Bu çalışma Doğa Bilimleri ve Kanada&#39;nın Mühendislik Araştırma Kurumu (hibe # 249669 ve # 482986) ve Kalp ve Kanada İnme Vakfı (hibe # 485508) tarafından finanse edilmektedir. Yazarlar deney protokolleri geliştirmek onların yardım için Youssef El-Hayek ve Dr Qi Wan ederim.

Hayvan taşıma içeren tüm prosedürler kurumsal düzenlemeler (protokol no. 20009387) uyarınca Toronto Hayvan Bakım Komitesince Üniversitesi tarafından onaylanmıştır. Aşağıdaki yöntemler uygulanabilir durumlarda bir laminer akış kaputu, steril alet ve teknikler kullanılarak yapılmalıdır. Aşağıdaki protokol metninde, ifade &quot;EFH-SFM&quot; epidermal büyüme faktörü, temel fibroblast büyüme faktörü ve heparin ile takviye serum serbest medya anlamına gelir. Bu mitojenler kendi ayrışmamış devlet 4 YZK&#39;lar korumak için farklılaşmamış NPCs galvanotaksis araştırılırken EFH-SFM kullanılır. Olgun hücre tipleri farklılaşması için indüklenebilir NPCs galvanotaksis araştırırken, &quot;FBS-SFM&quot;% 1 fetal sığır serumu serum serbest medya anlamına gelir. FBS olgun nöral fenotipleri 5 içine NPCs farklılaşma teşvik etmektedir. 1. Sinir Öncüleri izolasyonu ve Kültür (Değil) Videoda gösterilen <ol><li> Servikal dislokasyon yoluyla isofluorane ve özveri ile bir CD1 fare (6-8 haftalık) Anesteziyoloji. </li><li> % 70 etanol içinde baş söndürün ve keskin disseksiyon makas ile hayvan başını kesmek. </li><li> Cerrahi forseps ile kafa tutarken, kafatası maruz dorsal yüzeyi üzerinde cilt kaldırmak. </li><li> Bir neşter ve hiçbir kullanma. 11 bıçak, mediyolateral eksen boyunca ön sinüs de kafatası puan, ve aynı zamanda rostro yönde dikiş boyunca sagital. </li><li> Uzakta olmayan baş parietal kemikler Peel. 7 kavisli bir forseps, delmek için beyin dokusu umursamadı alıyor. </li><li> Beyincik altından başlayarak ve olfaktor doğru ilerleyen beyin altında ince bir spatula yerleştirin. Forseps ile yerde kafatası tutarken, yavaşça kafatası beyin çekin ve hemen buz yapay beyin omurilik sıvısı (aşağıda tarifleri bakınız) yerleştirin. </li><li> Bir diseksiyon mikroskobu altında, steril kullanarakDiseksiyon makas ve forseps orta hat boyunca yarım beyin kesti. Medial (kesim) yüzey yukarı bakacak şekilde her hemisferin döndürün. </li><li> Bir hemisfer seçin ve medial yüzeyi yukarı bakacak şekilde, korpus kallozum (KK posterior bölgesi) splenium bulun. </li><li> Korteks yüzeyinden dorsoventral ekseni boyunca korpus kallozum spleniumda bir kesi olun. </li><li> Lateral ventrikül medial ve lateral duvarları açığa koku ampul doğru kazılı korteks Peel. </li><li> Dorsal yüzeyi yukarı bakacak şekilde yarımkürede döndürün ve NPC&#39;ler 6 ikamet periventriküler bölge içerir maruz medial ve lateral duvarlar, kesip toplamak için kavisli microscissors kullanın. </li><li> Tekrar diğer yarımkürede için 1,8-1,11 adımları. </li><li> Bir 15 cc tüp içinde tripsin solüsyonu (aşağıda tarif bakınız) 7 ml içine izole edilmiş doku pipet, ve 37 ° C de bir rocker tüp yerleştirmek25 dakika inkübatör. </li><li> 5 dakika boyunca 1,500 rpm&#39;de santrifüje tüp, süpernatan aspire, ve (aşağıda tarif bakınız) tripsin inhibitörü çözelti 2 ml doku tekrar süspansiyon. </li><li> Yavaşça hava kabarcıkları önlemek için dikkatli bir küçük kuyu Pasteur pipeti 30-50 kez doku