Anthony Taş, in vivo hücre içi kayıt ve hücre içi boyama tekniği ilk geliştirme ile ilgili yardım için Brown ve David Maxwell yılında ilk eğitim için teşekkür ederim . M. Gümüş Ben collocalization makro yardım için teşekkür ederim. Birçok bilim adamı kiminle, R. Donga, M. Moritani, P. Luo, R. Ambalavanar dahil olmak üzere, bu tekniğin gelişimi sağlanan anlayış işbirliği var. Bu teknik, NIH hibe DE10132 DE15386 ve RR017971 önemli desteği ile geliştirildi.

1. Hayvan Hazırlık <ol><li> Pentobarbital sodyum (50mg/kg &#39;dan IP) ve bir ısıtma yastığı yer sıçan anestezisi. Hayvan kesme makineleri ile arka kafatası örten deri Tıraş. Hayvan ayak parmakları palpebral refleks yokluğu gibi sıkışmak yanı sıra zaman, bir geri çekilme refleksi ve seslendirme olmaması için cerrahi anestezi düzeyi seviyesi testi ile elde edilmiş olduğunu sağlamak için kontrol edin. Anestezi düzeyi her 15 dakikada bir kontrol edin ve sodyum pentobarbital 15mg/kg her 45 dakikada bir enjeksiyonu tarafından anesthetisa cerrahi bir düzeyde tutmak. </li><li> Aseptik teknik kullanın ve inguinal bölge, karın ve uyluk oluşan kırışık bir kesi hemen distalinde ve femoral vene kanül (1 mm çapında, Clay Adams) ve femoral arterden eklemek. Submandibular bölgede orta hat kesi yapmak, infrahyoid kasları yansıtmaktadır. Trakea küçük bir kesi yapın ve havalandırma sağlamak için bir kanül (2 mm çap) yerleştirin. Place.Monitor sistemik arteryel kanül ile diyastolik ve sistolik kan basıncı sabitlemek için cannual trachael etrafında bir sütür Tye. Şimdi geri çekilme ve palpebral refleks izlemeye ek olarak anestezi düzeyini ölçmek için kan basıncı monitör. Venöz kanül ile gerektiğinde ek anesteziklere. </li><li> Sıçan stereotaksik bir çerçevenin içine yerleştirin ve beyincik maruz kraniotomi gerçekleştirmek. Kemirgen ventilatör trakeal kanül takın. Nemlendirilmiş hava ile 100/min hızında 2 cm 3 hacmi ile hayvan havalandırınız. Akciğer çökmesini engellemek için 1 cm H 2 O bir pozitif ekspirasyon sonu basınç sağlanmalıdır. Her 15 dakikada atelektazi önlemek için akciğerlere hyperinflate. Sonra ısıtılmış mineral yağ (30 ° C) ile beynin yüzeyini kaplayan. Doğrudan parietal ve interparietal kemikler arasındaki birleşme altında bulunan büyük venöz sinüs önlemek için dikkatli olun. </li><li> Elektromanyetik bir vibratör ile birleştiğinde bir çubuk siyanoakrilat tutkal sürün ve çene diastema çubuk eklemek. Vibratör ya da A / D ve bilgisayar çıktılarını ya da sinyal üreteci Mandibula kullanarak komut sinyalleri taşıyın. </li><li> Kraniotomi bitişik cilt altında bir siliver-gümüş klorür topraklama elektrot yerleştirin. </li></ol> 2. Elektrot hazırlama <ol><li> Yatay bir mikroelektrot çektirmenin veya kuvars Alüminosilikattan cam kullanarak Mikroelektronlar Üretiyor. </li><li> 0.25m KCl ile 0,5 M Tris HCl tamponu (pH 7.6) veya serum fizyolojik içinde% 2 tetramethlyrhodamine (pH 6) çözünmüş% 5-10 biotinamide mikroelektrot doldurun. Elektrot empedansı bir elektrot test cihazı ile kontrol edin. 60-80M Ω empedans ile geniş çaplı aksonlar kaydetmek için elektrotlar yapmak, küçük akson ve internöronlar 80-150MΩ empedansı ile elektrotlar. </li><li> Elektrometre başkanı aşamaya mikroelektrot yerleştirin. , Hayvanın başının arkasında pozisyonda sabit küçük bir teleskop (20X kapalı bir dürbün ağı ile) ile elektrot gözünüzde canlandırın. Elektrotlar stereotaxically büyük bir hassasiyetle yerleştirilmiş sağlar çünkü teleskop kullanarak önemlidir. </li></ol> 3. Elektrofizyolojik kayıt ve hücre içi boyama <ol><li> Pia mater küçük bir açılış yapmak ve elektrot beyin dokunduğunda bir geri besleme sinyali üretilir, böylece kapasitans geribildirim telâfi. Bu beynin yüzeyine doğru konum sağlar. </li><li> Tekrarlanan mandibular deplasman üretmek ve bir step motor ile beyin içine elektrod ilerlemek. </li><li> DC potansiyel ani damla nöronal impalements