Bu çalışmada Ulusal Doğa Bilimleri Vakfı Çin tarafından (proje kod No 81373557; No 81403327) finanse edildi.

Bu çalışmada, Çin Çince tıbbi Bilimler Akademisi (referans numarası 20160014) Etik Komitesi tarafından kabul edildi. Tüm yordamları bakım ve laboratuvar hayvanlarının kullanımı (Ulusal Akademisi basın, Washington, DC, 1996) için Ulusal Sağlık Rehberi Enstitüleri uygun olarak gerçekleştirilmiştir. Dört yetişkin erkek rats (ağırlığı 250-280 g) Bu çalışmada kullanılmıştır. Bütün hayvanlar kontrollü sıcaklık ve nem ile 12 h açık/koyu döngüsünde yer alan ve gıda ve su ücretsiz erişim izni. Aletleri ve malzemeler mevcut çalışmada kullanılan şekil 1' de gösterdi.  Ameliyattan önce stereotaksik çerçeve ve cam pipet, gibi tüm araçları % 70 etanol kullanarak temizlenmiştir.
1. cerrahi işlemler
<ol><li>Stereotaksik atlas9 (şekil 2A) kullanarak VPM ilgi koordinat alanı belirleyin.</li>
	<li>1 µL mikro-cam micropipette (yaklaşık 10-20 µm ipucu çaplı) ile donatılmış şırınga hazırlamak (şekil 1 d) ve Sıvı parafin ile test edin.</li>
	<li>% 7 kloral hidrat (0.7 mL/100 g) farelerle mayi enjeksiyonu ile anestezi.</li>
	<li>Derin anestezi onaylandıktan sonra kuyruk ile pinch ve para çekme refleks pedal, hayvanın başının ile tıraş Tıraş ve cerrahi sitesi 3 kez sırasıyla % 70 etanol tarafından takip % 10 povidone iyot ile fırçalayın.</li>
	<li>Fareyi kulaklarına künt kulak çubuklarını yerleştirerek stereotaksik cihazın içine yerleştirin ve sıçan üst kesici dişler (şekil 1E) ağız yuvasına yerleştirin ve oftalmik merhem gözünü uygulayın.</li>
	<li>Tekrar % 70 etanol kullanarak cerrahi sitesinin baş cilt temizlemek. Havlu ve steril cerrahi eldiven steril koşullarda ameliyat korumak için kullanın.</li>
	<li>Cilt sagittal dikiş (şekil 1F) boyunca bir neşter ile sagittal bir kesi yapmak.</li>
	<li>Kas ve steril pamuklu uçlu uygulayıcıları (şekil 1F) kanama kontrol cerrahiye boyunca kullanarak kafatası uzak kapaklarini kazı.</li>
	<li>Bir Atlas (şekil 2A) önceden tanımlanmış koordinatları kullanarak, kranyotomi (şekil 1G) konumunu (-3.3 mm Bregma noktası, orta hat sağa 2.6 mm) belirleyin.</li>
	<li>Burr matkap ile yuvarlak uçlu biraz (#106) (Resim 1 H) kullanarak bir kranyotomi gerçekleştirmek ve meninkslerde (şekil 1I) ulaşan kadar bir kaç dakika içinde Delme hakkında 1 mm derinliği için devam edin.</li>
	<li>Serebral korteks enjeksiyon sitesi (şekil 1I) üzerinden ortaya çıkarmak için microforceps kullanarak dura mater tüketim.</li>
	<li>Sıvı parafin % 10 BDA (10.000 molekül ağırlığı, distile su içinde) çözüm (şekil 1J) mikro şırınga değiştirin.</li>
	<li>Şırınga mikroenjeksiyon aparatı monte ve bir mikro-pompa ile (şekil 1 K) bağlanın. Not: enjekte birimi mikro-pompa hızına bağlıdır. Burada 30 nL/dk (şekil 1 L) ayarlandı.</li>
	<li>Bir stereomicroscope altında VPM 5.8 mm (şekil 1 M) derinlikte beynin kortikal yüzeyi üzerinden el ile mikroenjeksiyon aparatı ile cam micropipette takın.</li>
	<li>Basınç enjekte-bir %100 10 nL BDA VPM 3 dk (35 nL/dk) bir mikro-pompa (şekil 1 L, M) ile bir süre içine.</li>
