Ayrıca Gabriel Hand eleştirel okuma için teşekkür ediyoruz.

Osaka Üniversitesi ve Japonya Nörobilim Derneği hayvan refahı komiteleri tarafından kurulan esaslarına göre tüm deneyler yapıldı.
1. Organotypic cocultures, talamus ve serebral korteks
Not: özgün yayınlar7,8,13ile ilgili ayrıntılı yordamlar için bakın. Tüm yordamları steril koşullarda gerçekleştirilmesi gerekiyor. Sprague-Dawley (SD) fareler nöronal kültürler için kullanılır.
<ol><li>
Reaktifler hazırlıkları Not: Aşağıdaki reaktifler hacimleri bir sıçan beyin için vardır.<ol>
<li>
Prepare 10 değiştirilmiş N2 ek x 13: insülin (50 μg/mL), progesteron (200 nM), hidrokortizon (200 nM), sodyum selenit (300 nM), transferrin (1 mg/mL), putrescine (1 mM), glikoz (60 mg/mL)</li>
		<li>
Prepare serum içeren kültür orta (10 mL): %85 DMEM/F12, %10 10 x değiştiren N2 ek, % 5 fetal sığır serum (FBS)</li>
		<li>
Prepare serum serbest kültür orta (20 mL): %90 DMEM/F12, %10 10 x değiştiren N2 ek</li>
		<li>Sıçan kuyruğu kollajen çözüm (0.5 mL) hazırlayın. Kısacası, steril koşullar altında bir sıçan kuyruk ihale lifleri al ve Asetik asit çözümde gecede kuluçkaya. Santrifüjü sonra süpernatant (bir kaç mg/mL) toplar ve bir buzdolabı13' te saklarız.</li>
	</ol>
</li>
	<li>
Kültür yemeklerin hazırlanması
	<ol>
<li>Bir membran eklemek bir 35 mm Petri kabına yerleştirin.</li>
		<li>Membran için 40-50 μL sıçan kuyruk kollajen çözüm ekleyin ve kollajen çözüm merkezi etrafında yayılmış.</li>
		<li>Hava Kuru membran tamamen temiz bir Bank içinde.</li>
		<li>1.5-2 mL serum içeren kültür orta çanağı içine koymak, böylece membran ile orta ıslatılır.</li>
		<li>Bir kuluçka (37 ° C, oksijen % 95 hava ve % 5 CO2) kaplama önce kültür çanak yerleştirin.</li>
	</ol>
</li>
	<li>
Kortikal dilimleri hazırlanması
	<ol>
<li>Tüm cerrahi aletler % 70 etanol ile 10 dakika veya daha uzun sterilize.</li>
		<li>Doğum sonrası gün (P) 2 üzerinden hızlı bir şekilde tüm beynini incelememize fare (-buz birkaç dakikalığına dalmış) buz gibi anestezi altında.</li>
		<li>Yer 100 mm Petri kabına 100 mL soğuk Hanks içeren beynine dengeli tuz solüsyonu.</li>
		<li>Pia sorun beyinden iyi Forseps ile kaldırın ve kortikal dilimler (300-400 mikron kalınlık) görsel ve somatosensor korteks mikrocerrahi makasla kesip (ayrıntı için bkz: şekil 1). Not: Kortikal dilim kalınlığı yaklaşık Binoküler mikroskop altında Izgaralar ölçeğini tarafından tahmin edilmektedir. Not: Alternatif olarak, kortikal dilimleri bir vibratome steril koşullarda yapılabilir.</li>
		<li>Birkaç kortikal dilim membran INSERT bir tek kullanımlık plastik pipet ile üzerine aktarın.</li>
		<li>Kültür INSERT (Ayrıntılar için bkz:8,13) yangın cilalı Pasteur pipet ile Merkezi yakın dilim konumlarını ayarlamak ve aşırı orta gelen Ekle Kaldır. Not: Dilimleri hem gaz hem de orta yeterli miktarda almak için biraz aşağıda dilimleri orta düzeyde ayarlayın. Bu hücre canlılık için çok önemlidir.</li>
		<li>Yalnız bir gün önce talamik bir blok alınır ve yanında yerleştirilir için oksijen % 95 hava ve % 5 CO2ve kültür ortamında 37 ° C'de serum içeren kültür orta dilim korumak.</li>
	</ol>
</li>
	<li>
Talamik blokları hazırlanması
	<ol>
<li>Tüm cerrahi aletler % 70 etanol ile 10 dakika veya daha uzun sterilize.</li>
		<li>A gebe iri fare (embriyonik gün 15) anestezi içeren Fentobarbital (50 mg/kg) mayi enjeksiyonu ile anestezi.</li>
		<li>Her embriyo rahim boynuzları üzerinden teşrih ve embriyoların bir 100 mm Petri kabına 100 mL soğuk Hanks dengeli tuz çözeltisi içeren yerleştirin.</li>
		<li>Binoküler mikroskop altında beyin her embriyo kaldırmak ve mikrocerrahi makas (bkz. şekil 1) kullanarak ventrobasal karmaşık ve yanal geniculate çekirdeği (yaklaşık 0.5 mm x 3)7 esas olarak içeren talamik blok kesme.</li>
	</ol>
</li>
	<li>
Kültür ve organotypic cocultures korumak
	<ol>
