This work was funded by the DFG (SFB 779 TPB8, Kr1879/3-1 MRK), DIP grant (MRK), EU FP7 MC-ITN NPlast (MRK), Center for Behavioral Brain Sciences (CBBS, Sahsen-Anhalt), (AK and SB), MM is a recipient of European Molecular Biology Organization (EMBO) Long-Term Fellowship (EMBO ALTF 884-2011) and Marie-Curie IEF.

Yetişkin sıçan beyninde gelen Akut Hipokampal Dilimleri hazırlanması 1. <ol><li> Izofluran ile sıçan anestezisi. DİKKAT: Kapalı bir exicator kullanarak prosedürü uygulayın, izofluran solumamanızı. Hayvan tamamen uyuşturulduktan emin olun. </li><li> Hızlı, sıçan başını kesmek beyin izole ve precarbonated (% 95 O 2/5% CO2 gaz karışımı), buz soğukluğunda mM Gey çözeltisi içinde (bileşim bekletmektir: 130 NaCl, 4.9 KCl, 1.5 CaCl2 · 2H 2 O , 0.3 MgSO 4 · 2H 2, O, 11 MgCI2 · 6H 2, O, 0.23 KH 2 PO 4, 0.8, Na 2 HPO 4 · 7H 2, O, 5 Glükoz · H2O, 25 HEPES, 22 NaHCO3, pH 7.32) 30 dakika boyunca 1,2,10,16. </li><li> Entorhinal korteks beyincik ve parçasını çıkarın. Daha sonra medial yüzeyi üzerinde her yarımkürede aşağı yerleştirin, bir mid-sagital kesim ile kortikal hemisferlerdir ayırın. Bundan sonra yapmakHer yarımkürede 13,14 dorsal kenarı boyunca 50-70 ° kesim (50-70 ° enine). </li><li> Kesit sistemin dilimleme platformda taze kesilmiş yüzeyi ile her yarımkürede Tutkal. Platform precarbogenated buz soğuk Gey çözümü ile kaplanmış olmalıdır. </li><li> Z-ekseni salınımı en aza indirmek için ayarlanmış bir vibratome kullanarak yan posterior anterior 350 mikron 50-70 ° enine dilim kesin. Hipokampal oluşumda, subiküler ve entorinal kortekste gibi hipokampusa Dorsolateral bulunan korteksleri deneyler 13,14 için kullanılan dilimlerin bir parçası olacaktır. </li><li> U-şekli ve batırılmış tipte inkübatöre hipokampal dilimler aktarın ve carbogenated yapay serebrospinal akışkan (ACSF mM içeren 32 ° C&#39;de en az 2 saat süreyle inkübe edin: 2H ​​2, O, 1,5 MgSO ^ · 110 NaCl, 2.5 KCl, 2.5 CaCl2 4 · 2H 2 O, 1.24 KH 2 PO 4, 10 Glukoz · H 2, O, 27.4 NaHCO 3, pH 7.3) 1,2,10,16. </li></ol> 2. Elektrotların Konumlandırma, Temel Kayıt ve LTP indüksiyonu <ol><li> Mikroskop altında monte edilmiş bir batırılmış tipte kayıt odasına hipokamp dilim aktarın. 32 ° C&#39;de en az 30 dakika boyunca carbogenated ACSF (6-7 ml / dak) perfüze. </li><li> (Uç direnci M? 3-5 olan) ACSF ile dolu cam kılcal Mikroelektronlar hazırlayın. </li><li> Uyarımı ve fEPSP için CA1 stratum radiatum kayıt 1,2,10 (Şekil 2) CA1 Schaffer kollateral-liflerde ACSF ile dolu bir cam Mikroelektronlar yerleştirin. Elektrotlar arasındaki mesafe yaklaşık 300 um olmalıdır. </li><li> 3-4 V arasındaki bir aralıkta iki fazlı dikdörtgen akım darbelerinin (200 ms / kutup) ile Schaffer kollateral liflerin-uyarılması ile alanını uyancı postsinaptik potansiyeller (fEPSPs) uyandırmak </li><li> Inp ölçerek maksimum stimülasyon testi gerçekleştirinut-çıkış ilişkisi ve elde edilen maksimum fEPSP-eğim değerlerinin% 40 olarak uyarım gücünü tanımlamak ve deney boyunca sabit tutun. </li><li> Maksimum uyarma testi sonra en az 15 dakika boyunca başlangıç ​​kayıt başlayın. Deney boyunca her dakika uyaranlara test yanıtları ölçün. Düşük frekanslı stimülasyon ile bazal kayıtları gerçekleştirin. </li><li> En az 30 dakika boyunca temel