çiğnemek. </li><li> 5 dakika boyunca 1,500 rpm&#39;de santrifüje tüp, süpernatan aspire ve pelet 3-5 kez triturating ile SFM 1-2 ml (aşağıda tarif bakınız) içinde tekrar süspansiyon haline getirin. </li><li> 3 dakika 1.500 rpm&#39;de tüp santrifüjleyin, SFM + EFH arasında bölgesindeki süpernatant ve tekrar süspansiyon 1 ml aspire. </li><li> SFM + EFH içinde başına 10 ul hücre yoğunluğunda bir T25 kültürü şişesi içinde bir haemocytometer ve plaka hücreler ile hücre yoğunluğu, canlı sayın. </li><li> Kültür NPC oluşan serbest yüzen birincil neurospheres verim örselenmemiş 7 gün büyümeye izin verin. </li></ol> 2. Galvanotaksis Odası Hazırlama <ol><li> Hayır 3 kare cam yerleştirin. 1 kapak camları (22 x 22 x 0.17 mm) iGecede 6N hidroklorik asit na şişe. </li><li> Ertesi gün, kare hiçbir gelen cam 6 dikdörtgen şeritler (22 x 5 x 0.17 mm) kesmek için bir elmas uçlu cam kesici kullanın. 1 kapak fişleri. </li><li> Bir laminer akış kaputun içine asitle yıkanmış kare fişleri ve dikdörtgen fişleri aktarın. Doku kültürü dereceli otoklava su ile, sonra% 70 etanol ile ilk dikdörtgen ve kare şeritler yıkayın ve silin bir Kim üzerinde kurumasına izin (ilave sterilite, cam hava kuru izin verilebilir). </li><li> Kare cam slaytlar bir yüzeyi çevre vakum gres sürün ve 60 mm plastik Petri kaplarının tabanına onları mühür. </li><li> Dikdörtgen cam şeritleri bir yüzeyi uzun ekseni boyunca vakum uygula yağ, ve bir merkezi oluk oluşturmak için kare cam slaytların zıt kenarları (birbirlerine paralel olduğu gibi) ile geri kapatın. </li><li> Laminer akış kaputu en az 15 dakika süreyle odaları UV sterilize. </li><li> Poli-L-lysi pipetle 250-300 ul2 saat oda sıcaklığında inkübe odaları ve merkezi oluk üzerine ne. </li><li> İnkübasyon süresinin sonunda önce yaklaşık 15 dakika, (aşağıda tarif bakınız) Matrigel solüsyon hazırlanır. </li><li> , Poli-L-lizin aspire otoklavlanmış 1 mL su, ve merkezi oluklar üzerine Matrigel solüsyonu bir pipet 250-300 ul ile merkezi çukurların yıkayın. </li><li> 1 saat boyunca 37 ° C&#39;de inkübe odaları. </li><li> Matrigel çözüm aspire ve yavaşça SFM 1-2 ml merkezi çukurların yıkayın. </li><li> Pipet 100 merkez çukurların üzerine EFH-SFM veya FBS-SFM ul ve sayma mikroskop sahneye galvanotaksis odaları aktarın. </li><li> Pipet 3-4 60 mm Petri kabı içine neurosphere içeren kültür ml ve sayma mikroskop aşamasına Petri kabı aktarın. </li><li> 5x görüntüleme amacı, her galvanotaksis merkezi çukur üzerine 5-8 bütün neurospheres (dört bir defada) aktarmak için bir P10 pipet kullanınOnları dissociating ve dikkatle Matrigel substrat aksatmadan merkez çukur etrafında neurospheres yayılmış olmadan oda. </li><li> Merkez oluklar üzerine EFH-SFM veya FBS-SFM ek 150-200 ul ekleyin. </li><li> 37 ° C,% 5 CO2, 17-20 saat süreyle% 100 nemlendirilmiş kuluçka makinesi içine galvanotaksis odaları aktarın (eğer farklılaşmamış analiz NPC) Şekil 1 &#39;de gösterildiği gibi neurospheres Matrigel substrata yapışır ve tek hücre halinde ayrıştırmak için izin vermek için . Farklılaşmış NPC analiz ise, kuluçka süresi, hücrelerin farklılaşması izin vermek için 69-72 saat için genişletilmelidir. </li></ol> 3. Hücre Time-Lapse Görüntüleme Canlı <ol><li> Canlı hücre görüntüleme sistemi 37, denge sağlaması için izin ° C,% 