	<li>Sonra enjeksiyon, pipet, yerde bir ek 5 min için tutun ve sonra yavaş yavaş geri.</li>
	<li>Yara steril vida (şekil 1N) dikiş. Öncesi ve ameliyat sonrası analjezi için senin yerel hayvan bakımı Komitesi yönergeleri izleyin.</li>
	<li>Bilincinin tekrar yerine geleceğinden ve tamamen iyileşti kadar fareyi bir sıcak kurtarma alanında koyun.</li>
	<li>Kurtarılan fareyi onun kafes için geri dönün.</li>
</ol>2. perfusions ve bölümler
<ol><li>Sonra bir yaşam süresi genellikle 10 günlük hayvan mayi enjeksiyonu ile ötenazi ikna etmek için % 10 üretan (2 mL/100 g) aşırı dozda enjekte et.</li>
	<li>Hood (şekil 1O) deneysel fareler sıvı.<ol>
<li>Bir kez nefes durur, makas ve forseps, kullanarak kalp erişmek için fareyi göğüs boşluğuna açın. Sol ventrikül aort doğru bir damar içi kateter takın ve doğru auricle açın.</li>
		<li>İlk % 0,9 fosfat tamponlu tuz (PBS), fizyolojik sıcaklığında (37 ° C) ile sıvı kadar kan kalpten çıktıktan 1-2 dk temizleyin ve 250-300 mL % 4 paraformaldehyde 0.1 M fosfat tampon (PB, pH 7,4) ile devam edin.</li>
	</ol>
</li>
	<li>Perfüzyon sonra baş cilt deşmek ve kafatasını açıp sıçan beyin incelemek. Oda sıcaklığında (26 ° C), o zaman cryoprotect içinde % 30 Sükroz 0.1 M PBS (pH 7,4) beyin çözüm (şekil 1 P) batırılır kadar 4 ° C'de 3 gün içinde 2 h için %4 paraformaldehyde disseke beyinde sonrası tamir et.</li>
	<li>Bir kez beyin çözümde batırılır, beyin üzerinde beyin matrisler (şekil 1Ç) Koronal yönde üç sokak bölün. Merkezi bloğu VPM ve S1 içerir.</li>
	<li>Koronal microtome sistemini sürgülü bir dondurucu sahnede merkezi bloğu 40 µm, beynin kesti. Bu bölümlerde düzenli bir 6-iyi yemek (rakamlar 1R, S) 0.1 M PBS (pH 7,4) ile toplamak.</li>
</ol>3. standart ABC boyama
Not: Beynin her üçüncü koronal bölümünden ücretsiz kayan bölümler BDA standart ABC yordamı10ile etiketleme görüntülenmesi için kullanılmıştır.
<ol><li>0.1 M PBS için yaklaşık 1 dakika bölümlerde durulayın.</li>
	<li>(PH 7,4) içeren %0,3 %1 0.1 M PB ABC çözümde bölümlerde kuluçkaya Triton X-100 oda sıcaklığında 1 h için.</li>
	<li>Sonraki bölümlerde 50 mM Tris arabellek (pH 7,4) üç kere yıkayın.</li>
	<li>%0,02 3, 3'-diaminobenzidine tetrahydrochloride (DAB) ve %0.01 H2O2 oda sıcaklığında yaklaşık 2-5 dakika için 50 mM Tris arabellek içeren bir çözüm bölümlerde leke.</li>
	<li>Sonraki bölümlerde 50 mM Tris arabellek (pH 7,4) üç kere yıkayın.</li>
	<li>Bölümler standart histochemical teknikleri (1T rakam; bkz: Ek Video dosyası III, perfüzyon ve bölümler) kullanarak mikroskop slaytlarda bağlayın.</li>
	<li>Gecede, oda sıcaklığında hava bölümlerde kuru.</li>
	<li>Alkol (% 50, % 70, % 95, % 100) çözümleri kısa bir süre içinde bir dizi bölümlerini kurutmak. Her çözüm için yaklaşık 15 slaytları daldırın s. Slaytlar arasında her adım kurumasına izin vermez.</li>
	<li>Ksilen bölümlerde üç kez, yaklaşık 20 dk temizleyin.</li>
	<li>2-3 damla balsam dilimleri koyun sonra coverslips bölümleri yerleştirin.</li>
</ol>4. çift floresan BDA ve serebral korteks nöral temel öğeleri için boyama