tanır ve devam eden sinaptik aktivite intrasomatic nöronal impalements belirlemek. Kapasitans karşılama elektrot vızıltı ya da nadiren başarılı bir hücreye nüfuz dokunarak. Çünkü elektrotlar yüksek empedans, nöronal yanıtları nadiren penetrasyon için önce gözlenir. </li><li> Nöronal yanıt rampa basılı tutun ve sinüzoidal çene hareketi ile karakterize bir nöron Impale ve penetrasyon stabil sayılır sonra, 5 Harita nöronun alıcı alanında iletken olmayan bir prob ile başının etrafında deri ve intra oral kavite problama tahta bir sopa gibi. Çalışma ile ilgili ise, nöron, kas kasılması ve zararlı uyaranlara gibi diğer işlevsel ilgili uyaranlara yanıt incelemek 6. Primer afferent nosiseptif nöronal reseptörler nosiseptif uyaranlara cevap. Genel anesthesetics pentobarbital sodyum gibi primer afferent nöronların aksonal iletim engellemek değil, sinaptik iletim deprese yok. Böylece birincil afferent nöron akson aksonal iletim korunur. </li><li> 15 70nA dakika olmak üzere toplam enjeksiyon süre akımı (DC, 1-4NA) enjekte edilir. Mevcut enjeksiyon sırasında elektrot penetrasyon Monitör ve membran potansiyeli-30mV daha olumlu hale gelirse durdurma.</li></ol> 4. Doku işleme <ol><li> (% 0.9 NaCl 38 durulama vasküler pentobarbital doz aşımı (140mg/kg IV) ve serpmek hayvan Euthanize ° C heparin 500 adet ve 1 ml% 2% 4 0.1M fosfat tamponu (pH 7.4) paraformaldehid takip xylocaine . </li><li> Ya da frontal, sagittal veya yatay düzlemde bir vibratome kullanarak 50-100μm beyin ve o bölümü çıkarın. Secions 25 ° C PBS içinde serbest dalgalı toplanır. </li><li> % 1-2 normal keçi serumu kuluçka ve% 1 Triton X 0.01m PBS-100 avidin biotin kompleksi inkübasyon (1:50 Elite Vectastain) tarafından takip DAB için beyin işleyin. H 2 O 2 ile nikel-DAB kullanarak bölümleri tepki. Texas Kırmızı, 4 geceleme PBS içinde avidin-biotin kompleksi (1:50) inkübe bölümleri ° C ve 4 ° C gecede PBST% 4 Texas Red avidin DCS kuluçkaya yatmaktadır. Işlemek için 23 ° C&#39;de bir floresan Nissl leke (NeuroTrace) 20min bölümler Counterstain </li><li> Nöron rodamin enjekte edildi, bir floresan mikroskobu (Ex-545nm) ile doğrudan enjekte nöron görselleştirmek. </li></ol> 5. Retrograd etiketleme, immünhistokimya, konfokal görüntüleme, kantitatif ko analiz yöntemi birleştiren Bu tekniğin diğer yöntemler ile birleştirerek başarı iyi bir hücre içi etiketleme üzerine büyük ölçüde bağımlı. <ol><li> Bu yöntem kolaylıkla retrograd nöronal etiketleme ile kombine edilebilir görüntülendi. 7-10 Bunu yapmak için, hayvan anestezi ve aseptik teknik kullanılarak, horseradish peroksidaz (% 20 horseradish peroksidaz Sigma VI, Sigma) gibi nöronal tracer enjekte ve% 1 buğday mikrop gibi masseter kası gibi periferik hedef bölgeye horseradish peroksidaz (Sigma) aggultinated. Bu nöronal tracer periferik aksonları içine alınır ve motonöron somata aksonal taşıma araçları ile taşınan olacaktır. Masseter kası için, izleyicinin 15μl steril 10 mikrolitrelik mikroenjektör kullanarak kas içine enjekte edilir. Hayvanlar daha sonra anestezi kurtarıldı ve daha sonra kurtarma için kafesler yerleştirilir kadar bir ısıtma yastığı yerleştirilir ve takip edilmektedir. 24 saat sonra, hayvanları uyutmak ve nöronlar karakterize fizyolojik ve hücre onlara leke. Sonra HRP olarak tetramethylbenzidine (TMB), kobalt sabitleyici ve yoğun olarak ve tungstat Sodyum siyanür kullanarak varlığı için yukarıda ve süreç doku kesitlerinde anlatıldığı gibi hayvan serpmek. % 1 sodyum Yeri bölümleri 0.1M PBS (pH 6.5) ve 0.0007% 15 mutlak etanol ve aseton içinde çözünmüş TMB çözünmüş tungstat ° C, 20 dk. Ardından 1.0 ml% 0.3, 60 dakika süreyle inkübasyon çözüm 100 ml H 2 O 2 ekleyerek doku bölümleri tepki . % 0.05 diaminobenzidin (pH 7.4),% 0.02 kobalt, ve% 0.01 0.1M 10 dakika süreyle PBS (pH 7.4) H 2 O 2 0.1M PBS (pH 6.5) yer doku ve durulayın. 