kaydedin. </li><li> Geç-LTP indüksiyonu için yüksek frekansı 5 dk diziler arası aralık ile 100 Hz üç 1 sn uyaran trenler oluşan 100 Hz tetanization geçerlidir. CA1 bölgesinde 5 uM bicuculline içinde stimülasyon alanını artırmak için tetanization önce, 2 dakika ile yıkanabilir ve hemen geçen tetanization sonra yıkanmalıdır. </li></ol> LTP ve Yapış Donma İndüksiyonunun sonra Dilimleri 3. Koleksiyonu <ol><li> Dur LTP 2 dakika ya da Geç-LTP uyaran tetanik stimülasyon üç trenlerin 30 dakika sonra kayıt. </li><li&gt; elektrotlar ve hızlı bir şekilde kuru buz üzerinde yerleştirilmiş metal bir platform üzerine precold dilim aktarın. U-şekilli inkübatör tutulan bir kontrol dilim için prosedürü tekrarlayın. DİKKAT: eldiven kullanın kuru buz ile çalışırken. </li><li> -80 ° C de, 1.5 ml tüp ve mağaza her dilim toplayın. </li></ol> Hippocampus CA1 Bölgesi&#39;nden Nükleer Zenginleştirilmiş Kesirler 4. İzolasyonu <ol><li> -80 ° C donmuş dilimleri, 1.5 ml&#39;lik Eppendorf tüpleri çıkarın ve buz üzerinde tutun. </li><li> Ekleme tüpüne taze soğuk TBS tampon çözeltisi ihtiva eden bir proteaz (PI) ve fosfataz (PS) inhibitörleri, 0.5 mi. 2-3 dakika inkübe ve stereomicroscope transfer. DİKKAT: koruyucu eldiven kullanın ve PI ve PS ile çalışırken bir laboratuvar önlüğü. </li><li> Iki iğne kullanılarak hipokampusun CA1 stratum radiatumdan, pyramidale bölgesini ayır. Stereomikroskop ve CA1 alanı kesmek için ikinci bir iğne altında tutulması için bir dilim iğne kullanın. </li><li> Disseke CA toplayın50 ul liziz tamponu ihtiva eden yeni bir 1.5 ml&#39;lik bir tüp içine (her bir grup için) 5 kesitlerden 1 bölgeleri (mM olarak içeren hipotonik liziz tamponu 1x: 10 HEPES, 1.5 MgCl2, 10 KCI, PI ve PS ile pH 7.9). Bu malzeme miktarı 2-3 imünoblotlarının çalıştırmak için yeterli olacağını unutmayın. </li><li> Aşağı dikkatli pipetleme up ve tarafından toplanan doku homojenize. 200 ul pipet kullanın. Hücre şişer izin vermek için 5-7 dakika boyunca buz üzerinde inkübe lizat. </li><li> , Lizatının 2 ul alır mikroskobik bir slayt üzerinde açılan ve parlak bir alan mikroskobu altında hücrelerin şişmesine görselleştirmek. Hücrelerin doğru şişmesi ile çekirdekler gibi yuvarlak sağlam yapıları görünür. </li><li> &#39;Homojenat fraksiyon &quot;olarak yeni bir 1.5 ml&#39;lik bir tüp içine 20 ul numune toplayın. 4 x SDS numune tamponu denatüre 8 ul ilave edin. </li><li> 1 dakika boyunca 11,000 rpm&#39;de santrifüje kalan lizatları. </li><li> Dikkatle, tr üst (&#39;Sitosolik kısım, M&#39;) &#39;süpernatan toplamakansfer yeni bir tüpe ve 4x numune tamponu 20 ul ekle. </li><li> , Hipotonik tampon maddesi, 60 ul pelletini ve 4x numune tamponu 20 ul ekle. Bu fraksiyon bir nükleer bakımından zengin kısım &quot;olarak adlandırılır. </li><li> Homojenat, sitosolik, nükleer veya zenginleştirilmiş kısımlar, daha sonra -80 ° C bağışıklık boyama için -20 ° C&#39;de saklanabilir. </li></ol> Synapto nükleer proteinlerin yarı kantitatif 5. Bağışıklık <ol><li> Örnekleri Defreeze ve 95 ° C&#39;de 5 dakika boyunca kaynatın. </li><li> Her parçadan 5 ul alın ve amido siyah testi veya BCA testi ile protein konsantrasyonu belirlemek. </li><li> SDS-PAGE üzerinde homojenattan protein örneklerinin eşit miktarda, sitoplazmik