all the way through the tissue, removing the eyes and upper jaw. Make a second incision all the way through the pharynx, removing the lower jaw (Figure 1B).</li>
	<li>Using standard forceps, remove all skin from the surface of a skull by peeling it away (Figure 1C) and remove frontal and parietal bones, revealing the brain.</li>
	<li>From a ventral aspect, remove pharyngeal cartilage and auxiliary mesenchyme.</li>
	<li>From a dorsal aspect, carefully remove the mesenchyme dorsal to the hindbrain, taking care not to damage pia, and separate the entire brain (Figure 1D).</li>
	<li>Make an incision all the way through the tissue between the midbrain and hindbrain and separate the hindbrain, including the cerebellum (Figure 1E).</li>
	<li>Remove the entire cerebellum by making incisions all the way through the tissue at both lateral junctions of the cerebellum and alar plate of the hindbrain, taking care to maintain the integrity of the pia throughout dissection (Figure 1F). Also, remove the forming choroid plexus (Figure 1G).</li>
	<li>Move the dissected cerebellum into ice-cold Hank&#39;s balanced salt solution (HBSS).</li>
</ol>
2. Slice Culture of e14 Cerebellum
<ol>
	<li>Transfer the whole cerebellum to the sterile platform of a Tissue Chopper using a spatula or a 3 ml Pasteur pipette with the tip cut away to widen the aperture. Remove excess liquid using a pipette.</li>
	<li>Cut entire whole cerebellum in the required orientation at 300 &#181;m thickness using the tissue chopper set with a cutting speed at 50% of the maximum. Tissue integrity is most easily preserved in sagittal section; however, orientation is also an important consideration for cell analysis (Purkinje cell dendrites are sagittally aligned, while granule cell axons run transversely, perpendicular to the plane of Purkinje cell dendrites).</li>
	<li>Using a 3 ml Pasteur pipette, cover the sliced cerebellum in ice-cold HBSS.</li>
	<li>Transfer the slices in HBSS to a 60 mm Petri dish containing ice-cold fresh HBSS using a 3 ml Pasteur pipette with a cut tip.</li>
	<li>Under a dissecting microscope illuminated with a fiber optic light source, ensure separation of individual slices using watchmaker forceps. 
	Note: Each dissection, from embryo to slice incubation in culture medium, takes around 10-20 min, depending upon experience. Perform dissections one at a time to ensure that the cerebella spends the minimum amount of time between decapitation and ex vivo culture.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>McIlwain tissue chopper</td>
			<td>Mickle Laboratory Engineering Ltd</td>
			<td></td>
			<td>Cut at 300&#956;m for best results.</td>
		</tr>
	</tbody>
</table>

obtaining cerebellum slices from an embryonic chick brain

Source: Hanzel, M., et al. <a href="https://app.jove.com/v/53421">Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors.</a> J. Vis. Exp. (2015).
The video demonstrates the preparation of cerebellum slices. It shows a detailed procedure for isolating the cerebellum from a chick embryo. The slices are then prepared from the dissected cerebellum using a tissue chopper for further experimental analysis.

<img alt="Figure 1" src="/files/ftp_upload/22616/22616_figure 1.jpg" /> 
Figure 1. Dissection of the cerebellum from E14 chick embryos. (A) Decapitate the chick in the egg and remove the head into a petri dish with ice-cold PBS. Remove the lower jaw and the eyes by making an incision behind the eyes and the pharynx (dashed line). (B) Remove the skin from the surface of the skull. (C) Remove the frontal and parietal bones, and (D) remove the brain from the mesenchyme ...

Obtaining Cerebellum Slices from an Embryonic Chick Brain

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Take a fertilized hen&#39;s egg at a suitable stage of development.
Cut the shell to access the embryo.
Locate and dissect out the head, then, transfer it to a Petri dish containing chilled phosphate buffer.
Remove the eyes and jaws.
Separate the skin from the surface of the skull.
Next, cut and remove the skull bones.
Clear the surrounding connective tissue to expose the brain.
Separate the forebrain.
Then, detach the cerebellum from the hindbrain and the blood vessel.
Transfer the cerebellum into a chilled salt buffer.
The embryonic cerebellum contains a layer of rapidly dividing neuronal progenitor cells, which makes it valuable for neurogenesis studies.
Place the cerebellum on the platform of a tissue chopper.
Remove excess buffer and slice the cerebellum.
Put chilled salt buffer on the slices.
Transfer the cerebellum slices to a Petri dish containing chilled salt buffer.
Under a microscope, separate individual slices for further analysis.

obtaining-cerebellum-slices-from-an-embryonic-chick-brain

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

35 Görüntüleme. Kaynak: Hanzel, M., et al. Civciv Serebellar Dilimlerinin Ex Vivo Kültürü ve Granül Hücre Öncülerinin Uzamsal Olarak Hedeflenmiş Elektroporasyonu. J. Vis. Uzm. (2015). Video, beyincik dilimlerinin hazırlanışını göstermektedir. Beyinciğin bir civciv embriyosundan izole edilmesi için ayrıntılı bir prosedür gösterir. Dilimler daha sonra daha fazla deneysel analiz için bir doku kıyıcı kullanılarak diseke edilmiş beyincikten hazırlanır. 

Video: Embriyonik Civciv Beyninden Beyincik Dilimleri Elde Edilmesi - Kavram

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

Faringeal Arches Ex Vivo Kültür Kalp ve kas atalarıdır ve Bunların Niche Eğitim için

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

JoVE Journal

Gelişim Biyolojisi

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

Embriyonik Fare Beyincisinden İzole Edilen Nöronların Birincil Kültürü

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...

Nörobilim

A Flexible Platform for Monitoring Cerebellum-Dependent Sensory Associative Learning

Climbing fiber inputs to Purkinje cells provide instructive signals critical for cerebellum-dependent associative learning. Studying these signals in head-fixed mice facilitates the use of imaging, electrophysiological, and optogenetic methods. Here, a low-cost behavioral platform (~$1000) was developed that allows tracking of associative learning in head-fixed mice that locomote freely on a running wheel. The platform incorporates two common associative learning paradigms: eyeblink conditioning and delayed tactile startle conditioning. Behavior is tracked using a camera and the wheel movement by a detector. We describe the components and setup and provide a detailed protocol for training and data analysis. This platform allows the incorporation of optogenetic stimulation and fluorescence imaging. The design allows a single host computer to control multiple platforms for training multiple animals simultaneously.

We have developed a single platform to track animal behavior during two climbing fiber-dependent associative learning tasks. The low-cost design allows integration with optogenetic or imaging experiments directed towards climbing fiber-associated cerebellar activity.

Beyinciğe Bağımlı Duyusal İlişkisel Öğrenmeyi İzlemek için Esnek Bir Platform

Climbing fiber inputs to Purkinje cells provide instructive signals critical for cerebellum-dependent associative learning. Studying these signals in ...

Obtaining Cerebellum Slices from an Embryonic Chick Brain

TRANSKRIPT

Obtaining Cerebellum Slices from an Embryonic Chick Brain