Bu eser Bilim Bakanlığı tarafından desteklenen ve teknoloji MOST104-2311-B-010-010-MY3, verir MOST106-2321-B-010-012, Ulusal Sağlık araştırma enstitüleri NHRI-EX106-10429NI ve seçme alanlarda araştırma merkezi programı hibe verin Eğitim Bakanlığı aracılığıyla beyin Araştırma Merkezi, Ulusal Yang Ming Üniversitesi Tayvan, ve doktora sonrası bursu MOST106-2811-B-010-031 (S.-Y.C.), MOST105-2811-B-010-036 ve MOST106-2811-B-010-030 (H.-Y.K.) verir.

Burada anlatılan hayvan iletişim kuralları hayvan bakımı ve kullanımı komiteler Ulusal Yang Ming Üniversitesi tarafından onaylanmıştır.
1. hazırlanması sahibi için yenidoğan Pups içinde stereotaksik aparatı
<ol><li>Baş tepsi olun: 1/5 Tüp duvarı kaldırarak yenidoğan pups Başkanı uygun şekil 1.5 mL santrifüj tüpü (15 mm uzun) alt kesti.</li>
	<li>Stereotaksik aparatı Kaide ile uygun doğru boyutta bir pipet ucu kutusu ve üst kapağı sökün. Adım 1.1 ve doku gömme kaset ipucu tepsi sıcak-erime yapıştırıcı ile temeli üzerine hazırlanan baş tepsi yapıştırmayın. 7 mm, PUPs boyun ve gövde baş-sabit konumda desteklemek için kullanılan doku gömme kaset yüksekliğidir.</li>
	<li>Tüm kurulum bir standart stereotaksik aparatı yerleştirin.</li>
</ol>2. 30G Enjeksiyon paslanmaz çelik iğneler hazırlanması
<ol><li>30 G şırınga iğneleri için 3 gün içinde kloroform ıslatarak önceden temiz.</li>
	<li>Forseps dikkatli ve yavaş bir kimyasal başlıklı Polipropilen hub üzerinden iğneyi çıkar için kullanın.</li>
	<li>Mutlak etanol iğnelerle bir shaker 50 RPM üzerinde % 70 etanol durular 3 × 10 dk ardından 20 dakika yıkayın. Kuru ve temiz bir kutu, oda sıcaklığında (RT) temizlenmiş iğneler depolamak iğneler hava kadar kullanmak izin.</li>
</ol>3. bir bağdaştırıcı mikroenjeksiyon boruları hazırlayın
<ol><li>Microliter şırınga 30 G Enjeksiyon iğne PE10 Polietilen boru (PE10 tüp) ile bağlayın. PE20 Polietilen boru (PE20 tüp) adaptör enjeksiyon iğne ve microliter şırınga köprülemesini için hazırlayın. PE10 tüp çapı 26 G microliter şırınga iğne daha küçük olduğu için adaptör kullanımı gereklidir.</li>
	<li>Microliter enjektör boruları hazırlayın: önceden temizlenmiş 30G Enjeksiyon iğne PE20 tüp ile 5 cm bağlanmak ve anlık tutkal ile birleşme mühür. Bu boru adaptörü yeniden kullanılabilir. Not: iğne mikroenjeksiyon şırınga 30 G ise, hazırlık (adım 3.1) ve bu adaptörü (adım 3.3) kullanımını gerekli değildir.</li>
	<li>(3.1 adımda hazırlanan) boru adaptörü microliter şırınga (10 µL) takın.</li>
	<li>PE10 (60 cm uzun) tüp PE20 boru adaptörü 30 G iğne üzerine bir ucunu bağlayın. Yeni 30 G Enjeksiyon iğne üzerine PE10 tüp diğer ucunu bağlayın.</li>
</ol>4. yük Autoclaved distile su, boya ve virüsler ile mikroenjeksiyon tüp
<ol><li>Microliter şırınga pistonu çıkarın. 25 G şırınga microliter şırınga ve onun bağlı PE boru boru gelen havayı çıkarmak için autoclaved distile su ile yüklemek için kullanın.</li>
	<li>Geri microliter şırınga fırlatıcıya yerleştirin ve pistonu 2 µL distile su kalır varil kadar itin. 2 µL düşük hale distile su hacmi izin vermeyin.</li>