<li>Pasteur pipet kullanarak, membran filtre üzerinde yerleştirilen kortikal dilimin ventrikül yüzey yanındaki blok talamik yerleştirin.</li>
		<li>Cocultures bir ortamda oksijen % 95 hava ve % 5 CO237 ° C'de serum içeren kültür ortamda korumak.</li>
		<li>Serum serbest kültür orta ile orta yarısı kültür iki gün sonra değişimi. Bundan sonra orta 2-3 günde değişimi. Not: Hücre hayatta kalmak için önemli olduğu gibi orta düzeyde orta değişiminden sonra kontrol edilmelidir.</li>
	</ol>
</li>
</ol>2. adım
Tüm yordamları steril koşullarda gerçekleştirilmesi gerekiyor.
Not: Elektroporasyon bir gün dekolmanı blokların önlemek için talamik blokları hazırlanması sonra gerçekleştirin.
<ol><li>
Plazmid
	<ol>
<li>Plazmid karışımı aşağıdaki gibi yapmak: pCAGGS-DsRed 2 μg/μL; pCAG-SYP-EGFP 3.5 μg/μL. Not: Endotoksin-Alerjik Maxiprep seti kullanarak her iki plazmid arındırmak ve Hanks dengeli tuz solüsyonu içinde çözülür.</li>
	</ol>
</li>
	<li>
Elektroporasyon ekipman kurulumu Şekil 2' de gösterildiği gibi bir uyarıcı ve bir izolatör gibi elektrikli aletleri bağlanması gerekir.<ol>
<li>Uyarıcı bifazik izolatör bağlayın.</li>
		<li>Bir elektrot (gümüş tel 0.2 mm çapında) izolatör negatif terminaline bağlayın.</li>
		<li>Bir elektrot (gümüş tel 1 mm çapında) serisi 100 Ω direnç ve izolatör pozitif terminal için bağlayın.</li>
		<li>Amplifikatör elektrik akımları ile amplifikatör gerilim ölçerek direnç iletilir olup olmadığını izlemek için osiloskop bağlayın.</li>
	</ol>
</li>
	<li>
Cam Pipetler hazırlanması Not: Plazmid çözüm çýkartýlmasýný ve elektrik darbeleri uygulanması için iki tür cam Pipetler kullanılır.<ol>
<li>Pipetler cam kılcal bir elektrot çektirme ile üzerinden yapmak.</li>
		<li>Fırlatma pipet için belgili tanımlık uç (uç çapı 20-50 mikron olmalıdır) bölünürler.</li>
		<li>Eziyet ve ateş-(iç çap 50-200 μm kalınlığında olmalı) parlatma tarafından takip zımpara ile elektrot pipet ucu Lehçe. Not: Doğrudan doku dokunuyor gibi elektrot uç düz ve pürüzsüz, olmalıdır.</li>
	</ol>
</li>
	<li>
Elektroporasyon prosedürü
	<ol>
<li>Fırlatma ve elektrot Pipetler için manipülatörler bağlayın.</li>
		<li>Gümüş tel (0.2 mm çap) elektrot pipet yerleştirin.</li>
		<li>Fırlatma pipet 1 mL şırınga ile bir plastik tüp takın.</li>
		<li>Almak yukarıya-> 5 μL plazmid çözüm içine şırınga emme tarafından fırlatma pipet.</li>
		<li>Elektroporasyon sahnede coculture koyun ve 1 mm çap elektrot kültür ortamı yerleştirin.</li>
		<li>Fırlatma pipet talamik explant yerleştirin.</li>
		<li>Talamik blok yüzeyine dokunduğundan sonra şırınga el ile itin. Not: Plazmid 0.5 μL hakkında çözüm genellikle bir talamik blok kullanılır.</li>
		<li>Elektrot pipet talamik blokta hemen fırlatma pipet retraksiyon sonra yerleştirin.</li>
		<li>Elektrik darbeleri (50-400 μA, 3-5 tren 1 ms süre 200 Hz'de 200 kare bakliyat) teslim. Genlik ve trenler osiloskop üzerinde sayısını izler. Not: Elektrik akımlarını genlik bir elektrot pipet13iç çap üzerinde bağlıdır.</li>
		<li>Elektrot pipet geri çek ve bulaşık kuluçka makinesi için dönün.</li>
	</ol>
</li>
	<li>
Mikroskobik gözlem
	<ol>
<li>EGFP için iki filtre kümesi ile donatılmış bir dik confocal mikroskop mikroskobik bir sahnede kültür çanak koymak (uyarma, 488 nm; emisyon, 515 nm) ve DsRed (uyarma, 514 nm; emisyon, 565 nm).</li>
		<li>10-25 optik bölümleri 1-7 µm-adım boyutlarında bir 20 X uzun çalışma mesafe objektif lens ile fotoğraf çekmek. Hem DsRed hem de SYP-EGFP hızlı tek tek ayırt etiketli aksonlar seçin. Not: Yaklaşık 0,7 x 0.7 mm (yani 1024 x 1024 piksele karşılık gelen, 0,68 mikron/piksel) gözlenen alandır.</li>
		<li>Esir alma imge her 24 saat için günlük düşsel. Oda sıcaklığında 10 dakika içinde tamamlamak ve kültürler için kuluçka dönün. Kısa-Aralık zaman atlamalı görüntüleme için kültür ortamının % 95 hava ve %5 37 ° C'de CO2 nemlendirilmelidir tutar bir kültür odasında devam et</li>
	</ol>
</li>
</ol>

