Name: visualization of thalamocortical axon branching and synapse formation in organotypic cocultures
Uploaded: 2018-03-28T12:30:00
Description: Watch this Scientific Journal Video about Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures at JoVE.com

Ayrıca Gabriel Hand eleştirel okuma için teşekkür ediyoruz.

Osaka Üniversitesi ve Japonya Nörobilim Derneği hayvan refahı komiteleri tarafından kurulan esaslarına göre tüm deneyler yapıldı.
1. Organotypic cocultures, talamus ve serebral korteks
Not: özgün yayınlar7,8,13ile ilgili ayrıntılı yordamlar için bakın. Tüm yordamları steril koşullarda gerçekleştirilmesi gerekiyor. Sprague-Dawley (SD) fareler nöronal k?...

<ol>
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Geçerli protokol de TC projeksiyon11dışında aksonlar büyüyen gelişim yönü eğitim için güçlü bir araçtır. Örneğin, kortikal dilim kültür ve elektroporasyon tekniği bir arada bireysel aksonal Morfoloji kortikal nöronlar ve uzun vadeli gözlem9,18görselleştirme sağlar.
Geçerli iletişim kuralını kullanarak, ilginç genler axon dallanma ve sinaps oluşumunda rol de floresan proteinler ve ...

Thalamocortical (TC) projeksiyon memeli beyinde axon rehberlik ve mekanizmaları hedefleme araştırmak için uygun bir sistemdir. Geliştirme sırasında duyusal TC aksonlar kortikal plaka ve formu şube ve sinapslarda tercihen içinde katman IV serebral korteks1,2birincil duyusal alanda büyümek. Temel bağlantılar kurulması sonra bile, aksonlar arbors ve sinaptik terminalleri çevresel değişiklikler3,4bağlı olarak yenilenmiş. Ancak, nasıl TC axon Morfoloji dinamik olarak değiştirilmez kötü anlaşılmaktadır. Bir tek hücre düzeyinde yapısal değişiklikleri gözlemlemek için yeterli bir teknik eksikliği ana nedenlerinden biri. Mikroskobu, İki fotonlu mikroskobu gibi son gelişmeler yaşayan kortikal nöronlar vivo içindedoğrudan gözlem izin vermiş, ancak genel TC yörüngeleri5, yakalamak için hala teknik sınırlamalar vardır 6. bu nedenle, vitro yöntemleri TC aksonlar canlı görüntüleme için axon dallanma ve synapse oluşumu yapısal analiz için güçlü araçlar sağlamak.
Bizim grup ilk kez bir statik dilim kültür yöntemi geçirgen membran7ile kurulmuş. Bu yöntemi kullanarak, bir sıçan kortikal dilim bir duyusal talamik blok ile cocultured ve lamina özgü TC bağlantıları bu organotypic cocultures7,8' recapitulated. Seyrek bir floresan protein ile etiketleme daha fazla TC axon büyüme ve dal oluşumu9,10,11gözlemlemek için bize izin. Son zamanlarda, dallanma, aynı anda görüntüleme için yeni bir yöntem geliştirdik ve bireysel TC aksonlar organotypic sinaps oluşumu12cocultures. TC akson ve presynaptic siteleri aynı anda görmek için DsRed ve EGFP etiketli synaptophysin (SYP-EGFP) talamik nöronların az sayıda organotypic coculture elektroporasyon tarafından ortak transfected. Geçerli yöntem TC aksonlar morfolojik analizi kolaylaştırır ve akson dallanma ve synapse oluşumu arasındaki nedensel ilişkiyi göstermek için kullanılan uzun vadeli gözlem için sağlar.