Not: Buna ek olarak, çift floresan boyama BDA etiketleme korelasyon gözlemleyerek için yapılmıştır ve yukarıdaki bitişik bölümleri üzerinde temel sinir öğelerini streptavidin-AF594 ile kullanılan ve floresan Nisl leke AF500/525 ile counterstained.
<ol><li>0.1 M PBS için yaklaşık 1 dakika bölümlerde durulayın.</li>
	<li>Streptavidin-AF594 (1:500) ve AF500/525 yeşil flüoresan Nisl leke (1:1, 000) 0.1 M PBS karışık bir çözümde bölümlerini (pH 7,4) içeren %0,3 kuluçkaya Triton X-100 oda sıcaklığında 2 h için.</li>
	<li>Sonraki bölümlerde 0.1 M PB (pH 7,4) üç kere yıkayın.</li>
	<li>Bölümler standart histochemical teknikleri kullanarak mikroskop slaytlarda bağlayın. Kuru havada yaklaşık 1 h için bölümler.</li>
	<li>Coverslips % 50 gliserin gözlem önce distile su ile floresan bölümlerine uygulanır.</li>
</ol>5. çift floresan BDA ve Interneurons serebral korteks için boyama
Not: Çift floresan boyama BDA etiketleme ve interneurons streptavidin-AF594 ve PV-İmmünokimya kortikal hedefle temsilcisi bölümlerde Tarih korelasyon gözlemleyerek için gerçekleştirilmiştir.
<ol><li>0.1 M PBS (pH 7,4) yaklaşık 1 dakika için temsilcisi bölümlerde durulayın.</li>
	<li>% 3 normal keçi serum ve %0,3 içeren bir engelleme çözüm bölümlerde kuluçkaya Triton X-100 0.1 M PBS için 30 dk içinde.</li>
	<li>Fare monoklonal anti-PV IgG (1:1, 000) 0.1 M PBS (pH 7,4) içeren % 1 normal keçi serum ve %0,3 bir çözüm içine bölümleri transfer Triton X-100 gecede 4 ° C'de için</li>
	<li>Ertesi gün, sonraki bölümlerde 0.1 M PBS (pH 7,4) üç kere yıkayın.</li>
	<li>Karışık bir çözüm bölümlerine keçi mouse-AF488 ikincil antikor (1:500), Serumda 0.1 M PBS (pH 7,4) içeren % 1 normal keçi streptavidin-AF594 (1:500) ve 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI, 1:40, 000) ve 0,3 açığa % Triton 1 h için X-100.</li>
	<li>4.3-4.5 adımları yineleyin.</li>
</ol>6. gözlem
<ol><li>VPM, thalamocortical akson ve corticothalamic nöronlar görüntülerini almak.<ol>
<li>Hedefleri lensler ile donatılmış bir confocal görüntüleme sistemi ile floresan örnekleri gözlemlemek (4 x, NA: 0,13; 10 x, NA: 0.40; ve 40 x, NA: 0.95). 405 (mavi), 488 (yeşil), uyarma ve emisyon dalga boylarında ve 559 (kırmızı) nm kullanın. Not: Burada, confocal iğne deliği 152 µm (4 x, 10 x) ve 105 µm (40 x). Görüntü yakalama Uzaysal çözünürlük 1024 × 1024 piksel (4 x, 10 x) ve 640 × 640 piksel (40 x) olduğunu.</li>
		<li>Yirmi görüntüler 40 µm (Z serisi) kalınlığı, her bölüm 2 µm ardışık çerçeveler içinde tutar.</li>
		<li>Görüntüleri tek bir odakta görüntü confocal görüntü işleme için aşağıdaki gibi üç boyutlu analiz yazılım sistemi ile entegre: ayarla başlat odak düzlemi → set sonunda odak düzlemi → set adım boyutu → derinlik desen → görüntü yakalama → Z serisi seçin.</li>
	</ol>
</li>
	<li>Bir dijital fotoğraf makinesi ile donatılmış bir ışık mikroskobu tarafından aydınlık alan görüntü alabilir (4 x, NA: 0,13; 10 x, NA: 0.40; ve 40 x, NA: 0.95 lensler). Bir çekim hızı 500 Bayan kullanım fotoğraf düzenleme yazılımı, parlaklık ve kontrast görüntülerin ve etiket eklemek için kullanın.</li>
</ol>