ve nükleer zenginleştirilmiş kısımlar yükleyin. Ve kontrol LTP kesitlerden örnekler doğrudan bir karşılaştırma için, aynı jel üzerine yerleştirilmelidir. </li><li> Standart Batı-kurutma işlemini gerçekleştirin (ıslak transferi önerilir). </li><li> T membranlar Probeo seçenek antikor. Nörona özgü enolase2 (NSE2) ve nükleer işaretleyici - - Neun bir yükleme kontrolü olarak bir p-aktin antikor daha sonra aynı blotlar (veya aynı anda paralel ölçüm), sitoplazmik bir işaretleyici ile problanabilir. </li></ol> 6. Veri Analizi <ol><li> LTP indüksiyon Verimliliği Clampfit yazılım ile analiz edilebilir. Bazal kayıtların ortalama eğim tetanization sonra yamaçları iki yönlü ANOVA, p kullanılması ile karşılaştırıldığında &lt;0.05 anlamlı farklılık olarak kabul edildi. FEPSP eğim değerleri ortalama ± SEM olarak şemalarda tasvir edildi </li><li> Immunoblotların ölçümü için, bir film ya da otoradyografik tarar ve bir Licor sistemi ile ImageJ veya flüoresan gruplar tarafından protein bantlarının yoğunluğu analiz entegre. Immunorekatif bantlar Değerleri yükleme ve kurutma kontrolü için normalize edilmelidir. Kontrolü karşılaştırması ve LTP gruplar için nonparametrical Mann-Whitney U-Testi kullanılmış olabilir. </li></ol><table border="0"cellpadding = &quot;0&quot; cellspacing = &quot;0&quot;&gt; <tbody><tr><td> Tamponun Adı </td><td> Reaktif </td><td> Konsantrasyon (mM) </td><td> Tutar </td><td> Yorumlar / Açıklama </td></tr><tr><td> Gey çözeltisi (pH: ~ 7.4 7.3) </td><td> NaCl </td><td> 130 </td><td> 7.6 gr </td><td> steril filtrasyon </td></tr><tr><td> 1000 mi </td><td> KCl </td><td> 4.9 </td><td> 0.37 gr </td><td></td></tr><tr><td></td><td> CaCl2 · 2H 2 O </td><td> 1.5 </td><td> 0.22 gr </td><td></td></tr><tr><td></td><td> MgSO 4 · 2H 2 O </td><td> 0.3 </td><td> 0.0739 g </td><td></td></tr><tr><td></td><td> MgCI2 · 6H 2 O </td><td> 11 </td><td> 2.24 g </td><td></td></tr><tr><td></td><td> KH 2 PO 4 </td><td> 0.23 </td><td> 0.0313 g </td><td></td></tr><tr><td></td><td> Na 2 HPO 4 · 7H 2 O </td><td> 0.8 </td><td> 0,2145 g </td><td></td></tr><tr><td></td><td> Glukoz · H2O </td><td> 5 </td><td> 0,9909 g </td><td></td></tr><tr><td></td><td> Hepes </td><td> 25 </td><td> 5.96 gr </td><td></td></tr><tr><td></td><td> NaHCO3 </td><td> 22 </td><td> 1.85 g </td><td></td></tr><tr><td> ACSF (pH: ~ 7.4 7.3) </td><td> NaCl </td><td> 110 </td><td> 6.428 g </td><td> steril filtrasyon </td></tr><tr><td> 1000 mi </td><td> KCl </td><td> 2.5 </td><td> 0,1865 g </td><td></td></tr><tr><td></td><td> CaCl2 · 2H 2 O </td><td> 2.5 </td><td> 0.368 g </td><td></td></tr><tr><td></td><td> MgSO 4 · 2H 2 O </td><td> 1.5 </td><td> 0.370 g </tD&gt; <td></td></tr><tr><td></td><td> KH 2 PO 4 </td><td> 1.24 </td><td> 0.169 gr </td><td></td></tr><tr><td></td><td> Glukoz · H2O </td><td> 10 </td><td> 1,9817 g </td><td></td></tr><tr><td></td><td> NaHCO3 </td><td> 27.4 </td><td> 2,3 g </td><td></td></tr><tr><td> 1x Hipotonik tamponu (pH: 7.9) </td><td> Hepes </td><td> 10 </td><td> 0.23 g </td><td></td></tr><tr><td> 100 mi </td><td> MgCI2 · 6H 2 O </td><td> 1.5 </td><td> 0.0304 g </td><td></td></tr><tr><td></td><td> KCl </td><td> 10 </td><td> 0,07455 g </td><td></td></tr></tbody></table> Tablo 1. Tamponlar.