37 ° C Açıklandığı gibi biotinamide enjekte nöron görünüm için doku Süreci ve etiketli duyusal nöron ve çeşitli motor nöronlar arasındaki ilişkilerin görselleştirmek. </li><li> Burada gösterilen tekniği de kolayca immnocytochemistry ile kombine edilebilir. 6. Bir örnek olarak, immunocytochemically sinaptofizin için doğru etiketli nöronların içinde sinaps (Şekil 3) bulmak için beyin süreci. Bunu yapmak için, 2 gün, 4 ° C ve daha sonra 23 1s için anti-fare FITC (1:400) inkübe ° C için fare anti-sinaptofizin antikor (1:10,000) beyin bölümleri inkübe </li><li> Floresan işaretleri ile etiketlenmiş veya floresan görüntüleme için işlenmiş Nöronlar konfokal görüntüleme için idealdir. Şekil 3, bir akson bouton aracılığıyla konfokal mikroskobu ile elde edilen optik bölümünde bir örnektir. (Şekil 3). Etiketli nöron (Şekil 4) ile birden fazla optik bölümleri satın alarak etiketli nöronların animasyonlar oluşturun . </li><li> Bu yöntem ile etiketlenmiş Nöronlar, kantitatif ko analiz için kullanılabilir. Bunu yapmak için, NIH Image ile birlikte kamuya açık bir yazılım makro (http://phy.ucsf.edu/ ° idl / colocalization.htm bulundu) kullanın. Sinaptofizin immünhistokimya ile hücre içi etiketleme birleştirerek tek bir akson terminalleri içinde sinaptofizin Localize 6 </li></ol> 6. Temsilcisi Sonuçlar: Bu yöntemi kullanarak elde edilebilir temsilcisi sonuçları genel bir bakış Şekil 1&#39;de gösterilmiştir. Bu tek beyin sapı nöron elektrofizyolojik açıkça görülebileceği gibi, alt çenenin hareketi sırasında kaydedilen ve hareketi sırasında, bu nöronun yanıt (Şekil 1 sola, açık mavi alt) modüle oldu. Bu nöron elektrofizyolojik karakterizasyonu sonra biotinamide ile enjekte edilir ve daha sonra görünüm için işlendi. Yeniden inşa nöron (Şekil 1 orta, yeşil) gerçek olabiliranatomik bir dönüm noktası Ted, bu durumda trigeminal motor çekirdeğinin (kırmızı anahat) atanmış. Nöron ve yeniden yapılanma hareketi sırasında nöronal yanıt dayanarak bu ikincil kas mili afferent nöron olarak tanımlanabilir. Şekil 2 çene yer değiştirme sırasında bir nöronun fizyolojik yanıt temsili bir örnek göstermektedir. Nöronun tepkisi ani ateş frekans olarak temsil edilmektedir. Nöronal yanıt mandibular yerinden bu özel nöron mandibular konumu ile ilgili duyusal feedback sağlar belirten yakından taklit eder. Şekil 3 Not sinaptofizin için boyama ve Nissl bir leke ile birlikte bir intraselüler lekeli akson yüksek bir büyütme görüntü. Akson bouton içinde sinaptofizin (sarı) ko unutmayın. Şekil 4 fizyolojik karakterize ve intraselüler etiketli, tek bir nöronun bir animasyon. <img src="/files/ftp_upload/2650/2650fig1.jpg" alt="Şekil 1"/> Şekil 1 Genel Bakış yöntemi. Üst sol: mandibular deplasman. Tek nöron (sarı) Orta Hücre içi kayıt (yeşil). Bu nöron morfolojisi, hücre içi kayıt ve enjeksiyondan sonra yeniden inşa edildi. Kırmızı anahat trigeminal motor çekirdeğinin yeri gösterir. Sol alt çene hareketi sırasında, bu nöronun fizyolojik yanıt. <img src="/files/ftp_upload/2650/2650fig2.jpg" alt="Şekil 2"/> Şekil 2 çenenin hareketi sırasında in vivo olarak kaydedilen tek bir kas duyusal nöron Temsilcisi fizyolojik yanıt. Çene deplasman nöronal yanıt benzerliği dikkat edin. <img src="/files/ftp_upload/2650/2650fig3.jpg" alt="Şekil 3"/> Şekil 3 problama kas sırasında yanıt intraselüler lekeli duyusal nöronun sinaptik BOUTONS (kırmızı şişlikler) Terminal aksonal arborization. Sinaptofizin için sonraki immünositokimyasal işleme aksonal bouton içinde sinaptofizin lokalizasyonu (sarı) gösterir. Yeşil floresan Nissl leke. Şekil 4, akson fizyolojik yanıt mandibuler hareketi sırasında in vivo kaydedildi kas mili primer afferent nöron akson Animasyon sonra intraselüler boyanmış ve görselleştirme için işlenmiş. <a href="/files/ftp_upload/2650/collateral4.avi">Şekil 4 yüksek çözünürlüklü video</a> <a href="/files/ftp_upload/2650/medres.mov">Şekil 4 orta çözünürlüklü video</a>