5 lik önce geçen zaman kaydının başlatılması için 30 dakika arasında, en az CO2. </li><li> Bir ucundan 1 mm çaplı gümüş tel, bobin ikisi 12 cm parçalar koparıp ve Clorox b koyunleach 20 dakika için Ag / AgCI elektrotlar oluşturur. </li><li> Bir sayma mikroskop sahneye galvanotaksis odaları aktarın ve aşağıdaki kriterlere göre canlı hücre görüntüleme göçü analiz için kullanılacak olan kamara seçin: i) neurospheres hücreleri sahip olmalıdır) tek hücreler ve ii içine neredeyse tamamen ayrılmış olmalıdır az-hayır süreçleri yuvarlak morfolojileri hücre gövdeleri uzanan. </li><li> Ayrı bir kare hayır birlikte, bir laminer akış kaputun içine seçilen galvanotaksis odasına aktarın. 1 cam kapak kayma ve vakum gres. </li><li> Kapak otoklavlanmış su ile, sonra% 70 etanol ile ilk kayma ve Kapak kayma iki paralel kenarlarında vakum gres bir şerit uygulayın yıkayın. </li><li> , Odasının merkezi çukur gelen kültür ortamı aspire sonra hızlı bir şekilde, iki paralel dikdörtgen cam şerit üzerinde kalan yağ şeritler, etkili bir çatı yaratılması da bu tür oda üzerine kapak slipi (yağ-yüzü aşağı bakacak şekilde) yerleştirmekodasına. </li><li> Kapiller eylem yoluyla merkezi çukur içine taze EFH-SFM veya FBS-SFM Pipet 100 ul. </li><li> Şekil 2&#39;de gösterildiği gibi, merkezi oluk her bir ucunda, kültür besiyeri havuzları için sınırlarını oluşturmak için vakum yağ kullanır. </li><li> PVC boru, iki 15 cm parçalar koparıp ve dikkatle hiçbir kabarcıklar tüpler oluşmaya sağlanması, boru içine agaroz enjekte çözüm için bir 18 gauge iğne ile 10 cc şırınga kullanmak ve jel 5 dakika boyunca katılaşmaya izin. </li><li> Agaroz jel tüpler, Ag / AgCI elektrodları, ve kültür ortamı rezervuar olarak kullanılmak üzere boş 60 mm Petri kapları bir çift ile birlikte, bir canlı hücre görüntüleme sistemi için galvanotaksis oda aktarın ve Ag / AgCI elektrot içerir. Galvanotaksis odası 37 içinde dinlenmek için izin ° C, 20-30 dakika süreyle% 5 CO 2 ortamında. </li><li> Bu süre zarfında, sondaj delikleri ile 2 boş Petri kaplarının kapakları ve galvanotaksis odasının en Petri kabı kapağı hazırlamakbunların Şekil 3 &#39;de gösterildiği gibi bir Dremel ya da benzer araç ile içine. </li><li> Pipet her boş Petri kabına merkez çukur iki yüzüne EFH-SFM veya FBS-SFM 1-1.5 ml ve SFM 7-8 ml. Her yemeğin içine galvanotaksis odasının merkezi çukur ve yerde bir Ag / AgCl elektrot her tarafında il birini Petri. Şekil 4&#39;te gösterildiği gibi agaroz jel köprüleri ile elektriksel süreklilik kurmak için galvanotaksis haznesi ve Petri kutularına arasındaki boşluğu. </li><li> Elektrik akımı ölçmek için seri bir ampermetre ile, harici bir güç kaynağına Ag / AgCl elektrot bağlayın ve güç kaynağını açın. Doğrudan merkezi çukur boyunca elektrik alanın gücünü ölçmek için bir voltmetre kullanın ve istenen elektrik alan şiddeti (Bu laboratuarda yapılan deneyler ile 250 mV / mm dcEF gücü kullanmak elde edilene kadar güç kaynağının çıkış ayarlayabilirsiniz 1 ve 1.5 mA arasında elektrik akımı). </li><li> Inisiyatifite time-lapse canlı hücre görüntüleme sistemi üzerinde modül ve deney süresi istenilen miktarda için çalışmasına izin. Tahlil tamamlanmasından sonra, standart bir immun analiz için% 4 paraformaldehit içinde hücreler düzeltmek. </li></ol>

