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.

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

Universidad de Cartagena UDEC

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

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

Bir Organotipik Beyin Dilim ve kanser hücrelerinin 3D Sferoid ile Coculture Sistemi

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

Tıp

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

Çok bölmesi MSS Nöron glia Co-kültür mikroakışkan Platformu

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Biyoloji

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed &#39;organoids&#39; or &#39;spheroids&#39;, for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures (&#39;asteroids&#39;) are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

Sinaptik Microcircuit modelleme 3D Cocultures Astrocytes ve Neurons insan Pluripotent kök hücre ile

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

Gelişim Biyolojisi

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Astrocyte ağlar gelişimsel mekanizmaları özetlemek ve sinir sistemi yeniden oluşturma işlemi kolaylaştırmak için tasarlanmış üç boyutlu doku uyumlu

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Biyomühendislik

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function.

We present detailed protocols for the generation and characterization of 2D and 3D human induced pluripotent stem cell (hIPSC)-based models of neocortical development as well as complementary methodologies enabling qualitative and quantitative analysis of primary cilium (PC) biogenesis and function.

Neokortik Gelişim Sırasında Primer Seyum Tutulumını Diseksiyona Yönelik 2D ve 3D İnsan Kaynaklı Pluripotent Kök Hücre Bazlı Modeller

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older ...

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

Using multiple angles to cut the mouse pup brain, we improve upon a previously-described acute brain slice which captures the connections between most of the major auditory midbrain and forebrain structures.

Bir Colliculo-talamokortikal Fare Beyin Dilim modifikasyonu, 3-D Odası Bileşenleri baskı ve Multi-ölçekli Optik Görüntüleme birleştirilerek

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

Nörobilim

Visualization of Cortical Modules in Flattened Mammalian Cortices

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and can be delineated by certain histochemical and immunohistochemical methods. In this study, we highlight a method to isolate the cortex from mammalian brains and flatten them to obtain sections parallel to the cortical sheet. We further highlight selected histochemical and immunohistochemical methods to process these flattened tangential sections to visualize cortical modules. In the somatosensory cortex of various mammals, we perform cytochrome oxidase histochemistry to reveal body maps or cortical modules representing different parts of the body of the animal. In the medial entorhinal cortex, an area where grid cells are generated, we utilize immunohistochemical methods to highlight modules of genetically determined neurons which are arranged in a grid-pattern in the cortical sheet across several species. Overall, we provide a framework to isolate and prepare layer-wise flattened cortical sections, and visualize cortical modules using histochemical and immunohistochemical methods in a wide variety of mammalian brains.

This article describes a detailed methodology to obtain flattened tangential sections from mammalian cortices and visualize cortical modules using histochemical and immunohistochemical methods.

Görselleştirme düzleştirilmiş memeli Cortices'deki kortikal modülleri

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and ...

A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s neuroprotective and therapeutic potential. Organotypic slices are prepared from explanted tissue and represent a complex multicellular ex vivo environment. They preserve the three-dimensional cytoarchitecture and local environment of brain cells, maintain the neuronal connectivity and the neuron-glia reciprocal interaction. Hippocampal organotypic slices are considered suitable to explore the basic mechanisms of epileptogenesis, but clinical research and animal models of epilepsy have suggested that the rhinal cortex, composed of perirhinal and entorhinal cortices, play a relevant role in seizure generation.
Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Recordings of spontaneous activity from the CA3 area under perfusion with complete growth medium, at physiological temperature and in the absence of pharmacological manipulations, showed that these slices depict evolving epileptic-like events throughout time in culture. Increased cell death, through propidium iodide uptake assay, and gliosis, assessed with fluorescence-coupled immunohistochemistry, was also observed. The experimental approach presented highlights the value of rhinal cortex-hippocampus organotypic slice cultures as a platform to study the dynamics and progression of epileptogenesis and to screen potential therapeutic targets for this brain pathology.

Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Under a gradual and controlled deprivation of serum, these slices depict evolving epileptic-like events and can be considered an ex vivo model of epileptogenesis. This system represents an excellent tool for monitoring the dynamics of spontaneous activity, as well as for assessing the progression of neuroinflammatory features throughout the course of epileptogenesis.

Renal Korteks-Hipokampus Organotipik Dilim Kültürlerinde Epileptogenez Modeli

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s ...

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

This protocol demonstrates a straightforward and robust method to study in situ axon growth and growth cone dynamics. It describes how to prepare ex vivo physiologically relevant acute brain slices and provides a user-friendly analysis pipeline.

Yerinde Akut Ex Vivo Embriyonik Beyin Dilimi Kültürlerinde Akson Büyümesi ve Büyüme Koni Dinamiğinin Görselleştirilmesi

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...

<meta charset="utf8"></head><body>Fare Duyusal Korteks Geliştirmede Nöronal Aktivite Haritalaması

In Vivo Visualization of Spontaneous Activity in Neonatal Mouse Sensory Cortex at a Single-Neuron Resolution

The mammalian brain undergoes dynamic developmental changes at both the cellular and circuit levels throughout prenatal and postnatal periods. Following the discovery of numerous genes contributing to these developmental changes, it is now known that neuronal activity also substantially modulates these processes. In the developing cerebral cortex, neurons exhibit synchronized activity patterns that are specialized to each primary sensory area. These patterns markedly differ from those observed in the mature cortex, emphasizing their role in regulating area-specific developmental processes. Deficiencies in neuronal activity during development can lead to various brain diseases. These findings highlight the need to examine the regulatory mechanisms underlying activity patterns in neuronal development. This paper summarizes a series of protocols to visualize primary sensory areas and neuronal activity in neonatal mice, to image the activity of individual neurons within the cortical subfields using two-photon microscopy in vivo, and to analyze subfield-related activity correlations. We show representative results of patchwork-like synchronous activity within individual barrels in the somatosensory cortex. We also discuss various potential applications and some limitations of this protocol.

<meta charset="utf8"></head><body>Yazar Spotlight: İki Foton Mikroskobu ve Tek Nöron Görüntülemeden Nöral Devre Oluşumunun Deşifre Edilmesi

Primary sensory areas in the neocortex exhibit unique spontaneous activities during development. This article describes how to visualize individual neuron activities and primary sensory areas to analyze area-specific synchronous activities in neonatal mice in vivo.

Yenidoğan Fare Duyusal Korteksinde Tek Nöron Çözünürlükte Spontan Aktivitenin İn vivo Görselleştirilmesi

The mammalian brain undergoes dynamic developmental changes at both the cellular and circuit levels throughout prenatal and postnatal periods. Following ...