<ol>
	<li>Cuellar, C. A., Tapia, J. A., Juarez, V., Quevedo, J., Linares, P., Marinez, L., Manjarrez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation+of+sinusoidal+electrical+waves+along+the+spinal+cord+during+a+fictive+motor+task.">Propagation of sinusoidal electrical waves along the spinal cord during a fictive motor task.</a> J. Neurosci. 29, 798-810 (2010).</li><li>Frigon, A., Gossard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+specialized+rhythm-generating+mechanisms+in+the+adult+mammalian+spinal+cord.">Evidence for specialized rhythm-generating mechanisms in the adult mammalian spinal cord.</a> J. Neurosci. 30, 7061-7071 (2010).</li><li>Wang, W., Chan, S. S., Heldman, D. A., Moran, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+cortical+representation+of+hand+translation+and+rotation+during+reaching.">Motor cortical representation of hand translation and rotation during reaching.</a> J. Neurosci. 30, 958-962 (2010).</li><li>Ma, C., He, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+investigating+cortical+control+of+stand+and+squat+in+conscious+behavioral+monkeys.">A method for investigating cortical control of stand and squat in conscious behavioral monkeys.</a> J. Neurosci. Meth. 192, 1-6 (2010).</li><li>Dessem, D., Donga, R., Luo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary-+and+secondary-like+jaw-muscle+spindle+afferents+have+characteristic+topographic+distributions.">Primary- and secondary-like jaw-muscle spindle afferents have characteristic topographic distributions.</a> J. Neurophysiol. 77, 2925-2944 (1997).</li><li>Dessem, D., Moritaini, A., Ambalavanar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nociceptive+craniofacial+muscle+primary+afferent+neurons+synapse+in+both+the+rostral+and+caudal+brain+stem.">Nociceptive craniofacial muscle primary afferent neurons synapse in both the rostral and caudal brain stem.</a> J. Neurophysiol. 98, 214-223 (2007).</li><li>Luo, P., Dessem, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inputs+from+identified+jaw-muscle+spindle+afferents+to+trigeminothalamic+neurons+in+the+rat:+a+double-labeling+study+using+retrograde+HRP+and+intracellular.">Inputs from identified jaw-muscle spindle afferents to trigeminothalamic neurons in the rat: a double-labeling study using retrograde HRP and intracellular.</a> J. Comp. Neurol. 353, 50-66 (1995).</li><li>Dessem, D., Luo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jaw-muscle+spindle+afferent+feedback+to+the+cervical+spinal+cord+in+the+rat.">Jaw-muscle spindle afferent feedback to the cervical spinal cord in the rat.</a> J. Comp. Neurol. 128, 451-459 (1999).</li><li>Luo, P., Wong, R., Dessem, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projection+of+jaw-muscle+spindle+afferents+to+the+caudal+brainstem+in+rats+demonstrated+using+intracellular+biotinamide.">Projection of jaw-muscle spindle afferents to the caudal brainstem in rats demonstrated using intracellular biotinamide.</a> J. Comp. Neurol. 358, 63-78 (1995).</li><li>Luo, P., Dessem, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+anatomy+of+physiologically+identified+jaw-muscle+spindle+afferent+terminations+onto+retrogradely+labeled+jaw-elevator+motoneurons+in+the+rat.">Ultrastructural anatomy of physiologically identified jaw-muscle spindle afferent terminations onto retrogradely labeled jaw-elevator motoneurons in the rat.</a> J. Comp. Neurol. 406, 384-401 (1999).</li><li>Hassani, O. K., Henny, P., Lee, M. G., Jones, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+neurons+intermingled+with+orexin+and+MCH+neurons+in+the+lateral+hypothalamus+discharge+maximally+during+sleep.">GABAergic neurons intermingled with orexin and MCH neurons in the lateral hypothalamus discharge maximally during sleep.</a> Eur. J. Neurosci. 32, 448-457 (2010).</li><li>Inokawa, H., Yamada, H., Matsumoto, N., Muranishi, M., Kimura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Juxtacellular+labeling+of+tonically+active+neurons+and+phasically+active+neurons+in+the+rat+striatum.">Juxtacellular labeling of tonically active neurons and phasically active neurons in the rat striatum.</a> Neuroscience. 168, 395-404 (2010).</li></ol>