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Yazarlar ifşa gerek yok.

Geçerli protokol de TC projeksiyon11dışında aksonlar büyüyen gelişim yönü eğitim için güçlü bir araçtır. Örneğin, kortikal dilim kültür ve elektroporasyon tekniği bir arada bireysel aksonal Morfoloji kortikal nöronlar ve uzun vadeli gözlem9,18görselleştirme sağlar.
Geçerli iletişim kuralını kullanarak, ilginç genler axon dallanma ve sinaps oluşumunda rol de floresan proteinler ve ...

Thalamocortical (TC) projeksiyon memeli beyinde axon rehberlik ve mekanizmaları hedefleme araştırmak için uygun bir sistemdir. Geliştirme sırasında duyusal TC aksonlar kortikal plaka ve formu şube ve sinapslarda tercihen içinde katman IV serebral korteks1,2birincil duyusal alanda büyümek. Temel bağlantılar kurulması sonra bile, aksonlar arbors ve sinaptik terminalleri çevresel değişiklikler3,4bağlı olarak yenilenmiş. Ancak, nasıl TC axon Morfoloji dinamik olarak değiştirilmez kötü anlaşılmaktadır. Bir tek hücre düzeyinde yapısal değişiklikleri gözlemlemek için yeterli bir teknik eksikliği ana nedenlerinden biri. Mikroskobu, İki fotonlu mikroskobu gibi son gelişmeler yaşayan kortikal nöronlar vivo içindedoğrudan gözlem izin vermiş, ancak genel TC yörüngeleri5, yakalamak için hala teknik sınırlamalar vardır 6. bu nedenle, vitro yöntemleri TC aksonlar canlı görüntüleme için axon dallanma ve synapse oluşumu yapısal analiz için güçlü araçlar sağlamak.
Bizim grup ilk kez bir statik dilim kültür yöntemi geçirgen membran7ile kurulmuş. Bu yöntemi kullanarak, bir sıçan kortikal dilim bir duyusal talamik blok ile cocultured ve lamina özgü TC bağlantıları bu organotypic cocultures7,8' recapitulated. Seyrek bir floresan protein ile etiketleme daha fazla TC axon büyüme ve dal oluşumu9,10,11gözlemlemek için bize izin. Son zamanlarda, dallanma, aynı anda görüntüleme için yeni bir yöntem geliştirdik ve bireysel TC aksonlar organotypic sinaps oluşumu12cocultures. TC akson ve presynaptic siteleri aynı anda görmek için DsRed ve EGFP etiketli synaptophysin (SYP-EGFP) talamik nöronların az sayıda organotypic coculture elektroporasyon tarafından ortak transfected. Geçerli yöntem TC aksonlar morfolojik analizi kolaylaştırır ve akson dallanma ve synapse oluşumu arasındaki nedensel ilişkiyi göstermek için kullanılan uzun vadeli gözlem için sağlar.

