Bu çalışmada Ulusal Doğa Bilimleri Vakfı Çin tarafından (proje kod No 81373557; No 81403327) finanse edildi.

Bu çalışmada, Çin Çince tıbbi Bilimler Akademisi (referans numarası 20160014) Etik Komitesi tarafından kabul edildi. Tüm yordamları bakım ve laboratuvar hayvanlarının kullanımı (Ulusal Akademisi basın, Washington, DC, 1996) için Ulusal Sağlık Rehberi Enstitüleri uygun olarak gerçekleştirilmiştir. Dört yetişkin erkek rats (ağırlığı 250-280 g) Bu çalışmada kullanılmıştır. Bütün hayvanlar kontrollü sıcaklık ve nem ile 12 h açık/koyu döngüsünde yer alan ve gıda ve su ücretsiz erişim izni. Aletleri ve malzemeler mevcut çalışmada kullanılan şekil 1' de gösterdi.  Ameliyattan önce stereotaksik çerçeve ve cam pipet, gibi tüm araçları % 70 etanol kullanarak temizlenmiştir.
1. cerrahi işlemler
<ol><li>Stereotaksik atlas9 (şekil 2A) kullanarak VPM ilgi koordinat alanı belirleyin.</li>
	<li>1 µL mikro-cam micropipette (yaklaşık 10-20 µm ipucu çaplı) ile donatılmış şırınga hazırlamak (şekil 1 d) ve Sıvı parafin ile test edin.</li>
	<li>% 7 kloral hidrat (0.7 mL/100 g) farelerle mayi enjeksiyonu ile anestezi.</li>
	<li>Derin anestezi onaylandıktan sonra kuyruk ile pinch ve para çekme refleks pedal, hayvanın başının ile tıraş Tıraş ve cerrahi sitesi 3 kez sırasıyla % 70 etanol tarafından takip % 10 povidone iyot ile fırçalayın.</li>
	<li>Fareyi kulaklarına künt kulak çubuklarını yerleştirerek stereotaksik cihazın içine yerleştirin ve sıçan üst kesici dişler (şekil 1E) ağız yuvasına yerleştirin ve oftalmik merhem gözünü uygulayın.</li>
	<li>Tekrar % 70 etanol kullanarak cerrahi sitesinin baş cilt temizlemek. Havlu ve steril cerrahi eldiven steril koşullarda ameliyat korumak için kullanın.</li>
	<li>Cilt sagittal dikiş (şekil 1F) boyunca bir neşter ile sagittal bir kesi yapmak.</li>
	<li>Kas ve steril pamuklu uçlu uygulayıcıları (şekil 1F) kanama kontrol cerrahiye boyunca kullanarak kafatası uzak kapaklarini kazı.</li>
	<li>Bir Atlas (şekil 2A) önceden tanımlanmış koordinatları kullanarak, kranyotomi (şekil 1G) konumunu (-3.3 mm Bregma noktası, orta hat sağa 2.6 mm) belirleyin.</li>
	<li>Burr matkap ile yuvarlak uçlu biraz (#106) (Resim 1 H) kullanarak bir kranyotomi gerçekleştirmek ve meninkslerde (şekil 1I) ulaşan kadar bir kaç dakika içinde Delme hakkında 1 mm derinliği için devam edin.</li>
	<li>Serebral korteks enjeksiyon sitesi (şekil 1I) üzerinden ortaya çıkarmak için microforceps kullanarak dura mater tüketim.</li>
	<li>Sıvı parafin % 10 BDA (10.000 molekül ağırlığı, distile su içinde) çözüm (şekil 1J) mikro şırınga değiştirin.</li>
	<li>Şırınga mikroenjeksiyon aparatı monte ve bir mikro-pompa ile (şekil 1 K) bağlanın. Not: enjekte birimi mikro-pompa hızına bağlıdır. Burada 30 nL/dk (şekil 1 L) ayarlandı.</li>
	<li>Bir stereomicroscope altında VPM 5.8 mm (şekil 1 M) derinlikte beynin kortikal yüzeyi üzerinden el ile mikroenjeksiyon aparatı ile cam micropipette takın.</li>
	<li>Basınç enjekte-bir %100 10 nL BDA VPM 3 dk (35 nL/dk) bir mikro-pompa (şekil 1 L, M) ile bir süre içine.</li>