<ol>
	<li>Veenman, C. L., Reiner, A., Honig, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotinylated+dextran+amine+as+an+anterograde+tracer+for+single-+and+double-labeling+studies.">Biotinylated dextran amine as an anterograde tracer for single- and double-labeling studies.</a> J Neurosci Methods. 41, 239-254 (1992).</li><li>Reiner, A., Veenman, C. L., Medina, L., Jiao, Y., Del Mar, N., Honig, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathway+tracing+using+biotinylated+dextran+amines.">Pathway tracing using biotinylated dextran amines.</a> J Neurosci Methods. 103, 23-37 (2000).</li><li>Ling, C., Hendrickson, M. L., Kalil, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+the+detailed+structure+of+cortical+and+thalamic+neurons+in+the+adult+rat+brain+with+refined+biotinylated+dextran+amine+labeling.">Resolving the detailed structure of cortical and thalamic neurons in the adult rat brain with refined biotinylated dextran amine labeling.</a> PLoS One. 7, e45886 (2012).</li><li>Zhang, W. J., 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=Anterograde+and+retrograde+tracing+with+high+molecular+weight+biotinylated+dextran+amine+through+thalamocortical+and+corticothalamic+pathways.">Anterograde and retrograde tracing with high molecular weight biotinylated dextran amine through thalamocortical and corticothalamic pathways.</a> Microsc Res Tech. 80, 260-266 (2017).</li><li>Han, X., 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=Biotinylated+dextran+amine+anterograde+tracing+of+the+canine+corticospinal+tract.">Biotinylated dextran amine anterograde tracing of the canine corticospinal tract.</a> Neural Regen Res. 7, 805-809 (2012).</li><li>Armstrong-James, M., Callahan, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thalamo-cortical+processing+of+vibrissal+information+in+the+rat.+II.+spatiotemporal+convergence+in+the+thalamic+ventroposterior+medial+nucleus+(VPm)+and+its+relevance+to+generation+of+receptive+fields+of+S1+cortical+&amp;#34;barrel&amp;#34;+neurones.">Thalamo-cortical processing of vibrissal information in the rat. II. spatiotemporal convergence in the thalamic ventroposterior medial nucleus (VPm) and its relevance to generation of receptive fields of S1 cortical &amp;#34;barrel&amp;#34; neurones.</a> J Comp Neurol. 303, 211-224 (1991).</li><li>Armstrong-James, M., Callahan, C. A., Friedman, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thalamo-cortical+processing+of+vibrissal+information+in+the+rat.+I.+Intracortical+origins+of+surround+but+not+centre-receptive+fields+of+layer+IV+neurones+in+the+rat+S1+barrel+field+cortex.">Thalamo-cortical processing of vibrissal information in the rat. I. Intracortical origins of surround but not centre-receptive fields of layer IV neurones in the rat S1 barrel field cortex.</a> J Comp Neurol. 303, 193-210 (1991).</li><li>Agmon, A., Yang, L. T., Jones, E. G., O'Dowd, D. 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. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracortical+connections+are+altered+after+long-standing+deprivation+of+dorsal+column+inputs+in+the+hand+region+of+area+3b+in+squirrel+monkeys.">Intracortical connections are altered after long-standing deprivation of dorsal column inputs in the hand region of area 3b in squirrel monkeys.</a> J Comp Neurol. 524, 1494-1526 (2016).</li></ol>