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Çıkar çatışması ilan etti.

Bu protokol, önceki çalışmalarda 7-9 köklü yöntemler adapte edilmiştir. Galvanotactic odaları hücre ekimi kısıtlanmasının veya merkez çukur 10,11 mikroimalat için CO2 lazer ablasyonu kullanmak için de ayrı bir cam yapım dahil olmak üzere farklı teknikler kullanarak çeşitli inşa edilebilir. Bazı teknikler diğerlerinden daha zahmetli ve masraflı olabilir. Biz genellikle çoğu hücre biyoloji laboratuvarlarında bulunan malzemeler kullanılarak bir NPC galvanotak...

<table border="1"><tbody><tr><td height="44"> Öğenin ismi </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td></td><td></td><td></td><td> Sinir Prekürsör Hücre İzolasyonu </td></tr><tr><td> 2M NaCl </td><td> Sigma </td><td> S5886 </td><td> 11.688 g 100 ml dH 2 O içerisinde çözündürüldü </td></tr><tr><td> 1M KCl </td><td> Sigma </td><td> P5405 </td><td> 7.456 g 100 ml dH 2 O içerisinde çözündürüldü </td></tr><tr><td> 1M MgCl2 </td><td> Sigma </td><td> M2393 </td><td> 20.33 g 100 ml dH 2 O içerisinde çözündürüldü </td></tr><tr><td> 155 mM NaHCO 3 </td><td> Sigma </td><td> S5761 </td><td> 1.302 g 100 ml dH 2 O içerisinde çözündürüldü </td></tr><tr><td> 0.5M Glikoz </td><td> Sigma </td><td> G6152 </td><td> 9.01 g 100 içinde çözülmüşml dH 2 O </td></tr><tr><td> 108 mM CaCl2 </td><td> Sigma </td><td> C7902 </td><td> 1.59 g 100 ml dH 2 O içerisinde çözündürüldü </td></tr><tr><td> Penisilin-streptomisin </td><td> Gibco </td><td> 15070 </td><td></td></tr><tr><td> Sığır pankreasın tripsin </td><td> Sigma </td><td> T1005 </td><td></td></tr><tr><td> Koyun hyaluronidaz testisler </td><td> Sigma </td><td> H6254 </td><td></td></tr><tr><td> Kynurenic asit </td><td> Sigma </td><td> K3375 </td><td></td></tr><tr><td> Ovomucoid tripsin inhibitörü </td><td> Worthington </td><td> LS003086 </td><td></td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 12100046 </td><td></td></tr><tr><td> F12 </td><td> Invitrogen </td><td> 21700075 </td><td></td></tr><tr><td> % 30 Glikoz </td><td> Sigma </td><td> G6152 </td><td></td></tr><tr><td> % 7.5 NaHCO3 </td><td> Sigma </td><td> S5761 </td><td></td></tr><tr><td> 1M Hepes </td><td> Sigma </td><td> H3375 </td><td> 23.83 g 100 ml dH 2 O içerisinde çözündürüldü </td></tr><tr><td> L-glutamin </td><td> Gibco </td><td> 25030 </td><td></td></tr><tr><td> EGF </td><td> Invitrogen </td><td> PMG8041 </td><td> 20 ul birimler halinde hormon karışımı ve kısım 1 ml sulandırın. </td></tr><tr><td> FGF </td><td> Invitrogen </td><td> PHG0226 </td><td> 20 ul birimler halinde hormon karışımı ve kısım 0.5 ml sulandırın. </td></tr><tr><td> Heparin </td><td> Sigma </td><td> H3149 </td><td></td></tr><tr><td> Apo-transferrin </td><td> R &amp; D Systems </td><td> 3188-AT </td><td> 0.1 g 4 ml dH 2 0 içine çözünmüş </td></tr><tr><td> Putressin </td><td> Sigma </td><td> P7505 </td><td> Apo-transferrin