(1985 revize Ulusal Sağlık Enstitüsü, Yayın No 86-23), Laboratuvar Hayvanları Bakım ve Kullanım Kılavuzu&#39;nda belirtilen ana hatlarına ve yönetmelik hükümleri uyarınca ve Maryland Üniversitesi Hayvan Bakım hayvanlar üzerinde deneyler yapılan ve Komite kullanın.

Burada gösterilen yöntem tek nöron fonksiyonu ile ilgili önemli bilgi sağlar ve nasıl bireysel nöronların yanıt nöronal devreleri katkıda güçlü bir tekniktir. 9 Bu bilgi sensorimotor fonksiyon anlamak için esastır. Bu tekniğin en büyük gücü, çok sayıda fizyoloji, morfoloji ve sinaptik morfolojisi ve dağıtım da dahil olmak üzere bir nöron ile ilgili parametrelerin belirlenmesi sağlar. Retrograd nöronal etiketleme nöronal devreleri gibi ek bilgi gibi diğer teknikleri ile kombine ...

<table border="1"><tbody><tr><th> Reaktif ya da ekipman Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar </th></tr><tr><td> elektromanyetik vibratör </td><td> Ling Dinamik Sistemler </td><td> V101 </td><td></td></tr><tr><td> sinyal üreteci </td><td> Geri Beslemeli Sistemlerin </td><td> PFG605 </td><td> Trapezoidal çıkış sinyali üretme yeteneğine sahip </td></tr><tr><td> elektrot cam </td><td> Sutter Aletleri </td><td> AF100-68-10 </td><td> filamanlı </td></tr><tr><td> elektrot çektirmenin </td><td> Sutter Aletleri </td><td> Model P-2000 veya P-80 </td><td></td></tr><tr><td> biotinamide </td><td> Vektör Laboratuvarları </td><td> SP-1120 </td><td> 4 saklanan ° C </td></tr><tr><td> Texas Red avidin DCS </td><td> Vektör Laboratuvarları </td><td> A-2016 </td><td></td></tr><tr><td> tetramethlyrhodamine </td><td> Moleküler Probları </td><td> D-3308 </td><td> 3000 molekül ağırlığı, lisin tamir edilebilir </td></tr><tr><td> fare anti-sinaptofizin antikor </td><td> Chemicon </td><td> MAB5258 </td><td></td></tr><tr><td> floresan Nissl leke </td><td> Neurotrace, Moleküler Probları </td><td> N-21480 </td><td></td></tr><tr><td> elektrot test </td><td> Winston Elektronik </td><td> BL-1000-B </td><td> elektrot empedansı ölçmek için </td></tr><tr><td> elektrometre </td><td> Akson Aletleri </td><td> Axoprobe 1A, Axoclamp 2B </td><td></td></tr></tbody></table>

physiological, morphological and neurochemical characterization of neurons modulated by movement