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		<col style="width:89pt" width="119" />
		<col style="width:84pt" width="112" />
		<col style="width:58pt" width="77" />
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	<tbody>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">DMEM/F12</td>
			<td class="xl20" style="width:89pt" width="119">GIBCO</td>
			<td class="xl20" style="width:84pt" width="112">11320-033</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Hanks&#8217; balanced salt solution (HBSS)</td>
			<td class="xl20" style="width:89pt" width="119">Nissui</td>
			<td class="xl21" style="width:84pt" width="112">5905</td>
			<td class="xl20" style="width:58pt" width="77"></td>
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		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Fetal bovine serum (FBS)</td>
			<td class="xl20" style="width:89pt" width="119">Thermo Scientific</td>
			<td class="xl20" style="width:84pt" width="112">SH30396-03</td>
			<td class="xl20" style="width:58pt" width="77">Hyclone</td>
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			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Insulin</td>
			<td class="xl20" style="width:89pt" width="119">Sigma</td>
			<td class="xl20" style="width:84pt" width="112">I6634</td>
			<td class="xl20" style="width:58pt" width="77"></td>
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		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Progesterone</td>
			<td class="xl20" style="width:89pt" width="119">Sigma</td>
			<td class="xl20" style="width:84pt" width="112">P8783</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Hydrocortisone</td>
			<td class="xl20" style="width:89pt" width="119">Sigma&#160;</td>
			<td class="xl20" style="width:84pt" width="112">H0888</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Sodium selenite</td>
			<td class="xl20" style="width:89pt" width="119">Wako Pure 
			Chemical Industries</td>
			<td class="xl20" style="width:84pt" width="112">192-10843</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Transferrin&#160;</td>
			<td class="xl20" style="width:89pt" width="119">Sigma</td>
			<td class="xl20" style="width:84pt" width="112">T1147</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Putrescine&#160;</td>
			<td class="xl20" style="width:89pt" width="119">Sigma</td>
			<td class="xl20" style="width:84pt" width="112">P5780</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Glucose</td>
			<td class="xl20" style="width:89pt" width="119">Wako 
			Pure Chemical Industries</td>
			<td class="xl20" style="width:84pt" width="112">16806-25</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">35 mm petri dishes</td>
			<td class="xl20" style="width:89pt" width="119">Falcon</td>
			<td class="xl21" style="width:84pt" width="112">351008</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Millicell-CM insert</td>
			<td class="xl20" style="width:89pt" width="119">Millipore</td>
			<td class="xl20" style="width:84pt" width="112">PICMORG50</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">100 mm petri dishes</td>
			<td class="xl20" style="width:89pt" width="119">BIO-BIK</td>
			<td class="xl20" style="width:84pt" width="112">I-90-20</td>
			<td class="xl20" style="width:58pt" width="77">petri dish sterrile</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">HiPure Plasmid Maxiprep Kit</td>
			<td class="xl20" style="width:89pt" width="119">Invitrogen</td>
			<td class="xl20" style="width:84pt" width="112">K210006</td>
			<td class="xl23" style="width:58pt" width="77"></td>
		</tr>
		<tr height="59" style="height:44.25pt">
			<td class="xl20" height="59" style="width:113pt;height:44.25pt" width="151">Disposable sterile plastic pipettes</td>
			<td class="xl20" style="width:89pt" width="119"></td>
			<td class="xl20" style="width:84pt" width="112">202-IS</td>
			<td class="xl20" style="width:58pt" width="77">transfer pipets sterile</td>
		</tr>
		<tr height="54" style="height:40.5pt">