içine 9,61 mg böylece çözülürgeçirdikleri evrim </td></tr><tr><td> Ensülin </td><td> Sigma </td><td> I5500 </td><td> 0.1N HCl 0.5 ml içine 25 mg çözülür ve dH 2 0 3.5 ml ekleyin </td></tr><tr><td> Selenyum </td><td> Sigma </td><td> S9133 </td><td></td></tr><tr><td> Progesteron </td><td> Sigma </td><td> P6149 </td><td></td></tr><tr><td> Standart Diseksiyon araçları </td><td> Güzel Bilim Araçları </td><td></td><td></td></tr><tr><td> Diseksiyon mikroskobu </td><td> Zeiss </td><td> Stemi 2000 </td><td></td></tr><tr><td></td><td></td><td></td><td> Galvanotaksis Odası Hazırlama </td></tr><tr><td> Kare cam kapak slaytlar </td><td> VWR </td><td> 16004 </td><td></td></tr><tr><td> 6N hidroklorik asit </td><td> VWR </td><td> BDH3204-1 </td><td></td></tr><tr><td> Yüksek vakum gres </td><td> Dow Corning </td><td></td><td></td></tr><tr><td> 60 mm Petri kapları </td><td> Fisher Scientific </td><td> 0875713A </td><td></td></tr><tr><td> Poli-L-lisin </td><td> Sigma </td><td> P4707 </td><td></td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 354234 </td><td> 150 ul birimler halinde çözülme ve kısım </td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 10082139 </td><td> NPC farklılaşma inducing Yalnızca kullanınız aksi SFM kullanımı + EFH kültür ortamı yukarıda da belirtildiği gibi </td></tr><tr><td> Mikroskop Sayma </td><td> Olimpos </td><td> CKX41 </td><td></td></tr><tr><td></td><td></td><td></td><td> Hücre Time-Lapse Görüntüleme Canlı </td></tr><tr><td> Gümüş tel </td><td> Alfa Aesar </td><td> 11434 </td><td></td></tr><tr><td> Ultrapure Agaroz </td><td> Invitrogen </td><td> 15510-027 </td><td></td></tr><tr><td> Isı Inactivated FBS </td><td> Sigma </td><td> 16140071 </td><td></td></tr><tr><td> PVC boru </td><td> Fisher Scientific </td><td> 80000006 </td><td> 3/32 &quot;ID x 5/32&quot; OD </td></tr><tr><td> Beyazlatıcı </td><td> Clorox </td><td></td><td></td></tr><tr><td> 10 cc şırınga </td><td> BD </td><td> 309604 </td><td></td></tr><tr><td> 18 gauge iğne </td><td> BD </td><td> 305195 </td><td></td></tr><tr><td> Dremel matkap </td><td> Dremel </td><td> Model 750 </td><td></td></tr><tr><td> Ters mikroskop nemlendirilmiş, İnkübe odası ile donatılmıştır </td><td> Zeiss </td><td> Axiovert-200M </td><td></td></tr><tr><td></td><td></td><td></td><td> Tarifler <table border="1"><tbody><tr><td> Madde </td><td> Hacim </td></tr><tr><td> 2M NaCl </td><td> 6.2 ml </td></tr><tr><td>1M KCl </td><td> 0.5 ml </td></tr><tr><td> 1M MgCl2 </td><td> 0.32 mi </td></tr><tr><td> 155mm NaHCO 3 </td><td> 16.9 ml </td></tr><tr><td> 1M Glikoz </td><td> 1 mL </td></tr><tr><td> 108 mM CaCl2 </td><td> 0,09256 mL </td></tr><tr><td> Penisilin-streptomisin </td><td> 1 mL </td></tr><tr><td> Otoklavlanmış su </td><td> 74 mL </td></tr></tbody></table> Yapay beyin omurilik sıvısı <table border="1"><tbody><tr><td> Madde </td><td> Hacim veya Yığın </td></tr><tr><td> Yapay beyin omurilik