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

Wir danken auch Gabriel Hand für kritisches Lesen.

Alle Versuche wurden nach den Richtlinien festgelegten Tierschutz Ausschüsse von Universität von Osaka und Japan Neurowissenschaftliche Gesellschaft durchgeführt.
(1) organotypischen Kokulturen des Thalamus und Großhirnrinde
Hinweis: Die detaillierte Vorgehensweise finden Sie in der ursprünglichen Publikationen7,8,13. Alle Verfahren sollte unter sterilen Bedingungen durc...

<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>

Das aktuelle Protokoll ist auch ein leistungsfähiges Werkzeug, Entwicklungsaspekte der wachsenden Axone anders als der TC Projektion11zu studieren. Zum Beispiel ermöglicht eine Kombination der kortikalen Stück Kultur und die Elektroporation Technik Visualisierung einzelner axonalen Morphologie der kortikalen Neuronen und langfristige Beobachtung9,18.
Mithilfe des aktuellen Protokolls können die Rollen von in...

Die Thalamocortical (TC) Projektion in das Gehirn von Säugetieren ist ein geeignetes System, Axon Guidance und targeting Mechanismen zu untersuchen. Während der Entwicklung wachsen sensorische TC Axone in die kortikale Platte und Form Zweige und Synapsen bevorzugt in Schicht IV der primären sensorischen Bereiche in der Großhirnrinde1,2. Auch nach Gründung der grundlegenden Verbindungen sind abhängig von Veränderungen der Umwelt3,4axonalen Lauben und synaptischen Terminals umgebaut. Jedoch wie TC Axon Morphologie dynamisch geändert ist schlecht verstanden. Einer der Hauptgründe ist das Fehlen einer angemessenen Technik, strukturelle Veränderungen auf eine einzelne Zellenebene zu beobachten. Obwohl jüngste Entwicklungen in der Mikroskopie, wie zwei-Photonen-Mikroskopie, direkten Beobachtung von lebenden kortikale Neuronen in Vivoerlaubt haben, gibt es noch technische Einschränkungen für die Erfassung der gesamten TC Trajektorien5, 6. daher, in-vitro- Methoden für live Aufnahmen von TC Axone böte leistungsstarke Tools für Strukturanalysen der Axon verzweigen und Synapse Formation.
Unsere Gruppe zum ersten Mal gegründet eine statische Slice-Kultur-Methode mit durchlässige Membran7. Mit dieser Methode eine Ratte kortikalen Scheibe war mit einem sensorischen Thalamische Block cocultured und Lamina-spezifische TC Verbindungen wurden in diesem organotypischen Kokulturen7,8zusammengefaßt. Spärliche Beschriftung mit einem fluoreszierenden Protein weiter konnten wir beobachten, TC Axon Wachstum und Zweig Bildung9,10,11. Vor kurzem haben wir eine neuartige Methode zur gleichzeitigen Darstellung von Verzweigungen und Synapse Bildung von individuellen TC Axone in die organotypischen cocultures12. Um TC Axone und präsynaptischen Websites gleichzeitig visualisieren, waren DsRed und Verschlagwortet mit EGFP Synaptophysin (SYP-EGFP) Co transfizierten in eine kleine Anzahl von Thalamische Neurone durch Elektroporation organotypischen Coculture. Die aktuelle Methode morphologische Analyse des TC Axone erleichtert und ermöglicht eine langfristige Beobachtung, die verwendet werden können, um den ursächlichen Zusammenhang zwischen Axon verzweigen und Synapse Bildung zu zeigen.

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			<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>
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			<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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			<td class="xl20" style="width:89pt" width="119">Sigma</td>
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			<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>
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			<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>
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		<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>
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			<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>
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		<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>
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		<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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			<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>
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			<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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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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		<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>
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		<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>
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		<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>
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		<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

Axon verzweigen und Synapse Bildung sind entscheidende Prozesse für die Festlegung der genauen neuronale Schaltkreise. Während der Entwicklung bilden sensorische Thalamocortical (TC) Axone Zweige und Synapsen in bestimmten Schichten der Großhirnrinde. Trotz der offensichtlichen räumliche Korrelation zwischen Axon verzweigen und Synapse Bildung ist die kausale Beziehung zwischen ihnen kaum verstanden. Um dieses Problem zu beheben, haben wir vor kurzem eine Methode für die gleichzeitige Darstellung von Verzweigungen und Synapse Bildung von individuellen TC Axone in organotypischen Kokulturen entwickelt.
Dieses Protokoll beschreibt eine Methode besteht aus einer Kombination von organotypischen Coculture und Elektroporation. Organotypischen Kokulturen des Thalamus und Großhirnrinde erleichtern, Genmanipulation und Beobachtung der axonalen Prozesse, Erhaltung der charakteristischen Strukturen wie laminar Konfiguration. Zwei unterschiedliche Plasmide Codierung DsRed und Verschlagwortet mit EGFP Synaptophysin (SYP-EGFP) wurden Co transfizierten in eine kleine Anzahl von Thalamische Neurone durch Elektroporation-Technik. Diese Methode erlaubt uns, individuelle axonalen Morphologien von TC Neuronen und ihren präsynaptischen Websites gleichzeitig visualisieren. Die Methode aktiviert auch Langzeit-Beobachtung, der den ursächlichen Zusammenhang zwischen Axon verzweigen und Synapse Bildung offenbart.

Das Experiment beschriebenen hier zielt darauf ab, die Beziehung zwischen TC Axon verzweigen und Synapse Bildung zu offenbaren. Um gleichzeitig visualisieren axonalen Flugbahnen und Standorte der präsynaptischen Seiten, einzelne oder wenige Thalamische Zellen in organotypischen Kokulturen wurden mit zwei Plasmide Codierung SYP-EGFP und DsRed mit Elektroporation transfiziert. In der zweiten Woche in Kultur wurden individuell unterscheidbar TC Axone durch DsRed (Abbild...

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

Dieses Protokoll beschreibt eine Methode für die gleichzeitige Darstellung von Thalamocortical Axon Verzweigung und Synapse Bildung in organotypischen Kokulturen des Thalamus und Großhirnrinde. Individuelle Thalamocortical Axone und ihre präsynaptischen Terminals werden durch eine einzelne Zelle Elektroporation Technik mit DsRed und GFP-Tags Synaptophysin visualisiert.

Visualisierung von Thalamocortical Axon Verzweigung und Synapse Bildung in organotypischen Kokulturen

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

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

Forschung

Neurowissenschaften

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Visualization of Proprioceptors in Drosophila Larvae and Pupae

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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

Gepaart ganze Zelle Recordings in Organotypische Hippocampus

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

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Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

Whole-Mount-Imaging von Mouse Embryo Sinnes Axon Projektionen

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

Kombinierte Methodiken Verwendung der Rolle der Plasmamembran Lieferung Während Axon Branching und Neuronale Morphogenese zu definieren

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

Quadruple Immunfärbung der olfaktorischen Glühbirne zur Visualisierung von olfaktorischen sensorischen Axon Molecular Identity Codes

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

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Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

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

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