			<td class="xl20" height="54" style="width:113pt;height:40.5pt" width="151">Glass capillary: OD 1.2 mm</td>
			<td class="xl20" style="width:89pt" width="119">Narishige</td>
			<td class="xl20" style="width:84pt" width="112">&#160;G-1.2</td>
			<td class="xl20" style="width:58pt" width="77">inner diameter, 1.2 mm</td>
		</tr>
		<tr height="53" style="height:39.75pt">
			<td class="xl20" height="53" style="width:113pt;height:39.75pt" width="151">Silver wire: 0.2 and 1 mm&#160;</td>
			<td class="xl20" style="width:89pt" width="119">Nilaco</td>
			<td class="xl20" style="width:84pt" width="112">AG-401265 (diameter, 0.2 mm), AG-401485 (diameter, 1.0 mm)</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">1 mL syringe</td>
			<td class="xl20" style="width:89pt" width="119">Terumo</td>
			<td class="xl20" style="width:84pt" width="112">SS-01T</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Stimulator&#160;</td>
			<td class="xl20" style="width:89pt" width="119">A.M.P.I</td>
			<td class="xl20" style="width:84pt" width="112">Master 8</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Biphasic isolator&#160;</td>
			<td class="xl20" style="width:89pt" width="119">BAK ELECTRONICS</td>
			<td class="xl20" style="width:84pt" width="112">BSI-2</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Amplifier&#160;</td>
			<td class="xl20" style="width:89pt" width="119">A-M Systems</td>
			<td class="xl20" style="width:84pt" width="112">Model 1800</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Oscilloscope</td>
			<td class="xl20" style="width:89pt" width="119">Hitachi</td>
			<td class="xl20" style="width:84pt" width="112">VC-6723</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Manipulator</td>
			<td class="xl20" style="width:89pt" width="119">Narishige</td>
			<td class="xl20" style="width:84pt" width="112">SM-15</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Micromanipulator</td>
			<td class="xl20" style="width:89pt" width="119">Narishige</td>
			<td class="xl20" style="width:84pt" width="112">MO-10</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Stereomicroscope&#160;</td>
			<td class="xl20" style="width:89pt" width="119">Olympus</td>
			<td class="xl20" style="width:84pt" width="112">SZ40</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Universal stand&#160;</td>
			<td class="xl20" style="width:89pt" width="119">Olympus</td>
			<td class="xl20" style="width:84pt" width="112">SZ-STU2</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Light illumination system&#160;</td>
			<td class="xl20" style="width:89pt" width="119">Olympus</td>
			<td class="xl20" style="width:84pt" width="112">LG-PS2, LG-DI, HLL301</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Electrode puller&#160;</td>
			<td class="xl20" style="width:89pt" width="119">Narishige</td>
			<td class="xl20" style="width:84pt" width="112">PC-10</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Confocal microscope</td>
			<td class="xl20" style="width:89pt" width="119">Nikon</td>
			<td class="xl20" style="width:84pt" width="112">Digital eclipse C1 laser</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">x20 objective</td>
			<td class="xl20" style="width:89pt" width="119">Nikon</td>
			<td class="xl20" style="width:84pt" width="112">ELWD 20x/0.45</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Culture chamber</td>
			<td class="xl20" style="width:89pt" width="119">Tokai Hit</td>
			<td class="xl20" style="width:84pt" width="112">UK A16-U</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Sprague-Dawley (SD) rat</td>
			<td class="xl20" style="width:89pt" width="119">Japan SLC and Nihon-Dobutsu</td>
			<td class="xl20" style="width:84pt" width="112"></td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:113pt;height:30.0pt" width="151">Microsurgery scissors</td>
			<td class="xl20" style="width:89pt" width="119">Natsume&#160;</td>
			<td class="xl20" style="width:84pt" width="112">MB-54-1</td>
			<td class="xl20" style="width:58pt" width="77"></td>
		</tr>
	</tbody>
</table>