sıvısı </td><td> 30 ml </td></tr><tr><td> Sığır pankreasın tripsin </td><td> 40 mg </td></tr><tr><td> Koyun hyaluronidaz testisler </td><td> 22.8 mg </td></tr><tr><td> Kynurenic asit </td><td> 5 mg </td></tr></tbody></table> &lt;strong&gt; Tripsin Çözüm <table border="1"><tbody><tr><td> Madde </td><td> Hacim veya Yığın </td></tr><tr><td> SFM </td><td> 15 ml </td></tr><tr><td> Ovomucoid tripsin inhibitörü </td><td> 10 mg </td></tr></tbody></table> Tripsin İnhibitör Çözüm <table border="1"><tbody><tr><td> Madde </td><td> Hacim </td></tr><tr><td> Otoklavlanmış su </td><td> 37 ml </td></tr><tr><td> 10X DMEM/F12 </td><td> 10 mL </td></tr><tr><td> % 30 Glikoz </td><td> 2 ml </td></tr><tr><td> % 7.5 NaHCO3 </td><td> 1.5 ml&#39;lik </td></tr><tr><td> 1M Hepes </td><td> 0.5 ml </td></tr><tr><td> Transferrin, putressin çözümü </td><td> 4 mL </td></tr><tr><td> 25 mg ensülin çözelti </td><td> 4 mL </td></tr><tr><td> Selenyum </td><td> 100 &amp; Mu; L </td></tr><tr><td> Progesteron </td><td> 100 ul </td></tr></tbody></table> Hormon Mix (-20 ° C&#39;de 100 ml toplam mağaza) <table border="1"><tbody><tr><td> Madde </td><td> Hacim </td></tr><tr><td> Otoklavlanmış su </td><td> 37.5 ml </td></tr><tr><td> 10X DMEM/F12 (3:1) </td><td> 5 ml </td></tr><tr><td> % 30 Glikoz </td><td> 1 mL </td></tr><tr><td> % 7.5 NaHCO3 </td><td> 0.75 ml </td></tr><tr><td> 1M Hepes </td><td> 0.25 ml&#39;lik </td></tr><tr><td> Hormon mix </td><td> 5 ml </td></tr><tr><td> L-glutamin </td><td> 0.5 ml </td></tr><tr><td> Penisilin-streptomisin </td><td> 0.5 ml </td></tr></tbody></table> Serum Ücretsiz Media EFH-SFM: EGF 10 ul, FGF 10 ul ve Heparin FBS-SFM 3.66 ul ekleyin: 0.5 ml FBS ekleyin <table border = &quot;1&quot;&gt; <tbody><tr><td> Madde </td><td> Hacim </td></tr><tr><td> Matrigel </td><td> 150 ul </td></tr><tr><td> SFM </td><td> 3.6 ml </td></tr></tbody></table> Matrigel Çözüm Matrigel kısım bir buz kutusuna yerleştirilir ve SFM ile karıştırılmadan önce viskoz bir sıvı oluşturmak için 4-5 saat içinde yavaş yavaş çözülme izin verilmelidir. Bu alt tabaka Matrigel düzgün bir tabaka oluşumunu temin edecektir. Yavaş yavaş çözülmüş değil, elde edilen substrat muhtemelen hücre göçü engelleyen, Matrigel kümeleri ihtiva edecektir. <table border="1"><tbody><tr><td> Madde </td><td> Hacim veya Yığın </td></tr><tr><td> Ultrapure Agaroz </td><td> 10 ml GKD 2 0 içinde 300 mg </td></tr><tr><td> SFM Isı İnaktive FBS </td><td> 8 ml 2 ml </td></tr></tbody></table> MatrigelÇözelti bir 15 cc şahin tüp içinde 2 ml ısı ile aktifliği giderilmiş FBS SFM 8 ml karıştırılır. Mix 10 ml GKD bir Erlenmeyer şişesinde 2 0 ile agaroz ve 10 sn aralıklarla 30 saniye mikrodalga ısı, her 10 sn aradan sonra mikrodalga çözüm kaldırmak ve iyice karışması sağlanır. Son 10 sn mikrodalga döneminin ardından, 57 ° C su banyosunda SFM / FBS çözüm ve mağaza ile agaroz çözüm karıştırmak. </td></tr></tbody></table>