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Uygun izleme aygıtı seçme başarılı sinirsel izleme deneme için kritik bir adımdır. BDA, yüksek molekül ağırlıklı BDA ailesi (10.000 moleküler ağırlık) düşük moleküler ağırlıklı BDA (3.000 moleküler ağırlık)2,3 aksine anterograd sinir yolu ile tercihen taşınması için tavsiye edilir , 11 , 12 , 13. ancak, birçok çalışma Ayr...

Yüksek molekül ağırlıklı BDA (10.000 moleküler ağırlık), son derece hassas bir izleyici, merkezi sinir sistemi üzerinde 20 yıl1için Sinirsel yollar izleme için kullanılmıştır. BDA kullanımı teknik izleme ortak bir sinir yolu olsa da, BDA etiketleme kalitesini hayvanlarda çeşitli faktörler1,2,3tarafından etkilenebilir. Bizim son çalışma BDA etiketleme en uygun yapısı sadece uygun sonrası enjeksiyon yaşam süresi ile ilgili ama aynı zamanda boyama yöntemi4ile korelasyon göstermiştir. Şimdi, geleneksel avidin-biotin-peroksidaz kompleksi (ABC), streptavidin floresein isothiocyanate ve streptavidin-AF594 kadar boyama yöntemleri BDA önceki çalışmalar2,3' teetiketleme ifşa için kullanılması, 4,5. Buna karşılık, floresan BDA için boyama kolayca gerçekleştirilebilir.
Yüksek molekül ağırlıklı BDA uygulanması genişletmek için rafine bir iletişim kuralı da çalışmanın kullanılmaya başlandı. Talamus sıçan beyin VPM içine BDA enjeksiyonu, BDA etiketleme ortaya standart ABC boyama düzenli yöntemi yanı sıra çift Floresan, hangi BDA etiketleme korelasyon gözlemleyerek için gerçekleştirildiği boyama ve temel Sinirsel öğeleri veya interneurons kortikal hedef streptavidin-AF594 ve floresan Nisl histochemistry veya PV-İmmünokimya, anılan sıraya göre. VPM ve Primer somatosensor korteks (S1)6,7,8arasında karşılıklı Sinirsel yollar, biz bizim gözlem BDA etiketleme öngörülen thalamocortical akson ve corticothalamic üzerinde duruldu S1 içinde öngörülen hücre somas. Bu işlem tarafından sadece sinirsel yüksek molekül ağırlıklı BDA ile etiketleme yüksek kalitesini elde etmek için ayrıntılı bir iletişim kuralı, aynı zamanda rafine bir protokol floresan BDA etiketleme ile birlikte ve diğer floresan sinirsel belirteçleri ile sağlamak bekleniyor histochemistry veya İmmünokimya. Bu yaklaşım yerel sinir devreleri ve confocal bir lazer mikroskobu tarama altında kimyasal özellikleri çalışma için tercih edilir.

<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

Yüksek molekül ağırlıklı biotinylated dextran Amin (BDA) son derece hassas bir nöroanatomik izleyici onlarca yıl için kullanılmıştır. Onun etiketleme kalitesini çeşitli etkenlere göre burada, etkilendi biz rafine bir protokol yüksek molekül ağırlıklı BDA uygulama için en uygun sinir merkezi sinir sistemi etiketleme çalışmak için sağlar. BDA farenin hassas cam pipet ile talamus ventral posteromedial çekirdeği (VPM) içine stereotaksik enjeksiyon sonra BDA floresan streptavidin ile-Alexa (AF) 594 lekeli ve floresan Nisl leke AF500/525 ile counterstained. Yeşil Nisl boyama arka plan üzerinde kırmızı BDA, nöronal hücre gövdeleri ve aksonal terminaller gibi etiketleme daha belirgin korteks somatosensor gösterilmiştir. Ayrıca, floresan BDA için boyama çift ve kalsiyum bağlayıcı protein parvalbumin (PV) korelasyon BDA etiketleme ve PV-pozitif interneurons sinirsel yerel çalışma fırsatı sağlayan kortikal hedefteki gözlemlemek için yapılmıştır Devreler ve kimyasal özellikleri. Böylece, rafine bu yöntem sadece yüksek kaliteli talamus ve serebral korteks arasında karşılıklı Sinirsel yollar aracılığıyla yüksek molekül ağırlıklı BDA ile etiketleme sinirsel görüntülenmesi için uygun değildir, ancak aynı zamanda eşzamanlı gösteri verecektir Floresan histochemistry veya İmmünokimya diğer sinir işaretleyicilerle.

BDA 10 gün sonrası enjeksiyon VPM içine yaşama yoğun nöral karşılık gelen kortikal alanlarda enjeksiyon yüz (Şekil 2) Ipsilateral etiketleme üretmek için yeterli idi. Hem geleneksel ABC ve yordamlar için BDA boyama floresan sinir anterogradely de dahil olmak üzere S1 üzerinde etiketleme benzer desen thalamocortical akson etiketli ve retrogradely (şekil 2C, D corticothalamic nöronlar etiketli ortaya </str...