	<li>Dikkatle microliter şırınga mikro akış hızı şırınga pompa üzerine monte.</li>
	<li>Küçük miktarlarda (% 0,9 serum içinde hazırlanan ve 0,22 µm filtre ile filtre) %0.1 hızlı yeşil boya ve bir parça parafilm virüs sıvı pipet.</li>
	<li>Az miktarda hava bir hava kabarcığı mikroenjeksiyon tüp sıvı akışını sınamak için 30 G Enjeksiyon iğne ve tüpün içine süzülmüş hızlı yeşil 0.7 µL yükleyerek takip PE10 tüp arasındaki kavşakta görünür yapmak için geri alıyorum.</li>
	<li>Mikroenjeksiyon boru içine virüs sıvılar yükleyerek ardından ikinci bir hava kabarcığı yapmak hava küçük bir miktar geri alıyorum.</li>
	<li>Takın ve 30 G mikroenjeksiyon iğne kolu stereotaksik aygıtının güvenli.</li>
</ol>5. anestezi hipotermi tarafından yenidoğan farelerin
<ol><li>Yavru bir lateks eldiven kol koyun ve 5 min için boyun kadar ezilmiş buz bırakın.</li>
	<li>Pups ayak forseps ile para çekme yanıt onun ayak emin olmak için çimdik.</li>
	<li>Lateks eldiven kol ile yavru baş tepsisine yerleştirin ve hipotermi anestezi için soğuk tutmak için lateks kol çevresinde bazı ezilmiş buz koyun.</li>
	<li>Veteriner merhem pups gözünü anestezi altında iken kuruluk mümkünse önlemek için geçerlidir.</li>
</ol>6. mikroenjeksiyon
<ol><li>Steril Cerrahi stereotaksik araç % 70 etanol ile iyice silerek hazırlayın. Cerrahi alet % 70 etanol çeker tarafından sterilize.</li>
	<li>Pups kafa % 70 etanol ile fırçalayın. Kafatasında Simgesel Yapı lambda bulun ve lambda marker kalemle işaretlemek. Lambda için iğne ucu amaç ve ön-arka (AP) ve medial-lateral (ML) koordinatları sıfır ayarlayın.</li>
	<li>Enjeksiyon kol hedef site X ve Y koordinatlarını göre hedef siteye taşımak. Doğum sonrası gün (P) 2 striatum için pups, Koordinatlar: AP, 2,4 mm anterior lambda; ML, ±1.0 mm orta hat yanal; dorsal-ventral (DV), kafatası mm'den −1.7. PE10 tüp hızlı yeşil boya bulunduğu bir kalemle işaretlemek.</li>
	<li>30 G Enjeksiyon iğne yavaş yavaş deri ve kafatası nüfuz ve kafatası yüzeyde durana kadar iğne ucu kadar alın. DV konumu sıfır ayarlayın.</li>
	<li>Hedef site DV koordinatı ulaşıncaya kadar 30 G Enjeksiyon iğne yavaşça. Sürdürme normal şeklini parankimi izin vermek 1 dakika bekleyin. Mikroenjeksiyon çalıştırın (100 nL/dk).</li>
	<li>Hızlı yeşil boya işareti virüs sıvı beyne enjekte edilir emin olmak için PE tüp hareketli olduğundan emin olun.</li>
	<li>Mikroenjeksiyon fesih sonra 1 dakika bekleyin ve sonra yavaş yavaş ve aşamalı olarak iğneyi DV derinliği 30 içinde 1/2 yüksekliğine getirmek s. Sonra 30 s, yavaş yavaş iğne pups başından çekilme.</li>
	<li>Microinjections tüm hedeflenen sitelerin tamamlanıncaya kadar 6.2-6,7 adımları yineleyin.</li>
</ol>7. ameliyat sonrası kurtarma yavru
<ol><li>Yavru için 33 ° C kuluçka 20 dk kadar sıcak. Yavru fok sternal recumbency korumak için yeterli bilinci yerine geldi kadar yavru fok kurtarma hipotermi anestezi her 5 dk dan kontrol edin.</li>
	<li>Yavru fok tamamen iyileşti sonra yavru fok baraja geri dönün.</li>
</ol>