	<li>Sonra enjeksiyon, pipet, yerde bir ek 5 min için tutun ve sonra yavaş yavaş geri.</li>
	<li>Yara steril vida (şekil 1N) dikiş. Öncesi ve ameliyat sonrası analjezi için senin yerel hayvan bakımı Komitesi yönergeleri izleyin.</li>
	<li>Bilincinin tekrar yerine geleceğinden ve tamamen iyileşti kadar fareyi bir sıcak kurtarma alanında koyun.</li>
	<li>Kurtarılan fareyi onun kafes için geri dönün.</li>
</ol>2. perfusions ve bölümler
<ol><li>Sonra bir yaşam süresi genellikle 10 günlük hayvan mayi enjeksiyonu ile ötenazi ikna etmek için % 10 üretan (2 mL/100 g) aşırı dozda enjekte et.</li>
	<li>Hood (şekil 1O) deneysel fareler sıvı.<ol>
<li>Bir kez nefes durur, makas ve forseps, kullanarak kalp erişmek için fareyi göğüs boşluğuna açın. Sol ventrikül aort doğru bir damar içi kateter takın ve doğru auricle açın.</li>
		<li>İlk % 0,9 fosfat tamponlu tuz (PBS), fizyolojik sıcaklığında (37 ° C) ile sıvı kadar kan kalpten çıktıktan 1-2 dk temizleyin ve 250-300 mL % 4 paraformaldehyde 0.1 M fosfat tampon (PB, pH 7,4) ile devam edin.</li>
	</ol>
</li>
	<li>Perfüzyon sonra baş cilt deşmek ve kafatasını açıp sıçan beyin incelemek. Oda sıcaklığında (26 ° C), o zaman cryoprotect içinde % 30 Sükroz 0.1 M PBS (pH 7,4) beyin çözüm (şekil 1 P) batırılır kadar 4 ° C'de 3 gün içinde 2 h için %4 paraformaldehyde disseke beyinde sonrası tamir et.</li>
	<li>Bir kez beyin çözümde batırılır, beyin üzerinde beyin matrisler (şekil 1Ç) Koronal yönde üç sokak bölün. Merkezi bloğu VPM ve S1 içerir.</li>
	<li>Koronal microtome sistemini sürgülü bir dondurucu sahnede merkezi bloğu 40 µm, beynin kesti. Bu bölümlerde düzenli bir 6-iyi yemek (rakamlar 1R, S) 0.1 M PBS (pH 7,4) ile toplamak.</li>
</ol>3. standart ABC boyama
Not: Beynin her üçüncü koronal bölümünden ücretsiz kayan bölümler BDA standart ABC yordamı10ile etiketleme görüntülenmesi için kullanılmıştır.
<ol><li>0.1 M PBS için yaklaşık 1 dakika bölümlerde durulayın.</li>
	<li>(PH 7,4) içeren %0,3 %1 0.1 M PB ABC çözümde bölümlerde kuluçkaya Triton X-100 oda sıcaklığında 1 h için.</li>
	<li>Sonraki bölümlerde 50 mM Tris arabellek (pH 7,4) üç kere yıkayın.</li>
	<li>%0,02 3, 3'-diaminobenzidine tetrahydrochloride (DAB) ve %0.01 H2O2 oda sıcaklığında yaklaşık 2-5 dakika için 50 mM Tris arabellek içeren bir çözüm bölümlerde leke.</li>
	<li>Sonraki bölümlerde 50 mM Tris arabellek (pH 7,4) üç kere yıkayın.</li>
	<li>Bölümler standart histochemical teknikleri (1T rakam; bkz: Ek Video dosyası III, perfüzyon ve bölümler) kullanarak mikroskop slaytlarda bağlayın.</li>
	<li>Gecede, oda sıcaklığında hava bölümlerde kuru.</li>
	<li>Alkol (% 50, % 70, % 95, % 100) çözümleri kısa bir süre içinde bir dizi bölümlerini kurutmak. Her çözüm için yaklaşık 15 slaytları daldırın s. Slaytlar arasında her adım kurumasına izin vermez.</li>
	<li>Ksilen bölümlerde üç kez, yaklaşık 20 dk temizleyin.</li>
	<li>2-3 damla balsam dilimleri koyun sonra coverslips bölümleri yerleştirin.</li>
</ol>4. çift floresan BDA ve serebral korteks nöral temel öğeleri için boyama