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The authors have nothing to disclose.

Yukarıdaki protokolün açıklanan adımlar, hipokampus akut genç ya da yetişkin farelerin dilimlenmiş hazırlamak dilim uyarılmış alanı neden ve kayıt LTP, hızla teşrih ve aktivite bağımlı protein dinamikleri incelemek için nükleer zenginleştirilmiş kısmını hazırlamak için nasıl rehberlik sağlamak. Bu yaklaşım, her biri birbirinden bağımsız bir şekilde kullanılan birçok farklı yöntemlerin kombinasyonu kaynaklanmaktadır. Biz bir iş akışını optimize edilmiş ve LTP indüksiyonu ü...

Sinaptik N-metil-D-aspartat-reseptörler (NMDARs) sinaptik plastisite ve hücre hayatta kalma ve nörodejenerasyonunun hücre ölümünü nasıl tetikleyebildiği extrasynaptic NMDARs aktivasyonu ise sinyal çok önemli bir rol oynar. Bu değişiklikler sıkı kontrol / düzenlenmiş bir faaliyet bağımlı gen ifadesine bağlı ve böylece aktif sinaps veya dendritler ve nükleus 7 arasında sürekli iletişim gerektirir. MAP ERK1 / 2 sinyalizasyon sinaptik NMDARs alt baş gerçekleştirmekle ve extrasynaptic NMDAR yoluyla sinyalleme yapan hiç ya da ERK1 / 2 aktivitesi 8,11 üzerinde inhibe edici bir etkiye sahiptir, oysa, NMDAR-aktivasyonu ile uyarılan gen sentezlenmesine katılan edilir kinaz. Distal dendrit ve çekirdeği arasındaki transfer gösterilmiştir proteinler vardır. Bu proteinlerin çoğu bir nükleer lokalizasyon sinyali içerir ve aktif olarak çekirdeğe bir 6,9 ve dinein importin bağımlı bir şekilde, mikrotübülün boyunca taşınmaktadır. InterestinglY, belirli bir sinaptik uyarıcıya tepki olarak çekirdeğe Bu taşıyıcı maddelerin bazıları için yalnızca bir geçiş. Örneğin, siklik AMP tepki elemanı bağlayıcı protein 2 (CREB2) retrograd nakil aracı kimyasal LTD, fakat LTp 12 tarafından indüklenir. Lokalize NMDAR-bağımlı uyarma sinaptik uzun vadeli plastisite hipokampal 4 katılır çekirdeği, bir translokasyon süreç içine CREB düzenlenmiş kopyalayıcı ortak aktifleştirici (CRTC1) tahrik eder. Son zamanlarda protein haberci Jacob hem sonra nükleus, sinaptik ve extrasynaptic NMDAR aktivasyonu için translocates ve CREB&#39;ye bağımlı gen transkripsiyonunu 5 düzenler olduğu gösterilmiştir. Sinyalinin sinaptik veya extrasynaptic kökenli Jacob bir sonrası modifikasyon kodlanmıştır. Sinaptik aktivitesi primer hipokampal kültürdeki çekirdeğe translokasyonu açısından daha sonra gerekli bir konumda olan 180 (pJacobS 180) açısından çok önemli bir serin bir Yakup&#39;un ERK1 / 2 bağımlı fosforilasyonunu uyanr. Ayrıca, in CA1 Schaffer kollateral LTP sonra çekirdeğe akut hipokampal dilimlerin pJacobS 180 translocates nöronlar değil 1,10 LTD. pS180 Jacob plastisite ilişkili genlerin ifadesindeki artışa neden olur ve bu gen ekspresyon sinaptik fonksiyon geri beslenir. Extrasynaptic NMDARs aktivasyon Ser180 fosforilatlanır değildir ve neden çekirdekte farklı bir protein kompleksi ile ilişkili olabilir sonra çekirdeğe translocates tezat, Jacob &#39;CREB&#39;ye kapatmak&#39; ve sinaptik bağlantıları 10 bir geri çekilme. Synapto-nükleer protein haberci nükleer ithalatına En yayınlanan çalışmalar, ayrışmış nöronal primer kültürlerinde yapılmıştır. Nöronal bağlantı ve fonksiyon çok daha iyi korunmuş olduğu, bu nedenle bu bulgular hipokampal dilimleri kullanarak fizyolojik daha uygun koşullar çoğaltılamaz görmek ilginç olacaktır. Burada LTP-de değerlendirmek için optimize edilmiş bir protokol mevcutimünoblotlama protein haberciler nükleer translokasyonunu pendent. Bu yöntem aynı zamanda bu basit bir nükleer fraksiyonda proteinin aktivitesine bağlı fosforilasyonunun analizi için uygundur. Özel olarak, mevcut protokolü akut, CA1, hipokampüse ait kesitler indüksiyonu ve LTP kayıt hazırlanmasını da kapsar. Sonraki CA1 bölgesi mikroskobik uyarılmış bölgeyi izole etmek için disseke edilir. Biz kombine ve Zhao ve arkadaşları 17 tarafından tanıtılan değişiklikler ile CellLytic nükleer Ekstraksiyon Kiti tarafından sağlanan nükleer izolasyonu için protokol değiştirilmiş. Optimize edilmiş prosedür, hücre şişmesi ve çekirdekler salımını sağlayan, hipotonik tampon maddesi içinde kesildi CA1 bölgelerin lızız ışlemını içermektedir. Hücre lizizi, ve çekirdekler morfolojisi mikroskopik inceleme ile tespit edilebilir. Nükleer zenginleştirme kısa bir santrifüjleme adımı ile elde edilir. Neun ve NSE2, nükleer veya sitosolik fraksiyonlar özel markörler, karşı antikorlar ile immunoblotting analizi bu yaklaşım hızlı bir şekilde kullanılabileceğini göstermektedirve tekrarlanabilir protokolü bu hücrealtı kesirler izole etmek ve protein fosforilasyonu gibi çok kararsız öteleme sonrası modifikasyonları çalışma. Buna ek olarak, bu yöntem, hipokampal dilimler kesilmiştir CA1 bölgelerinden kaynaklanan küçük bir doku numuneleri için avantajlıdır ve hipokampal dilimler immünohistokimya ile kombinasyon halinde de kullanılabilir.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1. Equipment</td>
		</tr>
		<tr>
			<td>CED 1401 AD/DA converter</td>
