Name: improving the application of high molecular weight biotinylated dextran amine for thalamocortical projection tracing in the rat
Uploaded: 2018-04-12T12:30:00
Description: Watch this Scientific Journal Video about Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat at JoVE.com

Undersøgelsen blev finansieret af National Natural Science Foundation of China (projekt kode nr. 81373557, nr. 81403327).

Denne undersøgelse blev godkendt af den etiske komité på Kina kinesisk medicinsk Videnskabernes Akademi (referencenummer 20160014). Alle procedurer blev gennemført i overensstemmelse med de nationale institutter sundhed Guide til pleje og anvendelse af forsøgsdyr (National Academy Press, Washington, D.C., 1996). Fire voksne mandlige rotter (vægt 250-280 g) blev brugt i denne undersøgelse. Alle dyr blev opstaldet i en 12 h lys/mørke cyklus med kontrolleret temperatur og luftfugtighed og gratis adgang til mad og va...

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topological+precision+in+the+thalamic+projection+to+neonatal+mouse+barrel+cortex.">Topological precision in the thalamic projection to neonatal mouse barrel cortex.</a> J Neurosci. 15, 549-561 (1995).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The rat brain in stereotaxic coordinates. , (1998).</li><li>Davidoff, M., Schulze, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+avidin-biotin-peroxidase+complex+(ABC)+staining+combination+of+the+peroxidase+anti-peroxidase+(PAP)-and+avidin-biotin-peroxidase+complex+(ABC)-techniques:+an+amplification+alternative+in+immunocytochemical+staining.">Standard avidin-biotin-peroxidase complex (ABC) staining combination of the peroxidase anti-peroxidase (PAP)-and avidin-biotin-peroxidase complex (ABC)-techniques: an amplification alternative in immunocytochemical staining.</a> Histochemistry. 93, 531-536 (1990).</li><li>Fritzsch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+axonal+diffusion+of+3000+molecular+weight+dextran+amines.">Fast axonal diffusion of 3000 molecular weight dextran amines.</a> J Neurosci Methods. 50, 95-103 (1993).</li><li>Kaneko, T., Saeki, K., Lee, T., Mizuno, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+retrograde+axonal+transport+and+subsequent+visualization+of+tetramethylrhodamine+(TMR)+-dextran+amine+by+means+of+an+acidic+injection+vehicle+and+antibodies+against+TMR.">Improved retrograde axonal transport and subsequent visualization of tetramethylrhodamine (TMR) -dextran amine by means of an acidic injection vehicle and antibodies against TMR.</a> J Neurosci Methods. 65, 157-165 (1996).</li><li>Medina, L., Reiner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efferent+projections+of+the+dorsal+and+ventral+pallidal+parts+of+the+pigeon+basal+ganglia,+studied+with+biotinylated+dextran+amine.">The efferent projections of the dorsal and ventral pallidal parts of the pigeon basal ganglia, studied with biotinylated dextran amine.</a> Neuroscience. 81, 773-802 (1997).</li><li>DE Venecia, R. K., Smelser, C. B., McMullen, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parvalbumin+is+expressed+in+a+reciprocal+circuit+linking+the+medial+geniculate+body+and+auditory+neocortex+in+the+rabbit.">Parvalbumin is expressed in a reciprocal circuit linking the medial geniculate body and auditory neocortex in the rabbit.</a> J Comp Neurol. 400, 349-362 (1998).</li><li>Ojima, H., Takayanagi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+two+anterograde+axon+tracers+to+label+distinct+cortical+neuronal+populations+located+in+close+proximity.">Use of two anterograde axon tracers to label distinct cortical neuronal populations located in close proximity.</a> J Neurosci Methods. 104, 177-182 (2001).</li><li>Kobbert, C., Apps, R., Bechmann, I., Lanciego, J. L., Mey, J., Thanos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+concepts+in+neuroanatomical+tracing.">Current concepts in neuroanatomical tracing.</a> Prog Neurobiol. 62, 327-351 (2000).</li><li>Vercelli, A., Repici, M., Garbossa, D., Grimaldi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+techniques+for+tracing+pathways+in+the+central+nervous+system+of+developing+and+adult+mammals.">Recent techniques for tracing pathways in the central nervous system of developing and adult mammals.</a> Brain Res Bull. 51, 11-28 (2000).</li><li>Liao, C. C., Reed, J. L., Kaas, J. H., Qi, H. 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At vælge en ordentlig tracer er en kritisk trin til en vellykket neurale sporing eksperiment. I familien af BDA, høj molekylvægt BDA (10.000 molekylvægt) blev anbefalet til fortrinsvis transporteres gennem anterograd neurale vej i modsætning til lavmolekylære BDA (3.000 molekylvægt)2,3 , 11 , 12 , 13. dog mange undersøgelser også antydet, at høj mole...