Not: Buna ek olarak, çift floresan boyama BDA etiketleme korelasyon gözlemleyerek için yapılmıştır ve yukarıdaki bitişik bölümleri üzerinde temel sinir öğelerini streptavidin-AF594 ile kullanılan ve floresan Nisl leke AF500/525 ile counterstained.
<ol><li>0.1 M PBS için yaklaşık 1 dakika bölümlerde durulayın.</li>
	<li>Streptavidin-AF594 (1:500) ve AF500/525 yeşil flüoresan Nisl leke (1:1, 000) 0.1 M PBS karışık bir çözümde bölümlerini (pH 7,4) içeren %0,3 kuluçkaya Triton X-100 oda sıcaklığında 2 h için.</li>
	<li>Sonraki bölümlerde 0.1 M PB (pH 7,4) üç kere yıkayın.</li>
	<li>Bölümler standart histochemical teknikleri kullanarak mikroskop slaytlarda bağlayın. Kuru havada yaklaşık 1 h için bölümler.</li>
	<li>Coverslips % 50 gliserin gözlem önce distile su ile floresan bölümlerine uygulanır.</li>
</ol>5. çift floresan BDA ve Interneurons serebral korteks için boyama
Not: Çift floresan boyama BDA etiketleme ve interneurons streptavidin-AF594 ve PV-İmmünokimya kortikal hedefle temsilcisi bölümlerde Tarih korelasyon gözlemleyerek için gerçekleştirilmiştir.
<ol><li>0.1 M PBS (pH 7,4) yaklaşık 1 dakika için temsilcisi bölümlerde durulayın.</li>
	<li>% 3 normal keçi serum ve %0,3 içeren bir engelleme çözüm bölümlerde kuluçkaya Triton X-100 0.1 M PBS için 30 dk içinde.</li>
	<li>Fare monoklonal anti-PV IgG (1:1, 000) 0.1 M PBS (pH 7,4) içeren % 1 normal keçi serum ve %0,3 bir çözüm içine bölümleri transfer Triton X-100 gecede 4 ° C'de için</li>
	<li>Ertesi gün, sonraki bölümlerde 0.1 M PBS (pH 7,4) üç kere yıkayın.</li>
	<li>Karışık bir çözüm bölümlerine keçi mouse-AF488 ikincil antikor (1:500), Serumda 0.1 M PBS (pH 7,4) içeren % 1 normal keçi streptavidin-AF594 (1:500) ve 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI, 1:40, 000) ve 0,3 açığa % Triton 1 h için X-100.</li>
	<li>4.3-4.5 adımları yineleyin.</li>
</ol>6. gözlem
<ol><li>VPM, thalamocortical akson ve corticothalamic nöronlar görüntülerini almak.<ol>
<li>Hedefleri lensler ile donatılmış bir confocal görüntüleme sistemi ile floresan örnekleri gözlemlemek (4 x, NA: 0,13; 10 x, NA: 0.40; ve 40 x, NA: 0.95). 405 (mavi), 488 (yeşil), uyarma ve emisyon dalga boylarında ve 559 (kırmızı) nm kullanın. Not: Burada, confocal iğne deliği 152 µm (4 x, 10 x) ve 105 µm (40 x). Görüntü yakalama Uzaysal çözünürlük 1024 × 1024 piksel (4 x, 10 x) ve 640 × 640 piksel (40 x) olduğunu.</li>
		<li>Yirmi görüntüler 40 µm (Z serisi) kalınlığı, her bölüm 2 µm ardışık çerçeveler içinde tutar.</li>
		<li>Görüntüleri tek bir odakta görüntü confocal görüntü işleme için aşağıdaki gibi üç boyutlu analiz yazılım sistemi ile entegre: ayarla başlat odak düzlemi → set sonunda odak düzlemi → set adım boyutu → derinlik desen → görüntü yakalama → Z serisi seçin.</li>
	</ol>
</li>
	<li>Bir dijital fotoğraf makinesi ile donatılmış bir ışık mikroskobu tarafından aydınlık alan görüntü alabilir (4 x, NA: 0,13; 10 x, NA: 0.40; ve 40 x, NA: 0.95 lensler). Bir çekim hızı 500 Bayan kullanım fotoğraf düzenleme yazılımı, parlaklık ve kontrast görüntülerin ve etiket eklemek için kullanın.</li>
</ol>

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Uygun izleme aygıtı seçme başarılı sinirsel izleme deneme için kritik bir adımdır. BDA, yüksek molekül ağırlıklı BDA ailesi (10.000 moleküler ağırlık) düşük moleküler ağırlıklı BDA (3.000 moleküler ağırlık)2,3 aksine anterograd sinir yolu ile tercihen taşınması için tavsiye edilir , 11 , 12 , 13. ancak, birçok çalışma Ayr...

Yüksek molekül ağırlıklı BDA (10.000 moleküler ağırlık), son derece hassas bir izleyici, merkezi sinir sistemi üzerinde 20 yıl1için Sinirsel yollar izleme için kullanılmıştır. BDA kullanımı teknik izleme ortak bir sinir yolu olsa da, BDA etiketleme kalitesini hayvanlarda çeşitli faktörler1,2,3tarafından etkilenebilir. Bizim son çalışma BDA etiketleme en uygun yapısı sadece uygun sonrası enjeksiyon yaşam süresi ile ilgili ama aynı zamanda boyama yöntemi4ile korelasyon göstermiştir. Şimdi, geleneksel avidin-biotin-peroksidaz kompleksi (ABC), streptavidin floresein isothiocyanate ve streptavidin-AF594 kadar boyama yöntemleri BDA önceki çalışmalar2,3' teetiketleme ifşa için kullanılması, 4,5. Buna karşılık, floresan BDA için boyama kolayca gerçekleştirilebilir.
Yüksek molekül ağırlıklı BDA uygulanması genişletmek için rafine bir iletişim kuralı da çalışmanın kullanılmaya başlandı. Talamus sıçan beyin VPM içine BDA enjeksiyonu, BDA etiketleme ortaya standart ABC boyama düzenli yöntemi yanı sıra çift Floresan, hangi BDA etiketleme korelasyon gözlemleyerek için gerçekleştirildiği boyama ve temel Sinirsel öğeleri veya interneurons kortikal hedef streptavidin-AF594 ve floresan Nisl histochemistry veya PV-İmmünokimya, anılan sıraya göre. VPM ve Primer somatosensor korteks (S1)6,7,8arasında karşılıklı Sinirsel yollar, biz bizim gözlem BDA etiketleme öngörülen thalamocortical akson ve corticothalamic üzerinde duruldu S1 içinde öngörülen hücre somas. Bu işlem tarafından sadece sinirsel yüksek molekül ağırlıklı BDA ile etiketleme yüksek kalitesini elde etmek için ayrıntılı bir iletişim kuralı, aynı zamanda rafine bir protokol floresan BDA etiketleme ile birlikte ve diğer floresan sinirsel belirteçleri ile sağlamak bekleniyor histochemistry veya İmmünokimya. Bu yaklaşım yerel sinir devreleri ve confocal bir lazer mikroskobu tarama altında kimyasal özellikleri çalışma için tercih edilir.

<table>
	<tbody>
		<tr>
			<td>Biotinylated dextran amine (BDA)</td>
			<td>Molecular Probes</td>
			<td>D1956</td>
			<td>10,000 molecular weight</td>
		</tr>
		<tr>
			<td>Streptavidin-Alexa Fluor 594</td>
			<td>Molecular Probes</td>
			<td>S32356</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>500/525 green fluorescent Nissl stain</td>
			<td>Molecular Probes</td>
			<td>N21480</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>Brain stereotaxis instrument</td>
			<td>Narishige</td>
			<td>SR-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezing microtome</td>
			<td>Thermo</td>
			<td>Microm International GmbH</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal imaging</td>
			<td>Olympus</td>
			<td>FV1200</td>
			<td></td>
		</tr>
		<tr>
			<td>system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Drill</td>
			<td>Saeyang Microtech</td>
			<td>Marathon-N7</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague Dawley</td>
			<td>Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences</td>
			<td>SCKX (JUN) 2012-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectastain ABC Kit</td>
			<td>Vector Laboratories</td>
			<td>PK-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>superfrost plus microscope slides</td>
			<td>Thermo</td>
			<td>#4951PLUS-001</td>
			<td>25x75x1mm</td>
		</tr>
		<tr>
			<td>Photoshop and Illustration</td>
			<td>Adobe</td>
			<td>CS5</td>
			<td></td>
		</tr>
	</tbody>
</table>

improving the application of high molecular weight biotinylated dextran amine for thalamocortical projection tracing in the rat

Yüksek molekül ağırlıklı biotinylated dextran Amin (BDA) son derece hassas bir nöroanatomik izleyici onlarca yıl için kullanılmıştır. Onun etiketleme kalitesini çeşitli etkenlere göre burada, etkilendi biz rafine bir protokol yüksek molekül ağırlıklı BDA uygulama için en uygun sinir merkezi sinir sistemi etiketleme çalışmak için sağlar. BDA farenin hassas cam pipet ile talamus ventral posteromedial çekirdeği (VPM) içine stereotaksik enjeksiyon sonra BDA floresan streptavidin ile-Alexa (AF) 594 lekeli ve floresan Nisl leke AF500/525 ile counterstained. Yeşil Nisl boyama arka plan üzerinde kırmızı BDA, nöronal hücre gövdeleri ve aksonal terminaller gibi etiketleme daha belirgin korteks somatosensor gösterilmiştir. Ayrıca, floresan BDA için boyama çift ve kalsiyum bağlayıcı protein parvalbumin (PV) korelasyon BDA etiketleme ve PV-pozitif interneurons sinirsel yerel çalışma fırsatı sağlayan kortikal hedefteki gözlemlemek için yapılmıştır Devreler ve kimyasal özellikleri. Böylece, rafine bu yöntem sadece yüksek kaliteli talamus ve serebral korteks arasında karşılıklı Sinirsel yollar aracılığıyla yüksek molekül ağırlıklı BDA ile etiketleme sinirsel görüntülenmesi için uygun değildir, ancak aynı zamanda eşzamanlı gösteri verecektir Floresan histochemistry veya İmmünokimya diğer sinir işaretleyicilerle.

BDA 10 gün sonrası enjeksiyon VPM içine yaşama yoğun nöral karşılık gelen kortikal alanlarda enjeksiyon yüz (Şekil 2) Ipsilateral etiketleme üretmek için yeterli idi. Hem geleneksel ABC ve yordamlar için BDA boyama floresan sinir anterogradely de dahil olmak üzere S1 üzerinde etiketleme benzer desen thalamocortical akson etiketli ve retrogradely (şekil 2C, D corticothalamic nöronlar etiketli ortaya </str...

Watch this Scientific Journal Video about Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat at JoVE.com

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

Burada, etkili bir floresan yöntemi aracılığıyla karşılıklı bir sinirsel yol boyama ile etiketleme biotinylated dextran Amin (BDA) ortaya çıkarmak için rafine bir iletişim kuralı mevcut. Bu güzel yapı çözümleniyor için uygundur, etiketleme ve confocal bir lazer tarama mikroskop altında sinirsel diğer öğelerinden ayırt BDA.

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 ...

improving-application-high-molecular-weight-biotinylated-dextran

Research

JoVE Journal

Neuroscience

8.9K Görüntüleme.  China Academy of Chinese Medical Sciences. Burada, etkili bir floresan yöntemi aracılığıyla karşılıklı bir sinirsel yol boyama ile etiketleme biotinylated dextran Amin (BDA) ortaya çıkarmak için rafine bir iletişim kuralı mevcut. Bu güzel yapı çözümleniyor için uygundur, etiketleme ve confocal bir lazer tarama mikroskop altında sinirsel diğer öğelerinden ayırt BDA.

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

Progressive-ratio Responding for Palatable High-fat and High-sugar Food in Mice

Foods that are rich in fat and sugar significantly contribute to over-eating and escalating rates of obesity. The consumption of palatable foods can produce a rewarding effect that strengthens action-outcome associations and reinforces future behavior directed at obtaining these foods. Increasing evidence that the rewarding effects of energy-dense foods play a profound role in overeating and the development of obesity has heightened interest in studying the genes, molecules and neural circuitry that modulate food reward1,2. The rewarding impact of different stimuli can be studied by measuring the willingness to work to obtain them, such as in operant conditioning tasks3. Operant models of food reward measure acquired and voluntary behavioral responses that are directed at obtaining food. A commonly used measure of reward strength is an operant procedure known as the progressive ratio (PR) schedule of reinforcement.4,5 In the PR task, the subject is required to make an increasing number of operant responses for each successive reward. The pioneering study of Hodos (1961) demonstrated that the number of responses made to obtain the last reward, termed the breakpoint, serves as an index of reward strength4. While operant procedures that measure changes in response rate alone cannot separate changes in reward strength from alterations in performance capacity, the breakpoint derived from the PR schedule is a well-validated measure of the rewarding effects of food. The PR task has been used extensively to assess the rewarding impact of drugs of abuse and food in rats (e.g.,6-8), but to a lesser extent in mice9. The increased use of genetically engineered mice and diet-induced obese mouse models has heightened demands for behavioral measures of food reward in mice. In the present article we detail the materials and procedures used to train mice to respond (lever-press) for a high-fat and high-sugar food pellets on a PR schedule of reinforcement. We show that breakpoint response thresholds increase following acute food deprivation and decrease with peripheral administration of the anorectic hormone leptin and thereby validate the use of this food-operant paradigm in mice.

The present report details the protocol employed to measure the rewarding effects of high-fat food in mice using a progressive ratio operant conditioning task.

Aşamalı-oranı Farelerde Palatable Yüksek yağ ve yüksek şeker Gıda tepki

Foods that are rich in fat and sugar significantly contribute to over-eating and escalating rates of obesity. The consumption of palatable foods can ...

Nörobilim

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling of auditory axons within the VIIIth cranial nerve would enhance the study of central auditory circuit development. This approach is challenging because multiple sensory organs of the inner ear contribute to the VIIIth nerve 1. Moreover, markers that reliably distinguish auditory versus vestibular groups of axons within the avian VIIIth nerve have yet to be identified. Auditory and vestibular pathways cannot be distinguished functionally in early embryos, as sensory-evoked responses are not present before the circuits are formed. Centrally projecting VIIIth nerve axons have been traced in some studies, but auditory axon labeling was accompanied by labeling from other VIIIth nerve components 2,3. Here, we describe a method for anterograde tracing from the acoustic ganglion to selectively label auditory axons within the developing VIIIth nerve. First, after partial dissection of the anterior cephalic region of an 8-day chick embryo immersed in oxygenated artificial cerebrospinal fluid, the cochlear duct is identified by anatomical landmarks. Next, a fine pulled glass micropipette is positioned to inject a small amount of rhodamine dextran amine into the duct and adjacent deep region where the acoustic ganglion cells are located. Within thirty minutes following the injection, auditory axons are traced centrally into the hindbrain and can later be visualized following histologic preparation. This method provides a useful tool for developmental studies of peripheral to central auditory circuit formation.

Here we describe a microdissection technique followed by fluorescent dye injection into the acoustic ganglion of early chick embryos for selective tracing of auditory axon fibers in the nerve and hindbrain.

Kuş Embriyonik vestibülokoklear sinir İşitsel Elyaf seçici Aydınger

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This approach has been most extensively used in the brain by utilizing the swine pathogen pseudorabies virus (PRV)1. PRV does not infect great apes, including humans, so it is most commonly used in studies on small mammals, especially rodents. The pseudorabies strain PRV152 expresses the enhanced green fluorescent protein (eGFP) reporter gene and only crosses functional synapses retrogradely through the hierarchical sequence of synaptic connections away from the infection site2,3. Other PRV strains have distinct microbiological properties and may be transported in both directions (PRV-Becker and PRV-Kaplan)4,5&#160;.&#160;This protocol will deal exclusively with PRV152. By delivering the virus at a peripheral site, such as muscle, it is possible to limit the entry of the virus into the brain through a specific set of neurons. The resulting pattern of eGFP signal throughout the brain then resolves the neurons that are connected to the initially infected cells. As the distributed nature of transsynaptic tracing with pseudorabies virus makes interpreting specific connections within an identified network difficult, we present a sensitive and reliable method employing biotinylated dextran amines (BDA) and cholera toxin subunit b (CTb) for confirming the connections between cells identified using PRV152. Immunochemical detection of BDA and CTb with peroxidase and DAB (3, 3&#39;-diaminobenzidine) was chosen because they are effective at revealing cellular processes including distal dendrites6-11.

Transsynaptic tracing has become a powerful tool for analyzing central efferents regulating peripheral targets through multi-synaptic circuits. Here we present a protocol that exploits the transsynaptic pseudorabies virus to identify and localize a functional brain circuit, followed by classical tract tracing techniques to validate specific connections in the circuit between identified groups of neurons.

Transsinaptik Kolera Toksin ve Biotinlenmiş Dekstran Aminler Çift Etiketleme Ardından Pseudorabies&#39;e Virüs ile Periferik Hedefler gelen İzleme

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This ...

Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic targeting for microcircuit dissection and disease modeling. The introduction of optogenetics, for example, has allowed for bidirectional manipulation of genetically-identified neurons, at an unprecedented temporal resolution. To capitalize on these tools and gain insight into dynamic interactions among brain microcircuits, it is essential that one has the ability to record from ensembles of neurons deep within the brain of this small rodent, in both head-fixed and freely behaving preparations. To record from deep structures and distinct cell layers requires a preparation that allows precise advancement of electrodes towards desired brain regions. To record neural ensembles, it is necessary that each electrode be independently movable, allowing the experimenter to resolve individual cells while leaving neighboring electrodes undisturbed. To do both in a freely behaving mouse requires an electrode drive that is lightweight, resilient, and highly customizable for targeting specific brain structures.
A technique for designing and fabricating miniature, ultralight weight, microdrive electrode arrays that are individually customizable and easily assembled from commercially available parts is presented. These devices are easily scalable and can be customized to the structure being targeted; it has been used successfully to record from thalamic and cortical regions in a freely behaving animal during natural behavior.

Understanding the neural substrates of behavior requires brain circuit ensemble recording. Because of its genetic tractability, the mouse offers a model for circuit dissection and disease mimicry. Here, a method of designing and fabricating miniaturized probes is described that is suitable for targeting deep brain structure in the mouse.

Farelerde Elektrofizyolojik Recordings Tasarım ve Ultralight Ağırlık Fabrikasyon, Ayarlanabilir Çoklu elektrot Probları

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic ...

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

Embriyonik Fare beyninde İzleme Koşullu Genetik transsinaptik

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) experiments. Determining the appropriate protocol to follow throughout the sample preparation process can be difficult as each step must be optimized to comply with the unique characteristics of the analytes of interest. This process involves not only finding a compatible matrix that can desorb and ionize the molecules of interest efficiently, but also selecting the appropriate matrix deposition technique. For example, a wet matrix deposition technique, which entails dissolving a matrix in solvent, is superior for desorption of most proteins and peptides, whereas dry matrix deposition techniques are particularly effective for ionization of lipids. Sublimation has been reported as a highly efficient method of dry matrix deposition for the detection of lipids in tissue by MALDI IMS due to the homogeneity of matrix crystal deposition and minimal analyte delocalization as compared to many wet deposition methods 1,2. Broadly, it involves placing a sample and powdered matrix in a vacuum-sealed chamber with the samples pressed against a cold surface. The apparatus is then lowered into a heated bath (sand or oil), resulting in sublimation of the powdered matrix onto the cooled tissue sample surface. Here we describe a sublimation protocol using 1,5-diaminonaphthalene (DAN) matrix for the detection and visualization of gangliosides in the rat brain using MALDI IMS.

A protocol for the sublimation of DAN matrix onto rat brain tissue for the detection of gangliosides using MALDI Imaging Mass Spectrometry is presented.

MALDI Görüntüleme Kütle Spektrometre Rat Beyin Dokusu içinde gangliositler Saptanmasında ve Görselleştirme DAN Matrix Sublime

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass ...

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of hemodynamics and the light scattering properties of the in vivo rat brain.

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system.

Serebral Hemodinamiğin ve Işık Saçılımının Eşzamanlı Değerlendirilmesi<em&gt; In Vivo</emÇokspektrumlu yaygın reflektans ımaging&#39;i kullanarak&gt; Sıçan Beyin

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

Embriyonik Motor Nöronlarının Aksonal Projeksiyon Paterninin Görselleştirilmesi<em&gt; Drosophila</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

Yüksek üretilen iş analiz Locomotor davranış Drosophila Adası tahlil

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes

In vivo microdialysis is a powerful technique to collect ISF from awake, freely-behaving animals based on a dialysis principle. While microdialysis is an established method that measures relatively small molecules including amino acids or neurotransmitters, it has been recently used to also assess dynamics of larger molecules in ISF using probes with high molecular weight cut off membranes. Upon using such probes, microdialysis has to be run in a push-pull mode to avoid pressure accumulated inside of the probes. This article provides step-by-step protocols including stereotaxic surgery and how to set up microdialysis lines to collect proteins from ISF. During microdialysis, drugs can be administered either systemically or by direct infusion into ISF. Reverse microdialysis is a technique to directly infuse compounds into ISF. Inclusion of drugs in the microdialysis perfusion buffer allows them to diffuse into ISF through the probes while simultaneously collecting ISF. By measuring tau protein as an example, the author shows how its levels are altered upon stimulating neuronal activity by reverse microdialysis of picrotoxin. Advantages and limitations of microdialysis are described along with the extended application by combining other in vivo methods.

In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes

In vivo microdialysis has enabled collection of molecules present in brain interstitial fluid (ISF) from awake, freely-behaving animals. In order to analyze relatively large molecules in ISF, the current article specifically focuses on the microdialysis protocol using probes with high molecular weight cut off membranes.

Vivo Microdialysis beyin interstisyel sıvı yüksek moleküler ağırlık kesme ile gelen büyük hücre dışı proteinler toplamak için yöntem probları

In vivo microdialysis is a powerful technique to collect ISF from awake, freely-behaving animals based on a dialysis principle. While microdialysis is an ...