Bireysel nöronlarda ve nöron devreleri fonksiyonu rol duyusal ve motor fonksiyonların nöronal mekanizmaları anlamak için esastır. Bir hayvan hareketi sırasında statik 1,2 veya kayıt ekstrasellüler nöronal aktivitenin ise sensorimotor mekanizmaları çoğu soruşturma nöron veya inceleme güveniyor. 3,4 sensorimotor fonksiyonu için temel bir arka plan bu çalışmalar varken, onlar da işlevsel bilgi değerlendirmek yok bir hareket sırasında ortaya çıkan ya da nöronun tam anatomi, fizyoloji ve nörokimyasal fenotip karakterize yeteneği sınırlıdır. Bir in vivo hareketi sırasında bireysel nöronların geniş karakterizasyonu olanak veren bir teknik burada gösterilir. Bu teknik, primer afferent nöronların çalışma değil, aynı zamanda motor nöronlar ve sensorimotor internöronlar karakterize etmek için kullanılabilir. Başlangıçta tek bir nöronun yanıt nöron için açık alanın belirlenmesi takip mandibula çeşitli hareketler sırasında elektrofizyolojik yöntemler kullanılarak kaydedilir. Nöronal tracer sonra intraselüler nöron içine enjekte edilir ve beynin nöron, ışık, elektron veya konfokal mikroskobu (Şekil 1) ile görülebilmesi şekilde işlenir. Nöronal morfoloji, nöron (Şekil 2,3) fizyolojik yanıt ile ilişkili olduğu ve böylece karakterize nöron ayrıntılı morfoloji sonra yeniden. Bu iletişimde, bu tekniğin başarılı bir şekilde uygulanması için önemli anahtar detaylar ve ipuçları verilmektedir. Bu yöntem diğer tekniklerle birleştirerek değerli ek bilgiler altında çalışma nöron tespit edilebilir. Retrograd nöronal etiketleme nöronlar belirlemek için kullanılabilir etiketli nöron sinaps; nöron devresi böylece ayrıntılı olarak belirlenmesi. İmmünositokimya etiketli nöron içinde nörotransmitterlerin incelemek ve etiketli nöron sinaps nöronların kimyasal fenotipleri belirlemek için bu yöntem ile kombine edilebilir. Etiketli nöron da etiketli nöron ultrastrüktürel özellikleri ve mikrodevreler belirlemek için elektron mikroskobu için işlenebilir. Genel olarak bu tekniği iyice böylece sensorimotor fonksiyonu nöron rolünü içine önemli fikir izin in vivo hareket sırasında nöronlar karakterize etmek için güçlü bir yöntemdir.

Watch this Scientific Journal Video about Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement at JoVE.com

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

Hareketi sırasında memeli nöronlar in vivo fizyolojik tepki ölçmek ve nöronal morfoloji, nörokimyasal fenotip ve sinaptik mikrodevreler nöron fizyoloji ilişkilendirmek için bir teknik tarif edilir.

Hareketi tarafından Modülasyonlu Nöronlar Fizyolojik, Morfolojik ve nörokimyasal Karakterizasyonu

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

physiological-morphological-neurochemical-characterization-neurons

Research

JoVE Journal

Neuroscience

13.7K Görüntüleme.  University of Maryland. Hareketi sırasında memeli nöronlar in vivo fizyolojik tepki ölçmek ve nöronal morfoloji, nörokimyasal fenotip ve sinaptik mikrodevreler nöron fizyoloji ilişkilendirmek için bir teknik tarif edilir.