			<td>Cambridge Electronics Design, UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Axopatch 200B amplifier</td>
			<td>Axon&#65292;USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td><a href="http://www.iciba.com/data_acquisition_system">Axon Digidata acquisition system</a></td>
			<td>Axon&#65292;USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clampex 10.0 Data acquisition and analysis software</td>
			<td>Axon&#65292;USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica microscope</td>
			<td>Leica TCS SP2&#65292;Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P-97 Standard microelectrode puller</td>
			<td>Sutter&#65292;USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td><a href="http://www.iciba.com/stereomicroscope">Stereomicroscope</a></td>
			<td>Leica</td>
			<td>Leica S4E, Germany</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrotome</td>
			<td>Leica VT1000S, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isolated pulse stimulator</td>
			<td>Model2100, A-M systmes, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific, HERAEUS, FRSCO17</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE system</td>
			<td>BIORAD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cooler</td>
			<td>JULABO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bright field Microscope</td>
			<td>NIKON ECLIPSE TS100</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>Thermo Electron Corporation, HERAEUS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Luigs &#38; Neumann, SM-5, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blotting chamber and electric power supplier</td>
			<td>Hoefer Scientific Instruments, San Francisco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cooler</td>
			<td>Julabo, F12</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific, Heraeus, FRESCO 17</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Submerged type Recording Chamber</td>
			<td>custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>U-shape and submerged type incubator&#160;</td>
			<td>custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small surgical scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thin spatula</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Pasteur pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic culture dish&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen beaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. Reagents</td>
		</tr>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>ROTH</td>
			<td>Art.-Nr.3957.1</td>
			<td>&#8805;99.5%, p.a.,ACS,ISO</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>ROTH</td>
			<td>Art.-Nr.6781.1</td>
			<td>&#8805;99.5%, p.a.,ACS,ISO</td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H2O</td>
			<td>MERCK</td>
			<td>1.02382.0500</td>
			<td>pro analysi</td>
		</tr>
		<tr>
			<td>MgSO4&#183;2H2O</td>
			<td>MERCK</td>
			<td>5886.05</td>
			<td>pro analysi</td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H2O</td>
			<td>AppliChem</td>
			<td>CAS-NO: 7791-18-6; EC-NO:2320946</td>
			<td>for Molecularbiology</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>MERCK</td>
			<td>12034.025</td>
			<td>for Molecularbiology</td>
		</tr>
		<tr>
			<td>Na2HPO4&#183;2H2O</td>
			<td>MERCK</td>
			<td>1.06574.1000</td>
			<td>extra pure</td>
		</tr>
		<tr>
			<td>Glucose&#183;H2O</td>
			<td>ROTH</td>
			<td>Art.-Nr.6887.1</td>
			<td>for Molecularbiology</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>ROTH</td>
			<td>Art.-Nr.9105.4</td>
			<td>PUFFERAN, &#8805;99.5%, p.a.</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>MERCK</td>
			<td>1.06329.1000</td>
			<td>pro analysi</td>
		</tr>
		<tr>
			<td>Protease inhibitor Coctail</td>
			<td>ROCHE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphostop</td>
			<td>ROCHE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bicuculline</td>
			<td>Tocris bioscience</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Isofluran</td>
			<td>Baxter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 4. Antibodies</td>
		</tr>
		<tr>
			<td>Primary antibodies</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Species</td>
		</tr>
		<tr>
			<td>pJac-s180</td>
			<td>Biogenes/ purified</td>
			<td></td>
			<td>rabbit (dil. 1:100)</td>
		</tr>
		<tr>
			<td>NeuN</td>
			<td>Milipore</td>
			<td>MAB377</td>
			<td>mouse (dil. 1:1,000)</td>
		</tr>
		<tr>
			<td>NSE</td>
			<td>Cell Signaling</td>
			<td>D20H2</td>
			<td>rabbit (dil. 1:1,000)</td>
		</tr>
		<tr>
			<td>beta-actin</td>
			<td>Sigma</td>
			<td>A-5441</td>
			<td>mouse (dil. 1:5,000)</td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
			<td>Species</td>
		</tr>
		<tr>
			<td>IgG HRP Conjugated</td>
			<td>DAKO</td>
			<td></td>
			<td>goat anti - mouse (1:5,000)</td>
		</tr>
		<tr>
			<td>IgG HRP Conjugated</td>
			<td>NEB</td>
			<td></td>
			<td>goat anti - rabbit (1:5,000)</td>
		</tr>
	</tbody>
</table>