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Yazarlar ifşa gerek yok.

Bu da çalışmanın, yenidoğan fare beyni striatum AAV virüs enjekte için basit ve güvenilir stereotaksik yöntem gösterilmektedir. Biz P2, Ai14 muhabir farelerin striatum içine AAV-eGFP-Cre virüs microinjected ve muhabir gen ekspresyonu P14, analiz ettik. AAV GFP pozitif hücreler boyunca striatum, rostrocaudal düzeyde transduced bulduk. Ayrıca, neredeyse tüm GFP pozitif hücreler AAV aracılı ifade Cre faaliyet tarafından indüklenen başarılı bir Cre-loxP DNA rekombinasyon düşündüren tdTomato muhabi...

Modern çalışmalar yapısı ve beyin fonksiyonu genellikle belirli genler nöronal hücrelerdeki genetik manipülasyon gerektirir. Farklı genler, transgenik fareler de dahil olmak üzere mutant gen taşıyan fonksiyonlar soruşturma için nakavt ve çakma allelleri düzenli olarak yeniden üretilmedi. Stereotaksik beyin ameliyatı yetişkin kemirgen için yerel olarak uyuşturucu, virüsler, izleyiciler ve diğer kimyasalları kemirgen beyin1,2özel bölgeler için teslim etmek için standart bir yöntemdir. Stereotaksik beyin ameliyatı transgenik fareler için uygulama bir genetik olarak gen fonksiyon ve fare beynin belirli nöron popülasyonları nöronal aktivite işlemek için izin verir. Hücre türüne özgü işleme karmaşık sinir devreleri beyin3,4,5nöronal işlevlerinde deşifre etmek için güçlü bir yaklaşım sağlar.
Sinir sisteminin sinirsel gelişme aşamalarında erken embriyonik başlar ve gelişim süreçleri Juvenil dönemine kadar doğumdan sonra devam edin. Postnatal olgunlaşma sinir sistemi beyin6fizyolojik ve bilişsel işlevler için temel sinir devreleri, hassas sinaptik kablolama içerir. Bu nedenle, yenidoğan zaman pencerelerde oluşan gelişimsel olaylarını eğitim sadece normal sinirsel gelişim anlamak için önemlidir, ancak de nörogelişimsel ve Nöropsikiyatrik bozukluklar7 patogenezinde anlayışlar sağlayabilir ,8. Stereotaksik beyin ameliyatı yöntemleri yetişkin kemirgen için hazır2,9, olmasına rağmen birkaç protokolleri stereotaksik beyin ameliyatı yenidoğan fareler10,11' deki yönergeleri için Internet üzerinde kullanılabilir. Yenidoğan yavru Başkanı standart stereotaksik aparatı düzeltilmesi için çok kırılgan olduğu için aslında, reaktifler stereotaksik microinjections yenidoğan fare pups beyinlerinin içine, zordur. Yine de, stereotaksik beyin ameliyatı uygulamaya transgenik farelerde neonatal fareler12için mümkün olabilir. Burada, yeni doğmuş fare pups içinde stereotaksik beyin ameliyatı gerçekleştirmek için ev yapımı bir kurulum ile basit bir yöntem açıklanmaktadır. Bu teknik bir muhabir gen fareler ve koşullu olarak floxed transgenik fareler striatum AAV ifade Cre DNA recombinase microinjecting tarafından floxed genler koşullu olarak silmek izin verdiğini göstermektedir. Bu teknik aynı zamanda reaktifler vahşi tipi farelerin striatum yenidoğan sunmak için geçerlidir.