a galvanotaxis assay for analysis of neural precursor cell migration kinetics in an externally applied direct current electric field

Nöral kök ve erişkin memeli beynindeki progenitor hücreler (toplu olarak adlandırılır sinir öncü hücreleri) (NPC) keşfedilmesi neuroregenerative stratejilerin geliştirilmesi için bu hücrelerin multipotent ve proliferatif özellikler kullanılarak yönelik araştırmalar bir gövde yol açmıştır. Bu tür stratejilerin başarısı için kritik bir adım eksojen Transplantasyon sonrası lezyon yerinde doğru veya MSS periventriküler bölgede bulunan endojen öncüleri yanıtı arttırmak için NPCs harekete geçirilmesidir. Buna göre, teşvik rehberlik ve NPC göç geliştirmek mekanizmaları anlamak için önemlidir. Galvanotaksis olarak bilinen bir olgu - Bizim iş tanıtmak için doğru akım elektrik alanların kullanımı (dcEFs) ve doğrudan NPC göç üzerinde duruluyor. Endojen fizyolojik elektrik alanları normal gelişimi ve yara onarımı sırasında hücre göçü için kritik ipuçları olarak işlev görür. Farmakolojik bozulmasıAxolotl embriyolar trans-nöral tüp potansiyeli ciddi gelişimsel malformasyonlar 1 neden olur. Yara iyileştirme bağlamında, yaralı, kornea tamir oranı doğrudan bu dcEF 2-3 farmakolojik olarak artırma ya da bozulma ile gösterildiği gibi, yaralanma sonrası ortaya çıkan epitel yara potansiyel büyüklüğü ile ilişkilidir. Biz bir şekilde dıştan uygulanan dcEF maruz kaldığında yetişkin subepandimal YZK&#39;lar in vitro hızlı ve yönetmenliğini cathodal göç tabi olduğunu göstermiştir. Bu protokolü biz yüksek çözünürlüklü, bir tek hücre düzeyinde yönettiği hücre gövdesi translokasyon (göç) uzun vadeli gözlem için basit ve etkili bir galvanotaksis tahlil oluşturmak için laboratuvar tekniklerini açıklar. Bu tahlil, transgenik veya nakavt fareleri kısa müdahale RNA, ya da özel bir reseptör agonistleri / antagonistleri kullanımı yoluyla hücre hareketliliği içine dcEF transdüksiyon düzenleyen mekanizma araştırılması için uygun olacaktır.