visualization of thalamocortical axon branching and synapse formation in organotypic cocultures

Axon dallanma ve synapse oluşumu kesin nöronal devreler kurmak için çok önemli işlemler vardır. Geliştirme sırasında duyusal thalamocortical (TC) aksonlar serebral korteksin belirli katmanlar halinde şube ve sinapslarda oluştururlar. Axon dallanma ve synapse oluşumu arasında bariz uzamsal ilişki rağmen aralarındaki nedensel ilişki kötü anlaşılmaktadır. Bu sorunu gidermek için son zamanlarda bireysel TC aksonlar organotypic cocultures içinde dallanma ve synapse oluşumu aynı anda görüntüleme için bir yöntem geliştirdi.
Bu iletişim kuralı bir organotypic coculture ve elektroporasyon birleşimini içerir bir yöntemini açıklar. Organotypic cocultures, talamus ve serebral korteks gen manipülasyon ve gözlem Laminer yapılandırması gibi karakteristik yapıları koruyarak aksonal süreçlerin kolaylaştırmak. DsRed ve EGFP etiketli synaptophysin (SYP-EGFP) kodlama iki ayrı plazmid talamik nöronların az sayıda bir adım tekniği ile birlikte transfected. Bu yöntem TC nöronların bireysel aksonal türleri morfoloji ve presynaptic siteleri aynı anda görselleştirmek izin verdi. Yöntemi Ayrıca axon dallanma ve synapse oluşumu arasındaki nedensel ilişkiyi ortaya uzun vadeli gözlem etkin.

Deneme amaçları TC axon dallanma ve synapse oluşumu arasındaki ilişkiyi ortaya çıkarmak için burada açıklanan. Aynı anda aksonal yörüngeler ve yerleri presynaptic siteleri, tek veya organotypic cocultures bir kaç talamik hücrelerde görselleştirmek için kodlama SYP-EGFP ve DsRed elektroporasyon kullanarak iki Plasmid'ler ile transfected. Kültür ikinci haftasında tek tek ayırt TC aksonlar açıkça DsRed (şekil 3) tarafından etiketli. Sa...

Watch this Scientific Journal Video about Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures at JoVE.com

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

Bu iletişim kuralı thalamocortical akson dallanma ve synapse oluşumu organotypic cocultures, talamus ve serebral korteks eşzamanlı görüntüleme için bir yöntemi açıklar. Bireysel thalamocortical akson ve presynaptic onların terminalleri bir tek hücre çoğalmasıyla tekniği ile DsRed ve synaptophysin GFP öğesini tarafından görüntülenmiştir.

Görselleştirme Thalamocortical Axon dallanma ve sinaps oluşumunda Organotypic Cocultures

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) ...

visualization-thalamocortical-axon-branching-synapse-formation

Research

JoVE Journal

Neuroscience

6.4K Görüntüleme.  Kanazawa University. Bu iletişim kuralı thalamocortical akson dallanma ve synapse oluşumu organotypic cocultures, talamus ve serebral korteks eşzamanlı görüntüleme için bir yöntemi açıklar. Bireysel thalamocortical akson ve presynaptic onların terminalleri bir tek hücre çoğalmasıyla tekniği ile DsRed ve synaptophysin GFP öğesini tarafından görüntülenmiştir.

Video: Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

Automated Interactive Video Playback for Studies of Animal Communication

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, parameter-based computer animation offers the opportunity to independently manipulate any number of behavioral, morphological, or spectral characteristics in the context of realistic, moving images of animals on screen. A major limitation of conventional playback, however, is that the visual stimulus lacks the ability to interact with the live animal. Borrowing from video-game technology, we have created an automated, interactive system for video playback that controls animations in response to real-time signals from a video tracking system. We demonstrated this method by conducting mate-choice trials on female swordtail fish, Xiphophorus birchmanni. Females were given a simultaneous choice between a courting male conspecific and a courting male heterospecific (X. malinche) on opposite sides of an aquarium. The virtual male stimulus was programmed to track the horizontal position of the female, as courting males do in the wild. Mate-choice trials on wild-caught X. birchmanni females were used to validate the prototype's ability to effectively generate a realistic visual stimulus.

Video playback is a widely used technique in animal behavior. We created and evaluated a program that applies rules-based, interactive playback of 3-D computer animations in response to real-time, automated data on subject behavior.