Høj molekylvægt BDA (10.000 molekylvægt), en meget følsom tracer, har været brugt til sporing nervebaner i det centrale nervesystem for over 20 år1. Selv om brugen af BDA er en fælles neurale tarmkanalen sporing teknik, kan kvaliteten af BDA mærkning blive påvirket i dyr af forskellige faktorer1,2,3. Vores seneste undersøgelse viste, at den optimale struktur af BDA mærkning er ikke kun forbundet med en ordentlig Post injektion overlevelsestid, men også korreleret med farvning metode4. Indtil nu, konventionelle begærlighed-biotin-peroxidase complex (ABC), streptavidin-fluorescein isothiocyanat og streptavidin-AF594 farvning metoder der er anvendt for at afsløre BDA mærkning i tidligere undersøgelser2,3, 4,5. I sammenligning, kan fluorescerende farvning for BDA let udføres.
For at udvide anvendelsen af høj molekylvægt BDA, blev en raffineret protokollen introduceret i den foreliggende undersøgelse. Efter injektion af BDA i VPM af thalamus i hjernen, rotte blev BDA mærkning afsløret ved den almindelige metode til standard ABC farvning samt ved dobbelt fluorescerende farvning, som blev gennemført for observere korrelation af BDA mærkning og grundlæggende neurale elementer eller interneurons i det kortikale mål med streptavidin-AF594 og fluorescerende Nissl histokemi eller PV-immunochemistry, henholdsvis. Gennem de gensidige nervebaner mellem VPM og primære somatosensoriske cortex (S1)6,7,8, vi har fokuseret vores observation på BDA mærkning i thalamocortical projiceres axoner og corticothalamic forventede celle svovldepositioner i S1. Ved denne proces, som vi forventes at levere en detaljeret protokol for at opnå den høje kvalitet af neurale mærkning med høj molekylvægt BDA, men også en raffineret protokol om kombinationen af fluorescerende BDA mærkning og andre fluorescerende neurale markører med histokemi eller immunochemistry. Denne tilgang er at foretrække at studere de lokale neurale kredsløb og deres kemiske egenskaber under en Konfokal laser scanning mikroskopi.

<table>
	<tbody>
		<tr>
			<td>Biotinylated dextran amine (BDA)</td>
			<td>Molecular Probes</td>
			<td>D1956</td>
			<td>10,000 molecular weight</td>
		</tr>
		<tr>
			<td>Streptavidin-Alexa Fluor 594</td>
			<td>Molecular Probes</td>
			<td>S32356</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>500/525 green fluorescent Nissl stain</td>
			<td>Molecular Probes</td>
			<td>N21480</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>Brain stereotaxis instrument</td>
			<td>Narishige</td>
			<td>SR-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezing microtome</td>
			<td>Thermo</td>
			<td>Microm International GmbH</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal imaging</td>
			<td>Olympus</td>
			<td>FV1200</td>
			<td></td>
		</tr>
		<tr>
			<td>system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Drill</td>
			<td>Saeyang Microtech</td>
			<td>Marathon-N7</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague Dawley</td>
			<td>Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences</td>
			<td>SCKX (JUN) 2012-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectastain ABC Kit</td>
			<td>Vector Laboratories</td>
			<td>PK-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>superfrost plus microscope slides</td>
			<td>Thermo</td>
			<td>#4951PLUS-001</td>
			<td>25x75x1mm</td>
		</tr>
		<tr>
			<td>Photoshop and Illustration</td>
			<td>Adobe</td>
			<td>CS5</td>
			<td></td>
		</tr>
	</tbody>
</table>

improving the application of high molecular weight biotinylated dextran amine for thalamocortical projection tracing in the rat

Høj molekylvægt biotinylated dextran Amin (BDA) har været brugt som en yderst følsom neuroanatomical sporstof i mange årtier. Da kvaliteten af dens mærkning blev ramt af forskellige faktorer, her, leverer vi en raffineret protokol til anvendelsen af høj molekylvægt BDA for at studere optimal neurale mærkning i centralnervesystemet. Efter stereotactic injektion af BDA i ventrale posteromedial kerne (VPM) af thalamus i rotte gennem en delikat glas pipette, var BDA farves med fluorescerende streptavidin-Alexa (AF) 594 og counterstained med fluorescerende Nissl pletten AF500/525. På baggrund af grønne Nissl farvning, blev den røde BDA mærkning, herunder neuronal celle organer og cytoskeletale terminaler, mere tydeligt demonstreret i den somatosensoriske cortex. Desuden dobbelt fluorescerende farvning for BDA og calcium-bindende protein parvalbumin (PV) blev udført for at observere korrelationen mellem BDA mærkning og PV-positive interneurons i den kortikale mål, giver lejlighed til at studere den lokale neurale kredsløb og deres kemiske egenskaber. Således, denne raffineret metode er ikke kun egnet til at visualisere høj kvalitet neurale mærkning med høj molekylvægt BDA gennem gensidige nervebaner mellem thalamus og hjernebarken, men også vil tillade samtidige demonstration af andre neurale markører med fluorescerende histokemi eller immunochemistry.

Overlevelse af 10 dage post injektion af BDA i VPM var tilstrækkelig til fremstilling af intens neurale mærkning på de tilsvarende kortikale områder ipsilaterale til injektion side (figur 2). Både konventionelle ABC og fluorescerende farvning procedurer for BDA afslørede den lignende mønster af neurale mærkning på S1, herunder anterogradely mærket thalamocortical axoner og retrogradely mærket corticothalamic neuroner (figur 2 c...

Watch this Scientific Journal Video about Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat at JoVE.com

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

Vi præsenterer her, en raffineret protokol for at effektivt afsløre biotinylated dextran Amin (BDA) mærkning med en fluorescerende farvning metode gennem en gensidig neurale vej. Det er egnet til at analysere fine strukturen af BDA mærkning og adskille det fra andre neurale elementer under en Konfokal laser scanning mikroskop.

Forbedring af anvendelsen af høj molekylvægt Biotinylated Dextran Amin for Thalamocortical projektion sporing i rotter

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

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