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

Burada, etkili bir floresan yöntemi aracılığıyla karşılıklı bir sinirsel yol boyama ile etiketleme biotinylated dextran Amin (BDA) ortaya çıkarmak için rafine bir iletişim kuralı mevcut. Bu güzel yapı çözümleniyor için uygundur, etiketleme ve confocal bir lazer tarama mikroskop altında sinirsel diğer öğelerinden ayırt BDA.

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

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

improving-application-high-molecular-weight-biotinylated-dextran

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

8.9K Görüntüleme.  China Academy of Chinese Medical Sciences. Burada, etkili bir floresan yöntemi aracılığıyla karşılıklı bir sinirsel yol boyama ile etiketleme biotinylated dextran Amin (BDA) ortaya çıkarmak için rafine bir iletişim kuralı mevcut. Bu güzel yapı çözümleniyor için uygundur, etiketleme ve confocal bir lazer tarama mikroskop altında sinirsel diğer öğelerinden ayırt BDA.

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

Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers

It is well known that anesthesia alters neural response properties in various regions of the brain.13. In the auditory system, fundamental response properties of brainstem neurons including threshold, frequency specificity, and inhibitory sidebands are altered in significant ways under anesthesia1-2. These observations prompted physiologists to seek ways to record from single neurons without the contaminating effects of anesthesia. One result was a decerebrate preparation, where the brainstem was completely transected at the level of the midbrain4. The drawbacks of this preparation are a formidable surgery, the elimination of descending projections from the forebrain, and an inability to use sensory stimulation to examine structures above the midbrain. A different strategy has been to implant electrode arrays chronically to record from single neurons and multiunit clusters while the animal is awake and/or behaving5,6. These techniques however are not compatible with injecting tracer dyes after first electrophysiologically characterizing a brain structure. To avoid altering neural response properties with anesthetics while recording electrophysiological response properties from single neurons, we have adapted a head restraint technique long used in bats7-9 to mouse10-12. Using this method, we are able to conduct electrophysiological recordings over several days in the unanesthetized mouse. At the end of the recording sessions, we can then inject a dye to reconstruct electrode positions and recording sites or inject a tracer so that pathways to and from the recording loci can be determined. This method allows for well isolated single neuron recordings over multiple days without the use anesthetics.

Electrophysiological characterization of neuronal responses is important for understanding brain function and for guiding the placement of dyes for pathway tracing. However, many studies are performed in anesthetized animals. To understand brain function without anesthetics, we developed a method to record neuronal response properties and inject dyes in awake mouse.

Sinir Yanıtları Kayıt ve Tarayıcıları enjekte için bir Awake Fare hazırlanması

It is well known that anesthesia alters neural response properties in various regions of the brain.13.  In the auditory system, fundamental response ...

Nörobilim

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Retrograd Floresan Etiketleme Belirlenen Nöronlar gelen Hedefli Hücre dışı Tek ünitesi Kayıt izin verir<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, direction5-7, ocular dominance8,9 and binocular disparity9. Orientation selectivity is the most widely studied feature and a continuous map with a quasi-periodic layout for preferred orientation is present across the entire primary visual cortex10,11. Integrating the synaptic, cellular and network contributions that lead to stimulus selective responses in these functional maps requires the hybridization of imaging techniques that span sub-micron to millimeter spatial scales. With conventional intrinsic signal optical imaging, the overall layout of functional maps across the entire surface of the visual cortex can be determined12. The development of in vivo two-photon microscopy using calcium sensitive dyes enables one to determine the synaptic input arriving at individual dendritic spines13 or record activity simultaneously from hundreds of individual neuronal cell bodies6,14. Consequently, combining intrinsic signal imaging with the sub-micron spatial resolution of two-photon microscopy offers the possibility of determining exactly which dendritic segments and cells contribute to the micro-domain of any functional map in the neocortex. Here we demonstrate a high-yield method for rapidly obtaining a cortical orientation map and targeting a specific micro-domain in this functional map for labeling neurons with fluorescent dyes in a non-rodent mammal. With the same microscope used for two-photon imaging, we first generate an orientation map using intrinsic signal optical imaging. Then we show how to target a micro-domain of interest using a micropipette loaded with dye to either label a population of neuronal cell bodies or label a single neuron such that dendrites, spines and axons are visible in vivo. Our refinements over previous methods facilitate an examination of neuronal structure-function relationships with sub-cellular resolution in the framework of neocortical functional architectures.