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			<td class="xl20" style="width:89pt" width="119">Narishige</td>
			<td class="xl20" style="width:84pt" width="112">PC-10</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Confocal microscope</td>
			<td class="xl20" style="width:89pt" width="119">Nikon</td>
			<td class="xl20" style="width:84pt" width="112">Digital eclipse C1 laser</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">x20 objective</td>
			<td class="xl20" style="width:89pt" width="119">Nikon</td>
			<td class="xl20" style="width:84pt" width="112">ELWD 20x/0.45</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Culture chamber</td>
			<td class="xl20" style="width:89pt" width="119">Tokai Hit</td>
			<td class="xl20" style="width:84pt" width="112">UK A16-U</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Sprague-Dawley (SD) rat</td>
			<td class="xl20" style="width:89pt" width="119">Japan SLC and Nihon-Dobutsu</td>
			<td class="xl20" style="width:84pt" width="112"></td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Microsurgery scissors</td>
			<td class="xl20" style="width:89pt" width="119">Natsume&#160;</td>
			<td class="xl20" style="width:84pt" width="112">MB-54-1</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
	</tbody>
</table>

visualization of thalamocortical axon branching and synapse formation in organotypic cocultures

Axon dallanma ve synapse oluşumu kesin nöronal devreler kurmak için çok önemli işlemler vardır. Geliştirme sırasında duyusal thalamocortical (TC) aksonlar serebral korteksin belirli katmanlar halinde şube ve sinapslarda oluştururlar. Axon dallanma ve synapse oluşumu arasında bariz uzamsal ilişki rağmen aralarındaki nedensel ilişki kötü anlaşılmaktadır. Bu sorunu gidermek için son zamanlarda bireysel TC aksonlar organotypic cocultures içinde dallanma ve synapse oluşumu aynı anda görüntüleme için bir yöntem geliştirdi.
Bu iletişim kuralı bir organotypic coculture ve elektroporasyon birleşimini içerir bir yöntemini açıklar. Organotypic cocultures, talamus ve serebral korteks gen manipülasyon ve gözlem Laminer yapılandırması gibi karakteristik yapıları koruyarak aksonal süreçlerin kolaylaştırmak. DsRed ve EGFP etiketli synaptophysin (SYP-EGFP) kodlama iki ayrı plazmid talamik nöronların az sayıda bir adım tekniği ile birlikte transfected. Bu yöntem TC nöronların bireysel aksonal türleri morfoloji ve presynaptic siteleri aynı anda görselleştirmek izin verdi. Yöntemi Ayrıca axon dallanma ve synapse oluşumu arasındaki nedensel ilişkiyi ortaya uzun vadeli gözlem etkin.

Deneme amaçları TC axon dallanma ve synapse oluşumu arasındaki ilişkiyi ortaya çıkarmak için burada açıklanan. Aynı anda aksonal yörüngeler ve yerleri presynaptic siteleri, tek veya organotypic cocultures bir kaç talamik hücrelerde görselleştirmek için kodlama SYP-EGFP ve DsRed elektroporasyon kullanarak iki Plasmid'ler ile transfected. Kültür ikinci haftasında tek tek ayırt TC aksonlar açıkça DsRed (şekil 3) tarafından etiketli. Sa...

Watch this Scientific Journal Video about Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures at JoVE.com

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

Bu iletişim kuralı thalamocortical akson dallanma ve synapse oluşumu organotypic cocultures, talamus ve serebral korteks eşzamanlı görüntüleme için bir yöntemi açıklar. Bireysel thalamocortical akson ve presynaptic onların terminalleri bir tek hücre çoğalmasıyla tekniği ile DsRed ve synaptophysin GFP öğesini tarafından görüntülenmiştir.

Görselleştirme Thalamocortical Axon dallanma ve sinaps oluşumunda Organotypic Cocultures

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) ...