isolation of ca1 nuclear enriched fractions from hippocampal slices to study activity-dependent nuclear import of synapto-nuclear messenger proteins

Öğrenim aktivitesine bağlı protein ekspresyonu, hücre-altı yer değiştirmesi, ya da fosforilasyon sinaptik plastisite altında yatan hücresel mekanizmaların anlaşılması önemlidir. Uzun vadeli potansiyelleri (LTP) ve akut hipokampal dilim bağlı uzun süreli depresyon (LTD) yaygın öğrenme ve hafızanın hücresel modelleri olarak kabul edilir. Etkinlik bağımlı protein dinamikleri görselleştirmek için canlı hücre görüntüleme veya immünohistokimya yaklaşımları kullanmak birçok çalışma bulunmaktadır. Bununla birlikte, bu yöntemler tek nöronlarda floresan-etiketli proteinlerin immünolojik hücre ya da aşırı dışavurumu için antikorların uygunluğu dayanır. Proteinlerin İmmünoblotting bulguların bağımsız onay veren bir alternatif yöntemdir. Bireysel tetanized hipokampal dilimlerde sub-hücresel fraksiyonları hazırlanmasında ilk sınırlayıcı faktör, malzemenin düşük miktarıdır. İkinci olarak, işleme yöntemi çok önemlidir l, hatta çok kısa bir küçük ve manipülasyon içindilimleri Iving belirli sinyal silsileleri uyarmamaktadır olabilir. Burada fare beyninden akut hippokampüs kesitlerinin CA1 bölgesinde yeterli saflıkta nükleer bakımından zengin kısım için yeterli malzeme elde etmek için bir optimum iş akışını açıklar. Temsili bir örnek olarak, synapto-nükleer protein haberci ERK1 / 2 fosforlu şekli Jacob aktif LTP indüksiyonu üzerine çekirdeğe doğru yer değiştirir ve CA1 nöronlarının bir nükleer bakımından zengin kısım tespit edilebileceğini göstermektedir.