Kinematik analizi bir 250 mV / mm dcEF varlığında, farklılaşmamış NPC katot karşı yönlendirilmiş ve çok hızlı galvanotaksis (Şekil 5A, Film 1) gösterdiğini ortaya koymaktadır. Bir dcEF yokluğunda, hücrelerin rasgele hareketi (Şekil 5B, Film 2) görülmektedir. Bu alan gücü az, farklılaşmamış NPCs&gt;% 98&#39;i yansıması olduğu için tüm 6-8 saat boyunca göç ve ölü hücreleri göç etmezler çünkü bu bir dcEF yokluğunda veya varlığında bu dönemde...

Watch this Scientific Journal Video about A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field at JoVE.com

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

Bu protokolü biz galvanotaksis sırasında nöral prekürsör hücre translokasyon edilen erişkin beyninin time-lapse görüntüleme sağlamak için bir doğru akım elektrik alanın uygulanmasına izin özel odaları oluşturmak nasıl gösterilmektedir.

Dıştan Uygulanan Doğru Akım Elektrik Alanında Nöral Prekürsör Hücre Göç Kinetik Analizi için galvanotaksis Assay

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

a-galvanotaxis-assay-for-analysis-neural-precursor-cell-migration

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

12.4K Görüntüleme.  University of Toronto. Bu protokolü biz galvanotaksis sırasında nöral prekürsör hücre translokasyon edilen erişkin beyninin time-lapse görüntüleme sağlamak için bir doğru akım elektrik alanın uygulanmasına izin özel odaları oluşturmak nasıl gösterilmektedir.

Video: A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

Hücre Testi Şekillendirme Sinir-Koloni: Sinir Progenitör Hücreler Bona Fide Sinir Kök Hücreler Ayırım Bir Testi

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Nörobilim

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

Bir Modifiye Zigmond Odası Testi kullanılarak Trunk Sinir Crest Hücre Göç Analizi

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Tüm Dağı Muscle Analizi ile tekrarlanabilir Fare Siyatik Sinir Ezilme ve Rejenerasyon daha sonraki Değerlendirme

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

Çapraz türler Nakli Sinir Crest Göç ve Farklılaşma Analiz

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Neuroblast Test: Akım Sitometri Kullanımı Farklılaşan Nöral Kök Hücre Döl nöronal Progenitör Hücrelerinin üretilmesi ve zenginleştirilmesi için bir Testi

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Embriyonik Mürin Nöral Tüp çıkan sinirsel Crest Hücre İzolasyonu ve Kültür

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

Kemirgen Enterik Sinir progenitörlerin için Kantitatif Hücre Göç Tahlili

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals&#39; access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal&#39;s body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal&#39;s body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.

We describe a set of techniques for studying spontaneous behavior of freely swimming weakly electric fish over an extended period of time, by synchronously measuring the animal&#39;s electric organ discharge timing, body position and posture both accurately and reliably in a specially designed aquarium tank inside a sensory isolation chamber.

Serbestçe Yüzme Zayıf Elektrik Balık Uzun vadeli Davranış Takip

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

Kortikal yerel alan potansiyelleri, Elektrokardiyogram, Electromyogram ve ritim serbestçe hareket bir sıçan gelen nefes eşzamanlı kayıt

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural circuits. Due to the fractional change in the light signal, it has been difficult to use this method as a quantitative assay. This article describes special optics and slice handling systems, which render this technique stable and reliable. The present article demonstrates the slice handling, staining, and recording of the VSD-stained hippocampal slices in detail. The system maintains physiological conditions for a long time, with good staining, and prevents mechanical movements of the slice during the recordings. Moreover, it enables staining of slices with a small amount of the dye. The optics achieve high numerical aperture at low magnification, which allows recording of the VSD signal at the maximum frame rate of 10 kHz, with 100 pixel x 100-pixel spatial resolution. Due to the high frame rate and spatial resolution, this technique allows application of the post-recording filters that provide sufficient signal-to-noise ratio to assess the changes in neural circuits.

We introduce a reproducible and stable optical recording method for brain slices using voltage-sensitive dye. The article describes voltage-sensitive dye staining and recording of optical signals using conventional hippocampal slice preparations.

Gerilim duyarlı boya kullanarak beyin dilimleri geniş alan tek foton optik kayıt

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural ...