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

Vi takker også Gabriel hånd for kritisk læsning.

Alle eksperimenter blev udført ifølge de retningslinier af dyrevelfærd udvalg af Osaka University og Japan neurovidenskab samfund.
1. Organotypic cocultures af thalamus og hjernebarken
Bemærk: For en detaljeret procedure, henvises til den originale publikationer7,8,13. Alle procedurer bør udføres under sterile forhold. Sprague-Dawley (SD) rotter bruges til neuronal kult...

<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>

Den nuværende protokol er også et kraftfuldt værktøj til at studere udviklingsmæssige aspekter af voksende axoner end TC projektion11. For eksempel, en kombination af kortikale skive kultur og elektroporation teknik giver mulighed for visualisering individuelle cytoskeletale morfologi af kortikale neuroner og langsigtet observation9,18.
Ved hjælp af den nuværende protokol, kan roller af interessante gener...

Thalamocortical (TC) projektion i pattedyr hjernen er et egnet system til at undersøge axon vejledning og målretning mekanismer. Under udvikling vokse sensoriske TC axoner i kortikale plade, og form grene og synapser fortrinsvis i lag IV af de primære sensoriske områder i hjernebarken1,2. Selv efter oprettelsen af grundlæggende forbindelser, er cytoskeletale Dorne og synaptiske terminalerne ombygget afhængigt af miljømæssige ændringer3,4. Men hvordan TC axon morfologi ændres dynamisk er dårligt forstået. En af hovedårsagerne er manglen på en passende teknik til at observere strukturelle ændringer på en enkelt celle niveau. Selv om den seneste udvikling i mikroskopi, såsom to-foton mikroskopi, har tilladt direkte observation af levende kortikale neuroner i vivo, er der stadig tekniske begrænsninger til at indfange den samlede TC baner5, 6. derfor, in vitro- metoder til live imaging af TC axoner ville give kraftfulde værktøjer for strukturelle analyser af axon forgrening og synapse dannelse.
Vores gruppe for første gang fastsat en statisk skive kultur metode med permeabel membran7. Brug denne metode, en rotte kortikale skive var cocultured med en sensoriske thalamic blok, og lamina-specifikke TC forbindelser blev sammenfattet i denne organotypic cocultures7,8. Sparsomme mærkning med en fluorescerende proteiner yderligere tillod os at observere TC axon vækst og gren dannelse9,10,11. For nylig, har vi udviklet en roman metode for samtidige billeddannelse af filialer og synapse dannelsen af individuelle TC axoner i organotypic cocultures12. For at visualisere TC axoner og præsynaptiske sites samtidigt, var DsRed og EGFP-mærket synaptophysin (SYP-EGFP) Co transfected ind i et lille antal thalamic neuroner ved elektroporation af organotypic coculture. Den nuværende metode letter morfologisk analyse af TC axoner og tillader langsigtet observation, som kan bruges til at vise årsagssammenhængen mellem axon forgrening og synapse dannelse.

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			<td class="xl20" style="width:84pt" width="112">11320-033</td>
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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>
			<td class="xl21" style="width:84pt" width="112">5905</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">Fetal bovine serum (FBS)</td>
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			<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>
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			<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">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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			<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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			<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" 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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			<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>
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		<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>
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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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		<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>
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		<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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			<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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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 forgrening og synapse dannelse er afgørende processer til at fastsætte præcise neuronal kredsløb. Under udvikling danner sensoriske thalamocortical (TC) axoner grene og synapser i særlige lag af hjernebarken. Trods åbenlyse rumlige korrelationen mellem axon forgrening og synapse dannelse, er årsagssammenhængen mellem dem dårligt forstået. For at løse dette problem, udviklet vi for nylig en metode til samtidige billeddannelse af forgrening og synapse dannelsen af individuelle TC axoner i organotypic cocultures.
Denne protokol beskriver en metode, som består af en kombination af en organotypic coculture og elektroporation. Organotypic cocultures af thalamus og hjernebarken lette genmanipulation og observation af cytoskeletale processer, bevare karakteristiske strukturer såsom laminar konfiguration. To særskilte plasmider kodning DsRed og EGFP-mærket synaptophysin (SYP-EGFP) var Co transfected ind i et lille antal thalamic neuroner ved en elektroporation teknik. Denne metode tillod os at visualisere individuelle cytoskeletale morfologier TC neuroner og deres præsynaptiske sites samtidigt. Metoden også aktiveret langsigtet observation, som afslørede årsagssammenhængen mellem axon forgrening og synapse dannelse.

Forsøget er beskrevet her har til formål at afsløre forholdet mellem TC axon forgrening og synapse dannelse. Du kan samtidig visualisere cytoskeletale baner og placeringer af præsynaptiske websteder, enkelt eller et par thalamic celler i organotypic cocultures var transfekteret med to plasmider kodning SYP-EGFP og DsRed ved hjælp af elektroporation. Under den anden uge i kultur, var individuelt skelnes TC axoner tydeligt mærket af DsRed (figur 3). Kun d...

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

Denne protokol beskriver en metode til samtidige billeddannelse af thalamocortical axon forgrening og synapse dannelse i organotypic cocultures af thalamus og hjernebarken. Enkelte thalamocortical axoner og deres præsynaptiske terminaler er visualiseret ved en enkelt celle elektroporation teknik med DsRed og normal god landbrugspraksis-mærket synaptophysin.

Visualisering af Thalamocortical Axon forgrening og Synapse dannelse i 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

Automated Interactive Video Playback for Studies of Animal Communication

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Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Forbundne Helcelle optagelser i Organotypiske hippocampusskiver

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

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Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

Hele-mount Imaging af museembryo Sensory Axon Fremskrivninger

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

Udnytte Kombineret Metoder til Definer rolle plasmamembranen Delivery Under Axon Forgrening og neuronal Morfogenese

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

Quadruple Immunostaining af Olfactory Bulb til Visualisering af Olfactory Sensory Axon Molecular Identity Codes

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

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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

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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 ...

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

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