Hayvan İletişim Araştırmaları Otomatik Etkileşimli Video Oynatma

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, ...

Nörobilim

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Içinde proprioseptörlere Görselleştirmesi<em&gt; Drosophila</em&gt; Larva ve Pupa

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

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 ...

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (&#947;-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA receptors (GABAARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials. 
During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAARs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other. 
To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAAR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAARs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts.

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

The molecular mechanisms that co-ordinate the formation of inhibitory GABAergic synapses during ontogeny are largely unknown. To study these processes,we have developed a co-culture model system which incorporates embryonic medium spiny GABAergic neurons cultured together with stably transfected human embryonic kidney 293 (HEK293) cells expressing functional GABAA receptors.

Co-kültür Model Birleşmeyle GABA&#39;erjik Orta Spiny Nöronlar ve HEK293 Hücreler inhibitör Synapse Oluşumu istikrarlı bir şekilde GABA ifade<sub&gt; A</sub&gt; Reseptörler

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

Fare Embriyo Duyu Akson Projeksiyonlar tüm montaj Görüntüleme

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

Kombine Metodolojileri kullanan Akson Dallanman ve Nöronal Morfogenez sırasında Plazma Membran Teslimat Rolü tanımlayın

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

Koku Meşfatının Olfaktör Duyusal Akson Moleküler Kimlik Kodlarının Görselleştirilmesi İçin Dörtlü İmmün Sulanması

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...

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

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of its labeling was affected by various factors, here, we provide a refined protocol for the application of high molecular weight BDA for studying optimal neural labeling in the central nervous system. After stereotactic injection of BDA into the ventral posteromedial nucleus (VPM) of the thalamus in the rat through a delicate glass pipette, BDA was stained with fluorescent streptavidin-Alexa (AF) 594 and counterstained with fluorescent Nissl stain AF500/525. On the background of green Nissl staining, the red BDA labeling, including neuronal cell bodies and axonal terminals, was more distinctly demonstrated in the somatosensory cortex. Furthermore, double fluorescent staining for BDA and the calcium-binding protein parvalbumin (PV) was carried out to observe the correlation of BDA labeling and PV-positive interneurons in the cortical target, providing the opportunity to study the local neural circuits and their chemical characteristics. Thus, this refined method is not only suitable for visualizing high quality neural labeling with the high molecular weight BDA through reciprocal neural pathways between the thalamus and cerebral cortex, but also will permit the simultaneous demonstration of other neural markers with fluorescent histochemistry or immunochemistry.

Here, we present a refined protocol to effectively reveal biotinylated dextran amine (BDA) labeling with a fluorescent staining method through a reciprocal neural pathway. It is suitable for analyzing the fine structure of BDA labeling and distinguishing it from other neural elements under a confocal laser scanning microscope.

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

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

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 ...

Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced tools currently used to study neurons, have only partly been taken on by oligodendrocyte researchers. Cell type-specific staining by viral transduction is a useful approach to study live organelle dynamics. This paper describes a protocol for visualizing oligodendrocyte mitochondria in organotypic brain slices by transduction with adeno-associated virus (AAV) carrying genes for mitochondrial targeted fluorescent proteins under the transcriptional control of the myelin basic protein promoter. It includes the protocol for making organotypic coronal mouse brain slices. A procedure for time-lapse imaging of mitochondria then follows. These methods can be transferred to other organelles and may be particularly useful for studying organelles in the myelin sheath. Finally, we describe a readily available technique for visualization of unstained myelin in living slices by Confocal Reflectance microscopy (CoRe). CoRe requires no extra equipment and can be useful to identify the myelin sheath during live imaging.

Myelinating oligodendrocytes promote rapid action potential propagation and neuronal survival. Described here is a protocol for oligodendrocyte-specific expression of fluorescent proteins in organotypic brain slices with subsequent time-lapse imaging. Further, a simple procedure for visualizing unstained myelin is presented.

Görselleştirme ve Oligodendrocyte organelleri Adeno ilişkili virüs ve Confocal mikroskobu kullanılarak Organotypic beyin dilimleri içinde canlı görüntüleme

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced ...

Görselleştirme Thalamocortical Axon dallanma ve sinaps oluşumunda Organotypic Cocultures

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