Daha önce synapto-nükleer protein haberci Jacob LTP indüksiyonu değil, LTD 1 Aşağıdaki çekirdekte birikmektedir göstermiştir. Ayrıca, sinaptik stimülasyonu sonrası Yakup&#39;un translokasyon Ser180 (Şekil 1) MAPK ERK1 / 2 aktivasyonunu ve Yakup&#39;un fosforilasyonu gerektirir. Fosforile edilmiş Jacob bir importin bağımlı bir şekilde çekirdeğe doğru yer değiştirir ve fosforile durum 15 αinternexin ara filaman ile birlikte (Şekil 1) uzun s?...

Watch this Scientific Journal Video about Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins at JoVE.c

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Biz, hipokampusun CA1 bölgesinde ve dilimin tetanized alan nükleer zenginleştirilmiş kısımların daha sonraki izolasyon Uzun süreli potansiyasyon indüksiyonu için ayrıntılı bir protokol sağlar. Bu yaklaşım, önemli aktivitesini öğrenme ve hafıza ile hücresel modellerinde bağımlı nükleer protein alma belirlemek için kullanılabilir.

Hipokampal Dilimleri adlı CA1 Nükleer Zenginleştirilmiş Kesirler izolasyonu Synapto-nükleer Messenger Proteinler Etkinliği-bağımlı Nükleer İthalat Okuyacak

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

isolation-ca1-nuclear-enriched-fractions-from-hippocampal-slices-to

Research

JoVE Journal

Neuroscience

12.0K Görüntüleme.  Leibniz Institute for Neurobiology.  Biz, hipokampusun CA1 bölgesinde ve dilimin tetanized alan nükleer zenginleştirilmiş kısımların daha sonraki izolasyon Uzun süreli potansiyasyon indüksiyonu için ayrıntılı bir protokol sağlar. Bu yaklaşım, önemli aktivitesini öğrenme ve hafıza ile hücresel modellerinde bağımlı nükleer protein alma belirlemek için kullanılabilir. 

Video: Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

Yaşlanma ve Amiloid Patoloji sırasında sinaptik değişiklikler Çalışması Sıçanlar ve Transgenik Farelerde akut Hipokampal Dilimleri hazırlanması

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

Nörobilim

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Kemirgen Beyin Dilimleri Nöronlar Vibrodissociation Sinaptik İletim ve Görüntü Presinaptik Terminalleri Eğitim

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Prenatal Fareler gelen Hipokampal Nöronlar izolasyonu ve Kültürü

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic cues. Their membranes are often juxtaposed at defined contact sites, which become hubs for the exchange of signaling molecules and membrane components1,2,3,4. The inter-organellar membrane microdomains that are formed between the endoplasmic reticulum (ER) and the mitochondria at the opening of the IP3-sensitive Ca2+ channel are known as the mitochondria associated-ER membranes or MAMs4,5,6. The protein/lipid composition and biochemical properties of these membrane contact sites have been extensively studied particularly in relation to their role in regulating intracellular Ca2+ 4,5,6. The ER serves as the primary store of intracellular Ca2+, and in this capacity regulates a myriad of cellular processes downstream of Ca2+ signaling, including post-translational protein folding and protein maturation7. Mitochondria, on the other hand, maintain Ca2+ homeostasis, by buffering cytosolic Ca2+ concentration thereby preventing the initiation of apoptotic pathways downstream of Ca2+ unbalance4,8. The dynamic nature of the MAMs makes them ideal sites to dissect basic cellular mechanisms, including Ca2+ signaling and regulation of mitochondrial Ca2+ concentration, lipid biosynthesis and transport, energy metabolism and cell survival 4,9,10,11,12. Several protocols have been described for the purification of these microdomains from liver tissue and cultured cells13,14. 
	Taking previously published methods into account, we have adapted a protocol for the isolation of mitochondria and MAMs from the adult mouse brain. To this procedure we have added an extra purification step, namely a Triton X100 extraction, which enables the isolation of the glycosphingolipid enriched microdomain (GEM) fraction of the MAMs. These GEM preparations share several protein components with caveolae and lipid rafts, derived from the plasma membrane or other intracellular membranes, and are proposed to function as gathering points for the clustering of receptor proteins and for protein&#x2013;protein interactions4,15.

Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

This procedure illustrates how to isolate from the adult mouse brain the mitochondria-associated ER membranes or MAMs and the glycosphingolipid-enriched microdomain fractions from MAMs and mitochondrial preparations.

Mitokondri ilişkili ER Membranlar (MAM) ve Glycosphingolipid zenginleştirilmiştir Microdomains (GEM&#39;lerle): Fare Beyin izolasyon

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Organotipik Hipokampal Dilimleri eşleştirilmiş Tüm Hücre Kayıtlar

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity is achieved in neurons within a specific time frame. These long-term plasticity-related processes are the leading candidate models to study the basis of memory formation and persistence at the cellular level. Both STC and cross-tagging involve two serial processes: (1) setting of the synaptic tag as triggered by a specific pattern of stimulation, and (2) synaptic capture, whereby the synaptic tag interacts with newly synthesized plasticity-related proteins (PRPs). Much of the understanding about the concepts of STC and cross-tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the laboratories are still unable to study these processes. Experimental conditions for the preparation of hippocampal slices and the recording of stable late-LTP/LTD are extremely important to study synaptic tagging/cross-tagging. This video article describes the experimental procedures to study long-term plasticity processes such as STC and cross-tagging in the CA1 pyramidal neurons using stable, long-term field-potential recordings from acute hippocampal slices of rats.

This video article describes experimental procedures to study long-term plasticity and its associative processes such as synaptic tagging, capture and cross-tagging in the CA1 pyramidal neurons using acute hippocampal slices from rodents.

Kemirgenler gelen akut Hipokampal Dilimleri kullanarak Synaptic Etiketleme / yakalama ve Çapraz yakalama İncelenmesi

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

Hipokampal kemirgen beyin dilimleri dansitesi Synapse değerlendirilmesi

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Implantation of Chronic Silicon Probes and Recording of Hippocampal Place Cells in an Enriched Treadmill Apparatus

An important requisite for understanding brain function is the identification of behavior and cell activity correlates. Silicon probes are advanced electrodes for large-scale electrophysiological recording of neuronal activity, but the procedures for their chronic implantation are still underdeveloped. The activity of hippocampal place cells is known to correlate with an animal&#39;s position in the environment, but the underlying mechanisms are still unclear. To investigate place cells, here we describe a set of techniques which range from the fabrication of devices for chronic silicon probe implants to the monitoring of place field activity in a cue-enriched treadmill apparatus. A micro-drive and a hat are built by fitting and fastening together 3D-printed plastic parts. A silicon probe is mounted on the micro-drive, cleaned, and coated with dye. A first surgery is performed to fix the hat on the skull of a mouse. Small landmarks are fabricated and attached to the belt of a treadmill. The mouse is trained to run head-fixed on the treadmill. A second surgery is performed to implant the silicon probe in the hippocampus, following which broadband electrophysiological signals are recorded. Finally, the silicon probe is recovered and cleaned for reuse. The analysis of place cell activity in the treadmill reveals a diversity of place field mechanisms, outlining the benefit of the approach.

We describe the diverse steps to implant chronic silicon probes and to record place cells in mice that are running head-fixed on a cue-enriched treadmill apparatus.

Kronik silikon sondalar ve kayıt bir zenginleştirilmiş koşu bandı aparat Hipokampal yer hücre implantasyonu

An important requisite for understanding brain function is the identification of behavior and cell activity correlates. Silicon probes are advanced ...

An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central nervous system and thus, can be used to analyze the diseased state of the brain or spinal cord without directly accessing these tissues. However, in mice it is difficult to obtain from the cisterna magna due to its closeness to blood vessels, which often contaminate samples. The area for CSF collection in mice is also difficult to dissect to and often only small samples are obtained (maximum of 5-7 &#181;L or less). This protocol describes in detail a technique that improves on current methods of collection to minimize contamination from blood and allow for the abundant collection of CSF (on average 10-15 &#181;L can be collected). This technique can be used with other dissection methods for tissue collection from mice, as it does not impact any tissues during CSF extraction. Thus, the brain and spinal cord are not affected with this technique and remain intact. With greater CSF sample collection and purity, more analyses can be used with this fluid to further aid neuroscience research and better understand diseases affecting the brain and spinal cord.

This protocol describes an improved technique for the abundant collection of cerebrospinal fluid (CSF) with no contamination from blood. With greater sample collection and purity, more analyses can be performed using CSF to further our understanding of diseases that affect the brain and spinal cord.

Beyin omurilik sıvısı imzalat fareler gelen topluluğu için geliştirilmiş bir yöntem

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.

The presented protocol describes the transport and preparation of resected human hippocampal tissue with the ultimate goal to use vital brain slices as a preclinical evaluation tool for potential antiepileptic substances.

Elektrofizyolojik Kayıtlar için Akut İnsan Hipokampal Dilimlerinin Hazırlanması

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for ...