The overall goal of this method is to study dynamic morphological changes of axon branching and synapse formation in the thalamocortical projection. This method can help answer key questions in the field of neurobiology, such as new neural circuit formation. The main advantage of this technique is simultaneous observation of axon growth and the synapse formation in individual living cells.This method can provide insight into the relationship between axon branching and synapse formation in the thalamocortical projections. It can also be applied to other neural circuits, such as intrinsic connection in the cerebral cortex. To begin this procedure, sterilize all the surgical instruments with 70%ethanol for 10 minutes or longer.Next, dissect the whole brain quickly from a postnatal day-two rat under ice-cold anesthesia. Then, place the brain into a 100-millimeter petri dish containing 40 milliliters of cold Hank's Balanced Salt Solution. Under a binocular microscope, remove the pia matter carefully with fine forceps, and cut the primary visual and somatosensory cortex into 300 to 400-micron thick slices using microsurgical scissors.After that, transfer the cortical slices onto the membrane filter of the culture insert using a disposable plastic pipette. Adjust the positions of the slices near the center of the culture insert with the disposable plastic pipette, and remove excess medium from the insert. Maintain the slices in the serum-containing culture medium at 37 degrees Celsius, and culture them alone for one day before placing a thalamic block from an E15 embryo next to it.Adjust the level of the medium slightly below the surface of the slice, so that the slices can receive sufficient supply of both the gas and the medium. This is crucial for the viability of the slices. In this step, make the glass capillaries with an electrode puller.Then break the tips of the glass capillaries for plasmid injection. For the electrode pipettes, grind and polish the tips of the glass capillaries with sandpaper. And then fire polish them to achieve an inner diameter of 50 to 200 microns.Next, attach the electrode pipette and the ejection pipette to the manipulators. Transfer five microliters of plasmid solution to a petri dish. Then draw the solution into the electrode pipette with capillary action.Insert the 0.2-millimeter diameter electrode into the electrode pipette. After connecting the ejection pipette to a one-milliliter syringe with a plastic tube, transfer five microliters of plasmid solution to a petri dish again. Then slowly draw the solution into the ejection pipette using the syringe.Following that, transfer the coculture on the electroporation stage and insert the one-millimeter diameter electrode into the culture medium. Subsequently, place the ejection pipette on the cultured thalamic block. Push the syringe manually after the tip touches the surface of the thalamic block.Place the electrode pipette on the thalamic block immediately after the retraction of the ejection pipette. Now, deliver the electrical pulses. Monitor the amplitude and the number of trains on the oscilloscope.Afterward, retract the electrode pipette and return the dish to the incubator. To observe the sample, place the culture dish on the stage of an upright confocal microscope equipped with two filter sets for EGFP and DsRed. Then take images of 10 to 25 optical sections, at one to seven-micron step sizes, with a 20x long working distance objective lens.Select individually-distinguishable labeled axons, which express both DsRed and Syp-EGFP. Shown here is an organotypic coculture of thalamic and cortical slices after 12 DIV. Here is a magnified image of the boxed region.And here is a DsRed-labeled thalamic cell extending an axon into the cortical slice, and forming branches in upper layers. These images show the DsRed-labeled thalamocortical axon and Syp-EGFP puncta. Discrete accumulation of Syp-EGFP was observed along a single thalamocortical axon.DsRed plus Syp-EGFP plasmids were introduced into cultured thalamic cells at one DIV using electroporation. This axon was imaged daily over four days. Axon branching and synapse formation were dynamic, with addition and elimination.Following these procedures, electroporation with interesting genes together with Syp-EGFP and DsRed can be performed in order to investigate the role of the gene in axon branching and synapse formations. This technique paves the way for researchers in the field of the neurobiology to explore the mechanisms of dynamic changes of neural circuits.

Research

JoVE Journal

Neuroscience

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Içinde proprioseptörlere Görselleştirmesi<em&gt; Drosophila</em&gt; Larva ve Pupa

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Nörobilim

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Organotipik Hipokampal Dilimleri eşleştirilmiş Tüm Hücre Kayıtlar

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

Co-kültür Model Birleşmeyle GABA&#39;erjik Orta Spiny Nöronlar ve HEK293 Hücreler inhibitör Synapse Oluşumu istikrarlı bir şekilde GABA ifade<sub&gt; A</sub&gt; Reseptörler

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

Fare Embriyo Duyu Akson Projeksiyonlar tüm montaj Görüntüleme

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

Kombine Metodolojileri kullanan Akson Dallanman ve Nöronal Morfogenez sırasında Plazma Membran Teslimat Rolü tanımlayın

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

Koku Meşfatının Olfaktör Duyusal Akson Moleküler Kimlik Kodlarının Görselleştirilmesi İçin Dörtlü İmmün Sulanması

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...

Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat

Yüksek molekül ağırlıklı Biotinylated Dextran Amin uygulama içinde sıçan izleme Thalamocortical projeksiyon için geliştirmek

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

Hipokampal kemirgen beyin dilimleri dansitesi Synapse değerlendirilmesi

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy

Görselleştirme ve Oligodendrocyte organelleri Adeno ilişkili virüs ve Confocal mikroskobu kullanılarak Organotypic beyin dilimleri içinde canlı görüntüleme

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced ...