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

我々 はまた、重要な読書のガブリエルの手を感謝します。

すべての実験は、大阪大学・日本神経科学学会動物愛護委員会によって確立されたガイドラインに従って行われました。
1. 脊髄共培養系視床と大脳皮質の
メモ: 手順については、オリジナルの出版物7,8,13を参照してください。すべてのプロシージャは、無菌条件の下で行わなけれ?...

<ol>
	<li>Kageyama, G. H., Robertson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+geniculocortical+projections+to+visual+cortex+in+rat:+evidence+early+ingrowth+and+synaptogenesis.">Development of geniculocortical projections to visual cortex in rat: evidence early ingrowth and synaptogenesis.</a> J. Comp. Neurol. 335 (1), 123-148 (1993).</li><li>Lopez-Bendito, G., Molnar, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thalamocortical+development:+how+are+we+going+to+get+there.">Thalamocortical development: how are we going to get there.</a> Nat. Rev. Neurosci. 4 (4), 276-289 (2003).</li><li>Espinosa, J. S., Stryker, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+plasticity+of+the+primary+visual+cortex.">Development and plasticity of the primary visual cortex.</a> Neuron. 75 (2), 230-249 (2012).</li><li>Portera-Cailliau, C., Weimer, R. M., De Paola, V., Caroni, P., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+modes+of+axon+elaboration+in+the+developing+neocortex.">Diverse modes of axon elaboration in the developing neocortex.</a> PLoS Biol. 3 (8), 272 (2005).</li><li>Holtmaat, A., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience-dependent+structural+synaptic+plasticity+in+the+mammalian+brain.">Experience-dependent structural synaptic plasticity in the mammalian brain.</a> Nat. Rev. Neurosci. 10 (9), 647-658 (2009).</li><li>Bhatt, D. H., Zhang, S., Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+spine+dynamics.">Dendritic spine dynamics.</a> Annu Rev Physiol. 71, 261-282 (2009).</li><li>Yamamoto, N., Kurotani, T., Toyama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+connections+between+the+lateral+geniculate+nucleus+and+visual+cortex+in+vitro.">Neural connections between the lateral geniculate nucleus and visual cortex in vitro.</a> Science. 245 (4914), 192-194 (1989).</li><li>Yamamoto, N., Yamada, K., Kurotani, T., Toyama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminar+specificity+of+extrinsic+cortical+connections+studied+in+coculture+preparations.">Laminar specificity of extrinsic cortical connections studied in coculture preparations.</a> Neuron. 9 (2), 217-228 (1992).</li><li>Uesaka, N., Hirai, S., Maruyama, T., Ruthazer, E. S., Yamamoto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+dependence+of+cortical+axon+branch+formation:+a+morphological+and+electrophysiological+study+using+organotypic+slice+cultures.">Activity dependence of cortical axon branch formation: a morphological and electrophysiological study using organotypic slice cultures.</a> J. Neurosci. 25 (1), 1-9 (2005).</li><li>Uesaka, N., Hayano, Y., Yamada, A., Yamamoto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+laminar+specificity+and+activity-dependent+mechanisms+of+thalamocortical+axon+branching.">Interplay between laminar specificity and activity-dependent mechanisms of thalamocortical axon branching.</a> J. Neurosci. 27 (19), 5215-5223 (2007).</li><li>Uesaka, N., Nishiwaki, M., Yamamoto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+electroporation+method+for+axon+tracing+in+cultured+slices.">Single cell electroporation method for axon tracing in cultured slices.</a> Dev. Growth Differ. 50 (6), 475-477 (2008).</li><li>Matsumoto, N., Hoshiko, M., Sugo, N., Fukazawa, Y., Yamamoto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapse-dependent+and+independent+mechanisms+of+thalamocortical+axon+branching+are+regulated+by+neuronal+activity.">Synapse-dependent and independent mechanisms of thalamocortical axon branching are regulated by neuronal activity.</a> Dev Neurobiol. 76 (3), 323-336 (2016).</li><li>Matsumoto, N., Sasaki, K., Yamamoto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+Method+for+Mammalian+CNS+Neurons+in+Organotypic+Slice+Cultures.">Electroporation Method for Mammalian CNS Neurons in Organotypic Slice Cultures.</a> Electroporation Methods in Neuroscience. , 159-168 (2015).</li><li>Molnar, Z., Blakemore, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+regional+specificity+for+connections+formed+between+thalamus+and+cortex+in+coculture.">Lack of regional specificity for connections formed between thalamus and cortex in coculture.</a> Nature. 351 (6326), 475-477 (1991).</li><li>Bolz, J., Novak, N., Staiger, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+specific+afferent+connections+in+organotypic+slice+cultures+from+rat+visual+cortex+cocultured+with+lateral+geniculate+nucleus.">Formation of specific afferent connections in organotypic slice cultures from rat visual cortex cocultured with lateral geniculate nucleus.</a> J. Neurosci. 12 (8), 3054-3070 (1992).</li><li>Yamamoto, 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=Inhibitory+mechanism+by+polysialic+acid+for+lamina-specific+branch+formation+of+thalamocortical+axons.">Inhibitory mechanism by polysialic acid for lamina-specific branch formation of thalamocortical axons.</a> J. Neurosci. 20 (24), 9145-9151 (2000).</li><li>Yamamoto, 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=Characterization+of+factors+regulating+lamina-specific+growth+of+thalamocortical+axons.">Characterization of factors regulating lamina-specific growth of thalamocortical axons.</a> J Neurobiol. 42 (1), 56-68 (2000).</li><li>Ohnami, S., 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=Role+of+RhoA+in+activity-dependent+cortical+axon+branching.">Role of RhoA in activity-dependent cortical axon branching.</a> J. Neurosci. 28 (37), 9117-9121 (2008).</li><li>Yamada, A., 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=Role+of+pre-+and+postsynaptic+activity+in+thalamocortical+axon+branching.">Role of pre- and postsynaptic activity in thalamocortical axon branching.</a> Proc Natl Acad Sci U S A. 107 (16), 7562-7567 (2010).</li></ol>

現在のプロトコルはまた TC 投影11以外の軸索の成長の発達的側面を研究する強力なツールです。例えば、皮質スライス培養とエレクトロポレーション技術の組み合わせは、皮質ニューロンと長期観測9,18の個々 の軸索形態を視覚化することができます。
現在のプロトコルを使用しては、蛍光タンパク質および?...

哺乳類の脳 (TC) 視床皮質投射は、軸索ガイダンスとターゲットのメカニズムを検討する適切なシステムです。開発時に TC の感覚軸索は皮質骨板やフォーム枝や大脳皮質1,2の一次感覚野のシナプスで優先的に層 IV で育ちます。基本的な接続の確立後も環境の変化3,4によって軸索のアーバーおよびシナプス終末が改造されています。ただし、TC 軸索形態を動的に変更する方法がよくわかっていません。主な理由の 1 つは、単一細胞レベルでの構造変化を観察する適切な方法がないです。2 光子顕微鏡などの顕微鏡の最近の進歩は、生活皮質ニューロンの生体内での直接観測を許可している、全体的な TC の軌跡5,をキャプチャするためまだ技術的な限界があります。6。 したがって、体外TC 軸索のライブ イメージング法は、軸索分岐およびシナプス形成の構造解析のための強力なツールを提供します。
初めて私たちのグループは、透過膜7静的スライス培養法を設立しました。このメソッドを使用すると、ラット脳スライスされた感覚視床ブロックと遊走し、ラミナ特異 TC 接続されたこの脊髄共培養系7,8で締めくくっています。蛍光タンパク質を持つスパース ラベリングさらに TC の軸索の成長や枝の形成9,10,11を観察するができました。最近では、我々 は分岐の同時可視化手法を開発しているし、切片における個々 の TC の軸索のシナプス形成 cocultures12。TC 軸索およびシナプス前のサイトを同時に視覚化するには、下流と EGFP タグ シナプトフィジン (SYP EGFP) は切片培養電気穿孔法による視床のニューロンの数が少ないに cotransfected でした。現在のメソッドは TC 軸索の形態素解析を容易に、軸索分岐およびシナプス形成の因果関係を示すため使用できます長期観察できます。

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		<col style="width:89pt" width="119" />
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			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">DMEM/F12</td>
			<td class="xl20" style="width:89pt" width="119">GIBCO</td>
			<td class="xl20" style="width:84pt" width="112">11320-033</td>
			<td class="xl20" style="width:58pt" width="77"></td>
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		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Hanks&#8217; balanced salt solution (HBSS)</td>
			<td class="xl20" style="width:89pt" width="119">Nissui</td>
			<td class="xl21" style="width:84pt" width="112">5905</td>
			<td class="xl20" style="width:58pt" width="77"></td>
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		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Fetal bovine serum (FBS)</td>
			<td class="xl20" style="width:89pt" width="119">Thermo Scientific</td>
			<td class="xl20" style="width:84pt" width="112">SH30396-03</td>
			<td class="xl20" style="width:58pt" width="77">Hyclone</td>
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		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Insulin</td>
			<td class="xl20" style="width:89pt" width="119">Sigma</td>
			<td class="xl20" style="width:84pt" width="112">I6634</td>
			<td class="xl20" style="width:58pt" width="77"></td>
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		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Progesterone</td>
			<td class="xl20" style="width:89pt" width="119">Sigma</td>
			<td class="xl20" style="width:84pt" width="112">P8783</td>
			<td class="xl20" style="width:58pt" width="77"></td>
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		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Hydrocortisone</td>
			<td class="xl20" style="width:89pt" width="119">Sigma&#160;</td>
			<td class="xl20" style="width:84pt" width="112">H0888</td>
			<td class="xl20" style="width:58pt" width="77"></td>
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		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Sodium selenite</td>
			<td class="xl20" style="width:89pt" width="119">Wako Pure 
			Chemical Industries</td>
			<td class="xl20" style="width:84pt" width="112">192-10843</td>
			<td class="xl20" style="width:58pt" width="77"></td>
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		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Transferrin&#160;</td>
			<td class="xl20" style="width:89pt" width="119">Sigma</td>
			<td class="xl20" style="width:84pt" width="112">T1147</td>
			<td class="xl20" style="width:58pt" width="77"></td>
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		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Putrescine&#160;</td>
			<td class="xl20" style="width:89pt" width="119">Sigma</td>
			<td class="xl20" style="width:84pt" width="112">P5780</td>
			<td class="xl20" style="width:58pt" width="77"></td>
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		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Glucose</td>
			<td class="xl20" style="width:89pt" width="119">Wako 
			Pure Chemical Industries</td>
			<td class="xl20" style="width:84pt" width="112">16806-25</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">35 mm petri dishes</td>
			<td class="xl20" style="width:89pt" width="119">Falcon</td>
			<td class="xl21" style="width:84pt" width="112">351008</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">Millicell-CM insert</td>
			<td class="xl20" style="width:89pt" width="119">Millipore</td>
			<td class="xl20" style="width:84pt" width="112">PICMORG50</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">100 mm petri dishes</td>
			<td class="xl20" style="width:89pt" width="119">BIO-BIK</td>
			<td class="xl20" style="width:84pt" width="112">I-90-20</td>
			<td class="xl20" style="width:58pt" width="77">petri dish sterrile</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">HiPure Plasmid Maxiprep Kit</td>
			<td class="xl20" style="width:89pt" width="119">Invitrogen</td>
			<td class="xl20" style="width:84pt" width="112">K210006</td>
			<td class="xl23" style="width:58pt" width="77"></td>
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		<tr height="59" style="height:44.25pt">
			<td class="xl20" height="59" style="width:113pt;height:44.25pt" width="151">Disposable sterile plastic pipettes</td>
			<td class="xl20" style="width:89pt" width="119"></td>
			<td class="xl20" style="width:84pt" width="112">202-IS</td>
			<td class="xl20" style="width:58pt" width="77">transfer pipets sterile</td>
		</tr>
		<tr height="54" style="height:40.5pt">
			<td class="xl20" height="54" style="width:113pt;height:40.5pt" width="151">Glass capillary: OD 1.2 mm</td>
			<td class="xl20" style="width:89pt" width="119">Narishige</td>
			<td class="xl20" style="width:84pt" width="112">&#160;G-1.2</td>
			<td class="xl20" style="width:58pt" width="77">inner diameter, 1.2 mm</td>
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		<tr height="53" style="height:39.75pt">
			<td class="xl20" height="53" style="width:113pt;height:39.75pt" width="151">Silver wire: 0.2 and 1 mm&#160;</td>
			<td class="xl20" style="width:89pt" width="119">Nilaco</td>
			<td class="xl20" style="width:84pt" width="112">AG-401265 (diameter, 0.2 mm), AG-401485 (diameter, 1.0 mm)</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">1 mL syringe</td>
			<td class="xl20" style="width:89pt" width="119">Terumo</td>
			<td class="xl20" style="width:84pt" width="112">SS-01T</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">Stimulator&#160;</td>
			<td class="xl20" style="width:89pt" width="119">A.M.P.I</td>
			<td class="xl20" style="width:84pt" width="112">Master 8</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">Biphasic isolator&#160;</td>
			<td class="xl20" style="width:89pt" width="119">BAK ELECTRONICS</td>
			<td class="xl20" style="width:84pt" width="112">BSI-2</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">Amplifier&#160;</td>
			<td class="xl20" style="width:89pt" width="119">A-M Systems</td>
			<td class="xl20" style="width:84pt" width="112">Model 1800</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">Oscilloscope</td>
			<td class="xl20" style="width:89pt" width="119">Hitachi</td>
			<td class="xl20" style="width:84pt" width="112">VC-6723</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">Manipulator</td>
			<td class="xl20" style="width:89pt" width="119">Narishige</td>
			<td class="xl20" style="width:84pt" width="112">SM-15</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">Micromanipulator</td>
			<td class="xl20" style="width:89pt" width="119">Narishige</td>
			<td class="xl20" style="width:84pt" width="112">MO-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">Stereomicroscope&#160;</td>
			<td class="xl20" style="width:89pt" width="119">Olympus</td>
			<td class="xl20" style="width:84pt" width="112">SZ40</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">Universal stand&#160;</td>
			<td class="xl20" style="width:89pt" width="119">Olympus</td>
			<td class="xl20" style="width:84pt" width="112">SZ-STU2</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">Light illumination system&#160;</td>
			<td class="xl20" style="width:89pt" width="119">Olympus</td>
			<td class="xl20" style="width:84pt" width="112">LG-PS2, LG-DI, HLL301</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">Electrode puller&#160;</td>
			<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

軸索分岐とシナプスの形成は、精密な神経回路を確立するための重要なプロセスです。開発中には、感覚視床皮質 (TC) 軸索は大脳皮質の特定のレイヤーで枝とシナプスを形成します。にもかかわらず、軸索分岐とシナプス形成間明白な相関、因果関係はよくわかっていません。この問題に対処するため、我々 は最近脊髄共培養系における軸索 TC 個々 の分岐とシナプス形成の同時イメージング法を開発しました。
このプロトコルでは、切片培養とエレクトロポレーションの組み合わせから成っている方法について説明します。層構成などの特徴的な構造を維持する軸索のプロセスの遺伝子操作と観測の視床と大脳皮質脊髄共培養系を促進します。エンコードした DsRed と EGFP タグ シナプトフィジン (SYP EGFP) 2 つの明瞭なプラスミドはエレクトロポレーション法による視床のニューロンの数が少ないに cotransfected だったこのメソッドでは、個々 の軸索組織の TC ニューロンとシナプス前サイトを同時に可視化することができました。メソッドには、軸索分岐およびシナプス形成の因果関係を明らかにした長期的な観測も有効になります。

ここで TC 軸索分岐とシナプス形成との関係を明らかにすることを目的に記載されているの実験。同時に脊髄共培養系における少数の視床細胞やシナプス前のサイトは、単一の場所軸索の軌跡を視覚化するには、SYP EGFP とした DsRed エレクトロポレーションを使用して 2 つのプラスミドを導入させた。文化の第 2 週の間に軸索 TC 個別に区別はっきりした DsRed (<strong class=...

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

このプロトコルでは、視床皮質軸索分岐およびシナプス形成の脊髄共培養系における視床と大脳皮質の同時イメージング法について説明します。個々 の視床皮質軸索とその神経終末は、下流と GFP タグ シナプトフィジン単細胞エレクトロポレーション法による可視化します。

視床皮質軸索分岐とシナプスの可視化脊髄共培養系における形成

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

Automated Interactive Video Playback for Studies of Animal Communication

アニマルコミュニケーションの研究のための自動化されたインタラクティブなビデオ再生

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Visualization of Proprioceptors in Drosophila Larvae and Pupae

でProprioceptorsの可視化<em&gt;ショウジョウバエ</em&gt;幼虫および蛹

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 ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

器官型海馬スライスにおけるペア全細胞記録

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

共培養モデルの組み込みGABA作動性中型有棘ニューロンおよびHEK293細胞における抑制性シナプスの形成を安定的にGABAを表現<sub&gt; A</sub&gt;受容体

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

マウスの胚感覚軸索投影のホールマウントイメージング

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

軸索分岐と神経形態形成中に形質膜配信の役割を定義するために組み合わせる方法論を活用

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

嗅覚神経軸索分子識別コードの視覚化のための嗅球の四重免疫染色

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

トレースのラットにおける視床皮質投射のビオチン標識デキストランの高分子量アミンの運用の改善

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

海馬の齧歯動物の脳スライスにおけるシナプス密度の評価

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

可視化とアデノ随伴ウイルスと共焦点顕微鏡を用いた脊髄脳スライスにおけるオリゴデンドロ サイト オルガネラのライブ イメージング

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