<table border="0" cellpadding="0" cellspacing="0" width="1134">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">30G PrecisionGlide Needle</td>
			<td style="width:273px;">Becton Dickinson</td>
			<td style="width:199px;">REF 305106</td>
			<td style="width:395px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Chloroform</td>
			<td style="width:273px;">JT Baker</td>
			<td style="width:199px;">9180-03</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Hamilton MICROLITER Syringe</td>
			<td style="width:273px;">Hamilton&#160;</td>
			<td style="width:199px;">80300</td>
			<td style="width:395px;">30G needle fit for PE10 tube; 26G needle needs a PE20 adaptor</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Polyethylene tubing PE20</td>
			<td style="width:273px;">Becton Dickinson</td>
			<td style="width:199px;">427406</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Polyethylene tubing PE10</td>
			<td style="width:273px;">Becton Dickinson</td>
			<td style="width:199px;">427401</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Micro Flow Rate Syringe Pump</td>
			<td style="width:273px;">Longer Precision Pump Co.</td>
			<td style="width:199px;">TJ-2A (Controller) and L0107-2A (Drive Unit)</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">25G syringe</td>
			<td style="width:273px;">Becton Dickinson</td>
			<td style="width:199px;">REF 302105</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Fast green</td>
			<td style="width:273px;">Sigma-Aldrich</td>
			<td style="width:199px;">F-7252</td>
			<td style="width:395px;">0.1%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Standard Stereotaxic Instruments</td>
			<td style="width:273px;">RWD Life Science</td>
			<td style="width:199px;">68037</td>
			<td style="width:395px;">Without using 68030 Mouse/Neonatal Rat Adaptor</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Anti-FOXP2 antibody</td>
			<td style="width:273px;">Abcam</td>
			<td style="width:199px;">ab16046</td>
			<td style="width:395px;">Rabbit polyclonal to FOXP2, 1:4K</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Anti-RFP antibody</td>
			<td style="width:273px;">Abcam</td>
			<td style="width:199px;">ab65856</td>
			<td style="width:395px;">Mouse monoclonal to RFP, 1:1K</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">BX63 microscope</td>
			<td style="width:273px;">Olympus</td>
			<td style="width:199px;">BX63</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">LSM 880 confocal microscope</td>
			<td style="width:273px;">Zeiss</td>
			<td style="width:199px;">LSM 880</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Goat anti-rabbit conjugated Alexa fluor594</td>
			<td style="width:273px;">Jackson lmmunoReserch Laboratories Inc.</td>
			<td style="width:199px;">111-585-003</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">AAV9.hSynapsin.HI.eGFP-Cre.WPRE.SV40</td>
			<td style="width:273px;">Penn Vector Core</td>
			<td style="width:199px;">AV-9-PV1848</td>
			<td style="width:395px;">Lot # CS0987, 5.506x1013 (GC/mL)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">AAV9.chicken actin-eGFP</td>
			<td style="width:273px;">AAV core, Institute of Biomedical Sciences, Academia Sinica, Taiwan</td>
			<td style="width:199px;">N/A</td>
			<td style="width:395px;">1x1014 (GC/ml)</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:59px;width:269px;">B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J</td>
			<td style="width:273px;">The Jackson Labtorary&#160;</td>
			<td style="width:199px;">007914</td>
			<td>Ai14</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">B6(Cg)-Foxp2tm1.1Sfis/CfreJ</td>
			<td style="width:273px;">The Jackson Labtorary&#160;</td>
			<td style="width:199px;">026259</td>
			<td style="width:395px;">Foxp2fl/fl</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Dulbecco&#8217;s phosphate buffered saline</td>
			<td style="width:273px;">Corning&#160;cellgro</td>
			<td style="width:199px;">21-030-CVR</td>
			<td></td>
		</tr>
	</tbody>
</table>

stereotaxic surgery for genetic manipulation in striatal cells of neonatal mouse brains

Birçok genler embriyonik beyinlerinde ifade edilir ve bazıları sürekli doğumdan sonra beyinde ifade edilir. Israrla ifade böyle genler için gelişimsel süreci ve/veya neonatal beynindeki fizyolojik fonksiyon düzenlemek için çalışmayabilir. Beyindeki belirli genlerin nörobiyolojik işlevleri araştırmak için beyindeki genleri devre dışı bırakabilirsiniz esastır. Burada, striatum gen ifadesinde transgenik fareler yenidoğan zaman Windows devre dışı bırakabilirsiniz için basit bir stereotaksik yöntem açıklanmaktadır. AAV-eGFP-Cre virüs Ai14 muhabir gen fareler striatum Doğum sonrası gün (P) 2 stereotaksik beyin ameliyatı tarafından microinjected. TdTomato muhabir gen ekspresyonu P14 striatum, başarılı bir Cre-loxP DNA rekombinasyon AAV transduced striatal hücrelerdeki aracılı düşündüren tespit edildi. Daha fazla bu teknik AAV-eGFP-Cre virüs P2Foxp2fl/fl fareler microinjecting tarafından doğrulanmış. Çift etiketleme GFP ve Foxp2 GFP pozitif hücreler P9 striatum, AAV-eGFP-Cre transduced striatal hücrelerdeki Foxp2 protein kaybı düşündüren Foxp2 immunoreactivity yoksun gösterdi. Birlikte ele alındığında, bu sonuçlar yenidoğan beynindeki belirli nöron popülasyonları floxed transgenik fareler Stereotaxic microinjected AAV-eGFP-Cre virüsler tarafından etkili bir genetik silme işlemini göstermektedir. Sonuç olarak, bizim stereotaksik tekniği yenidoğan fare beynindeki genetik manipülasyon için kolay ve basit bir platform sağlar. Bu teknik sadece genler, yenidoğan beyinlerinin belirli bölgelerde silmek için kullanılamaz, ancak farmakolojik ilaçlar, nöronal izleyiciler, genetiği değiştirilmiş optogenetics ve chemogenetics proteinler, nöronal aktivite göstergelerini enjekte etmek için de kullanılabilir ve diğer reaktifler striatum, yenidoğan fare beyinlerinin içine.