Video: Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

Eye Movement Monitoring of Memory

Explicit (often verbal) reports are typically used to investigate memory (e.g. "Tell me what you remember about the person you saw at the bank yesterday."), however such reports can often be unreliable or sensitive to response bias 1, and may be unobtainable in some participant populations. Furthermore, explicit reports only reveal when information has reached consciousness and cannot comment on when memories were accessed during processing, regardless of whether the information is subsequently accessed in a conscious manner. Eye movement monitoring (eye tracking) provides a tool by which memory can be probed without asking participants to comment on the contents of their memories, and access of such memories can be revealed on-line 2,3. Video-based eye trackers (either head-mounted or remote) use a system of cameras and infrared markers to examine the pupil and corneal reflection in each eye as the participant views a display monitor. For head-mounted eye trackers, infrared markers are also used to determine head position to allow for head movement and more precise localization of eye position. Here, we demonstrate the use of a head-mounted eye tracking system to investigate memory performance in neurologically-intact and neurologically-impaired adults. Eye movement monitoring procedures begin with the placement of the eye tracker on the participant, and setup of the head and eye cameras. Calibration and validation procedures are conducted to ensure accuracy of eye position recording. Real-time recordings of X,Y-coordinate positions on the display monitor are then converted and used to describe periods of time in which the eye is static (i.e. fixations) versus in motion (i.e., saccades). Fixations and saccades are time-locked with respect to the onset/offset of a visual display or another external event (e.g. button press). Experimental manipulations are constructed to examine how and when patterns of fixations and saccades are altered through different types of prior experience. The influence of memory is revealed in the extent to which scanning patterns to new images differ from scanning patterns to images that have been previously studied 2, 4-5. Memory can also be interrogated for its specificity; for instance, eye movement patterns that differ between an identical and an altered version of a previously studied image reveal the storage of the altered detail in memory 2-3, 6-8. These indices of memory can be compared across participant populations, thereby providing a powerful tool by which to examine the organization of memory in healthy individuals, and the specific changes that occur to memory with neurological insult or decline 2-3, 8-10.

Eye movement monitoring (or eye tracking) reveals where in space the eyes linger, when and for how long. Here, we demonstrate how eye tracking can be used to investigate the integrity of memory in multiple participant populations, without requiring verbal, or otherwise explicit, reports.

Bellek Eye Movement Kontrolü

Explicit (often verbal) reports are typically used to investigate memory (e.g. "Tell me what you remember about the person you saw at the bank ...

Nörobilim

Coherence between Brain Cortical Function and Neurocognitive Performance during Changed Gravity Conditions

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and focused on psychological aspects. Until now, objective observation of neurophysiological parameters has not been carried out - undoubtedly because the technical and methodological means have not been available -, investigations into the neurophysiological effects of weightlessness are in their infancy (Schneider et al. 2008).
While imaging techniques such as positron emission tomography (PET) and magnetic resonance imaging (MRI) would be hardly applicable in space, the non-invasive near-infrared spectroscopy (NIRS) technique represents a method of mapping hemodynamic processes in the brain in real time that is both relatively inexpensive and that can be employed even under extreme conditions. The combination with electroencephalography (EEG) opens up the possibility of following the electrocortical processes under changing gravity conditions with a finer temporal resolution as well as with deeper localization, for instance with electrotomography (LORETA).
Previous studies showed an increase of beta frequency activity under normal gravity conditions and a decrease under weightlessness conditions during a parabolic flight (Schneider et al. 2008a+b). Tilt studies revealed different changes in brain function, which let suggest, that changes in parabolic flight might reflect emotional processes rather than hemodynamic changes. However, it is still unclear whether these are effects of changed gravity or hemodynamic changes within the brain. Combining EEG/LORETA and NIRS should for the first time make it possible to map the effect of weightlessness and reduced gravity on both hemodynamic and electrophysiological processes in the brain. Initially, this is to be done as part of a feasibility study during a parabolic flight. Afterwards, it is also planned to use both techniques during medium- and long-term space flight.
It can be assumed that the long-term redistribution of the blood volume and the associated increase in the supply of oxygen to the brain will lead to changes in the central nervous system that are also responsible for anaemic processes, and which can in turn reduce performance (De Santo et al. 2005), which means that they could be crucial for the success and safety of a mission (Genik et al. 2005, Ellis 2000).
Depending on these results, it will be necessary to develop and employ extensive countermeasures. Initial results for the MARS500 study suggest that, in addition to their significance in the context of the cardiovascular and locomotor systems, sport and physical activity can play a part in improving neurocognitive parameters. Before this can be fully established, however, it seems necessary to learn more about the influence of changing gravity conditions on neurophysiological processes and associated neurocognitive impairment.

The effect of weightlessness and hypergravity on both hemodynamic and electrophysiological processes in the brain is going to be followed during parabolic flight by EEG and NIRS techniques. 
A feasibility study of a more complex experiment, which is planned to carry out during medium- and long-term space flight.

Beyin Kortikal Fonksiyon ve değiştirildi Yerçekimi Koşullar sırasında Nörobilişsel Performansı arasındaki uyum,

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and ...