A method is described for labeling neurons with fluorescent dyes in predetermined functional micro-domains of the neocortex. First, intrinsic signal optical imaging is used to obtain a functional map. Then two-photon microscopy is used to label and image neurons within a micro-domain of the map.

Belirli Fonksiyonel İçsel Sinyal birleştiren tarafından neokorteks Mikro-etki ve İki foton Görüntüleme nöronlar Hedefli Etiketleme

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, ...

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling of auditory axons within the VIIIth cranial nerve would enhance the study of central auditory circuit development. This approach is challenging because multiple sensory organs of the inner ear contribute to the VIIIth nerve 1. Moreover, markers that reliably distinguish auditory versus vestibular groups of axons within the avian VIIIth nerve have yet to be identified. Auditory and vestibular pathways cannot be distinguished functionally in early embryos, as sensory-evoked responses are not present before the circuits are formed. Centrally projecting VIIIth nerve axons have been traced in some studies, but auditory axon labeling was accompanied by labeling from other VIIIth nerve components 2,3. Here, we describe a method for anterograde tracing from the acoustic ganglion to selectively label auditory axons within the developing VIIIth nerve. First, after partial dissection of the anterior cephalic region of an 8-day chick embryo immersed in oxygenated artificial cerebrospinal fluid, the cochlear duct is identified by anatomical landmarks. Next, a fine pulled glass micropipette is positioned to inject a small amount of rhodamine dextran amine into the duct and adjacent deep region where the acoustic ganglion cells are located. Within thirty minutes following the injection, auditory axons are traced centrally into the hindbrain and can later be visualized following histologic preparation. This method provides a useful tool for developmental studies of peripheral to central auditory circuit formation.

Here we describe a microdissection technique followed by fluorescent dye injection into the acoustic ganglion of early chick embryos for selective tracing of auditory axon fibers in the nerve and hindbrain.

Kuş Embriyonik vestibülokoklear sinir İşitsel Elyaf seçici Aydınger

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This approach has been most extensively used in the brain by utilizing the swine pathogen pseudorabies virus (PRV)1. PRV does not infect great apes, including humans, so it is most commonly used in studies on small mammals, especially rodents. The pseudorabies strain PRV152 expresses the enhanced green fluorescent protein (eGFP) reporter gene and only crosses functional synapses retrogradely through the hierarchical sequence of synaptic connections away from the infection site2,3. Other PRV strains have distinct microbiological properties and may be transported in both directions (PRV-Becker and PRV-Kaplan)4,5&#160;.&#160;This protocol will deal exclusively with PRV152. By delivering the virus at a peripheral site, such as muscle, it is possible to limit the entry of the virus into the brain through a specific set of neurons. The resulting pattern of eGFP signal throughout the brain then resolves the neurons that are connected to the initially infected cells. As the distributed nature of transsynaptic tracing with pseudorabies virus makes interpreting specific connections within an identified network difficult, we present a sensitive and reliable method employing biotinylated dextran amines (BDA) and cholera toxin subunit b (CTb) for confirming the connections between cells identified using PRV152. Immunochemical detection of BDA and CTb with peroxidase and DAB (3, 3&#39;-diaminobenzidine) was chosen because they are effective at revealing cellular processes including distal dendrites6-11.

Transsynaptic tracing has become a powerful tool for analyzing central efferents regulating peripheral targets through multi-synaptic circuits. Here we present a protocol that exploits the transsynaptic pseudorabies virus to identify and localize a functional brain circuit, followed by classical tract tracing techniques to validate specific connections in the circuit between identified groups of neurons.

Transsinaptik Kolera Toksin ve Biotinlenmiş Dekstran Aminler Çift Etiketleme Ardından Pseudorabies&#39;e Virüs ile Periferik Hedefler gelen İzleme

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This ...

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

A surgical method is described to expose the ventral skull in neonate rats. Using this approach it is possible to open a craniotomy to perform acute electrophysiology and two-photon microscopy experiments in the brainstem of anesthetized pups.

Yenidoğan Kemirgenlerin Ventral Kafatası kranyotomi olun Bir Yöntem

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain ...

Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive functions including decision making, sensory-guided behavior or memory. To understand how such intricate neuronal networks perform such tasks, a crucial step is to determine the function (or electrical activity) of individual cell types within the network, preferentially when the animal is performing a relevant cognitive task. Additionally, it is equally important to determine the anatomical structure of the network and the morphological architecture of the individual neurons to allow reverse engineering the cortical network. Technical breakthroughs available today allow recording cellular activity in awake, behaving animals with the valuable option of post&#160;hoc identifying the recorded neurons. Here, we demonstrate the juxtasomal biocytin labeling technique, which involves recording action potential spiking in the extracellular (or loose-patch) configuration using conventional patch pipettes. The juxtasomal recording configuration is relatively stable and applicable across behavioral conditions, including anesthetized, sedated, awake head-fixed, and even in the freely moving animal. Thus, this method allows linking cell-type specific action potential spiking during animal behavior to reconstruction of the individual neurons and ultimately, the entire cortical microcircuit. In this video manuscript, we show how individual neurons in the juxtasomal configuration can be labeled with biocytin in the urethane-anaesthetized rat for post&#160;hoc identification and morphological reconstruction.

To understand the structure of neuronal networks, functional and morphological characterization of individual neurons is a necessity. Here, we demonstrate juxtasomal biocytin labeling, which allows electrophysiological recordings in the extracellular configuration, yet maintaining the ability to intracellularly label the neuron for post&#160;hoc reconstruction of dendritic and axonal architecture.

Juxtasomal Biocytin Etiketleme Bireysel Kortikal nöronlar yapı-fonksiyon ilişkisi Çalışması için

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive ...

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

Wholemount Sıçan Retina Immunohistochemical ve Kalsiyum Görüntüleme Yöntemleri

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Laser-guided Neuronal Tracing In Brain Explants

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake through electroporation in brain explants with targeted laser illumination and matching filter goggles during the procedure. The described technique of in vitro electroporation by itself yields relatively good visual control for targetting certain areas of the brain. By combining it with laser excitation of fluorescent genetic markers and their read-out through band-passing filter goggles, which can pick up the emissions of the genetically labeled cells/nuclei and the fluorescent tracing dye, a researcher can substantially increase the accuracy of injections by finding the area of interest and controlling for the dye-spread/uptake in the injection area much more efficiently. In addition, the laser illumination technique allows to study the functionality of a given neurocircuit by providing information about the type of neurons projecting to a certain area in cases where the GFP expression is linked to the type of transmitter expressed by a subpopulation of neurons.

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

Lazer güdümlü Nöronal İzleme Beyin Eksplantlarındaki

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a premise for determining the role of neuron types within a neural circuit, and their significance in information encoding relative to learning. Likewise, the method can be used to test the physiological significance of two or more connected brain regions in awake and anesthetized animals. The current study demonstrates how VTA glutamate neurons modulate the firing rate of putative pyramidal neurons in the CA1 (hippocampus) of anesthetized mice. This protocol employs adeno-associated virus (AAV)-dependent labeling of VTA glutamate neurons for the tracing of VTA presynaptic glutamate terminals in the layers of the hippocampus. Expression of light-controlled opsin (channelrhodopsin; hChR2) and fluorescence protein (eYFP) harbored by the AAV vector permitted anterograde tracing of VTA glutamate terminals, and photostimulation of VTA glutamate neuron cell bodies (in the VTA). High-impedance acute silicon electrodes were positioned in the CA1 to detect multi-unit and single-unit responses to VTA photostimulation in vivo. The results of this study demonstrate the&#160;layer-dependent distribution of presynaptic VTA glutamate terminals in the hippocampus (CA1, CA3, and DG). Also, the photostimulation of VTA glutamate neurons increased the firing and burst rate of putative CA1 pyramidal units in vivo.

The current protocol demonstrates a simple method for tracing of ventral tegmental area (VTA) glutamate projections to the hippocampus. Photostimulation of VTA glutamate neurons was combined with CA1 recording to demonstrate how VTA glutamate terminals modulate CA1 putative pyramidal firing rate in vivo.

Hipokampustaki Ventral Tegmental Alan Glutamat Terminallerinin Kombine In Vivo Anatomik ve Fonksiyonel İzlenmesi

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a ...

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

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