Deney ilk kümesi için biz 200 microinjected nL Cre DNA recombinase hızlı AAV9.hSynapsin.HI.eGFP-Cre.WPRE.SV40 virüs (AAV-eGFP-Cre, Dulbecco'nın fosfat tamponlu tuz 1/10 seyreltme) erimiş GFP ile Ai14 farelerin P2 striatum. Ai14 fareler tdTomato muhabir gen Cre-aracılı silme (floxed) loxP çevrili STOP kaset (2F rakam) üzerine hızlı. Beyin P14 GFP ve tdTomato immunostaining için hasat edildi. Birçok AAV GFP pozitif hücreler transduced hediye (<s...

Watch this Scientific Journal Video about Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains at JoVE.com

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Yenidoğan fare beyin Striatal hücrelerdeki genetik manipülasyon için stereotaksik cerrahi

Biz yenidoğan fare beyni striatum microinjecting reaktifler için ev yapımı bir baş-sabit aygıt ile bir protokol stereotaksik cerrahi açıklamak. Bu teknik, yenidoğan fare beyni, özel bölgelerin nöronal hücrelerin genetik manipülasyon sağlar.

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

stereotaxic-surgery-for-genetic-manipulation-striatal-cells-neonatal

Research

JoVE Journal

Neuroscience

14.8K Görüntüleme.  National Yang-Ming University. Biz yenidoğan fare beyni striatum microinjecting reaktifler için ev yapımı bir baş-sabit aygıt ile bir protokol stereotaksik cerrahi açıklamak. Bu teknik, yenidoğan fare beyni, özel bölgelerin nöronal hücrelerin genetik manipülasyon sağlar.

Video: Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Rodent Stereotaxic Surgery and Animal Welfare Outcome Improvements for Behavioral Neuroscience

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to investigate the effects of locally manipulated neurotransmitter and signaling pathways in awake, behaving animals. Moreover, the stereotaxic implantation of electrodes for electrophysiological stimulation and recording studies has been instrumental to our current understanding of neuroplasticity and brain networks in behaving animals. Ever-increasing knowledge about optimizing surgical techniques in rodents1-4, public awareness concerning animal welfare issues and stringent legislation (e.g., the 2010 European Union Directive on the use of laboratory animals5) prompted us to refine these surgical procedures, particularly with respect to implementing new procedures for oxygen supplementation and the continuous monitoring of blood oxygenation and heart rate levels during the surgery as well as introducing a standardized protocol for post-surgical care. Our observations indicate that these modifications resulted in an increased survival rate and an improvement in the general condition of the animals after surgery (e.g. less weight loss and a more active animal). This video presentation will show the general procedures involved in this type of stereotaxic surgery with special attention to our several modifications. We will illustrate these surgical procedures in rats, but it is also possible to perform this type of surgery in mice or other small laboratory animals by using special adaptors for the stereotaxic apparatus6.

Stereotaxic surgery on rodents allows for targeted administration of drugs or electrical stimulation and recordings in awake, behaving animals. In this video presentation we will demonstrate recent procedural refinements to this long-standing procedure that successfully improved survival rate and reduced post-surgical weight loss.

Behavioral Neuroscience Kemirgen Stereotaksik Cerrahi ve Hayvan Refahı Sonuç İyileştirmeler

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to ...

Nörobilim

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or &beta;III tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

Fare Dopaminerjik ve Üç Boyutlu Collagen Matrix Tahliller striatal Eksplantlarından diseksiyonu ve Kültür

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

Yetişkin Rat spesifik Beyin Alanlarının eksitotoksik Lezyon için Stereotaksik Cerrahi

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Kullanarak Fare Serebral Korteks ve Hippocampus Görselleştirme ve Dendritler ve Spine Genetik Manipülasyon<em&gt; Utero olarak</em&gt; Elektroporasyon

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

Fare Omurilik Koşullu Gen Manipülasyon için bir Viral Vector Stereotaksik Enjeksiyon

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

Fare Genetik Manipülasyon yoluyla Hipotalamus geliştirilmesi<em&gt; Utero olarak</em&gt; Elektroporasyon

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Anjiyogenez Araştırma Yenidoğan Fare Retina Tüm Dağı Immunofluorescent Boyama<em&gt; In vivo</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Serebellar Granül Nöronlar Genetik Manipülasyon<em&gt; In Vitro</em&gt; Ve<em&gt; In Vivo</em&gt; Nöronal morfoloji ve Göç Eğitim için

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

Kalıcı ve yaygın Nöronal İletim için Yenidoğan Fare Beyin intraserooroventriküler Viral Enjeksiyon

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Optik Koherens tomografi: Fare retina Ganglion hücreleri Vivo içinde görüntüleme

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...