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

Diyabetik Sıçanlarda Termal Nosiseptif Eşik Hızlı Belirlenmesinde

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

Emme Pipet Tekniği kullanılarak Olfaktör Reseptör Nöronlar Kaydedilmiş odorant bağlı Cevapları

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

Immünopresipitasyon ve Açil-Biotin Borsası (ABE) tarafından Yetiştirilmiş Hipokampal Nöronlar Protein Palmitoylation tespiti

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique known as electron probe microanalysis (EPMA) are described. Intramitochondrial calcium is a particular focus because of the critical role that mitochondrial calcium overload plays in neurodegenerative diseases. The method is based on the analysis of X-rays generated in an electron microscope (EM) by interaction of an electron beam with the specimen. In order to maintain the native distribution of diffusible elements in electron microscopy specimens, EPMA requires &#34;cryofixation&#34; of tissue followed by the preparation of ultrathin cryosections. Rapid freezing of cultured cells or organotypic slice cultures is carried out by plunge freezing in liquid ethane or by slam freezing against a cold metal block, respectively. Cryosections nominally 80 nm thick are cut dry with a diamond knife at ca. -160 &#176;C, mounted on carbon/pioloform-coated copper grids, and cryotransferred into a cryo-EM using a specialized cryospecimen holder. After visual survey and location mapping at &#8804;-160 &#176;C and low electron dose, frozen-hydrated cryosections are freeze-dried at -100 &#176;C for ~30 min. Organelle-level images of dried cryosections are recorded, also at low dose, by means of a slow-scan CCD camera and subcellular regions of interest selected for analysis. X-rays emitted from ROIs by a stationary, focused, high-intensity electron probe are collected by an energy-dispersive X-ray (EDX) spectrometer, processed by associated electronics, and presented as an X-ray spectrum, that is, a plot of X-ray intensity vs. energy. Additional software facilitates: 1) identification of elemental components by their &#34;characteristic&#34; peak energies and fingerprint; and 2) quantitative analysis by extraction of peak areas/background. This paper concludes with two examples that illustrate typical EPMA applications, one in which mitochondrial calcium analysis provided critical insight into mechanisms of excitotoxic injury and another that revealed the basis of ischemia resistance.

This paper describes the application of cryoanalytical electron microscopy to the quantitative measurement of total calcium content and distribution at subcellular resolution in physiologically defined biological specimens.

Elektron Probe X-ışını Mikroanaliz tarafından Nöronlar Toplam Kalsiyum Ölçümü

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

Kaynaklı Nöronlar içine Yetişkin İnsan Beyninin perisit türetilmiş Hücreler Lineage yeniden programlama

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

Akut Beyin Dilimleri nöronal Mikro şemalar Elektrofizyolojik ve Morfolojik Karakterizasyonu Eşleştirilmiş Patch-Kelepçe Kayıtlar Kullanma

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

Frenik Motor Nöronlar tarafından Diyafram İNNERVASYONU Fonksiyonel ve Morfolojik Değerlendirmesi

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing patterns. The inclusion of biocytin/neurobiotin in the recording electrode permits post-hoc recovery of morphological details, which are necessary to determine the dendritic arborization and the regions targeted by the axons of the recorded neurons. However, given the presence of morphologically similar neurons with distinct neurochemical identities and functions, immunohistochemical staining for cell-type-specific proteins is essential to definitively identify neurons. To maintain network connectivity, brain sections for physiological recordings are prepared at a thickness of 300 &#181;m or greater. However, this thickness often hinders immunohistological postprocessing due to issues with antibody penetration, necessitating the resectioning of the tissue. Resectioning of slices is a challenging art, often resulting in the loss of tissue and morphology of the cells from which electrophysiological data was obtained, rendering the data unusable. Since recovery of morphology would limit data loss and guide in the selection of neuronal markers, we have adopted a strategy of recovering cell morphology first, followed by secondary immunostaining. We introduce a practical approach to biocytin filling during physiological recordings and subsequent serial immunostaining for the recovery of morphology, followed by the restaining of sections to determine the neurochemical identity. We report that sections that were filled with biocytin, fixed with paraformaldehyde (PFA), stained, and coverslipped can be removed and restained with a second primary antibody days later. This restaining involves the removal of the coverslip, the washing of sections in a buffer solution, and the incubation of primary and secondary antibodies to reveal the neurochemical identity. The method is advantageous for eliminating data loss due to an inability to recover morphology and for narrowing down the neurochemical markers to be tested based on morphology.

This protocol presents a method for the morphological recovery of neurons patched during electrophysiological recordings using biocytin filling and subsequent immunohistochemical postprocessing. We show that thick biocytin-filled sections that were stained and coverslipped can be restained with a second primary antibody days&#160;or months later.

Nörokimyasal markerleri Biocytin dolu ve İşlenmiş Bölümler immün

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing ...