Bu çalışma Ulusal Sağlık Enstitüleri hibe tarafından finanse edildi: EY015748, EY017964 (MEZ) ve EY019517 (ASV). Bu çalışma aynı zamanda Göz Hastalıkları Anabilim Dalı ve Orta New York Lions Körlük Serbest Grant Engellenir Research tarafından desteklenmiştir. Biz de hayvanların onun mükemmel bakım için ve bu video yıldız için adım için bizim hayvan teknisyeni, Matthew Mellini, teşekkür etmek istiyorum.

Hayvan Bakımı Xenopus Bu çalışmada kullanılan tadpoles Laboratuar Hayvanlarının Bakımı ve Kullanımı için Upstate Medical University IACUC ve Kılavuz tarafından onaylanan prosedürlere uygun olarak, yükseltilmiş yetiştirilen ve tedavi edildi laevis. . 1. Ön davranış Kurulum: Tadpoles <ol><li> Bir ticari olarak temin edilebilen bir kaynaktan, döllenmiş embriyolar Xenopus elde edilir ya da daha önce tarif edildiği gibi 6, in vitro olarak, oosit döller. </li><li> 20 mM KCI;; 20 mM CaCl 2; 50 mM HEPES 0.1x MMR [10 X Marc&#39;ın Modifiye Ringer (MMR) çözelti stok (10 mM MgCl2 içeren 60 mm Petri kapları 18 ˚ C&#39;de nöral plak sahne embriyolar blastula yerleştirin; 1 M NaCI, 50 ug / ml gentamisin antibiyotik]), pH 7.5 &#39;e ayarlandı ve otoklavda, oda sıcaklığında saklandı. Her gün 0.1x MMR değiştirin ve herhangi bir ölü embriyolar kaldırmak. </li><li> Embriyolar ~ 27 Nieuwkoop ve Faber aşamaya ulaştıklarında - 30 7, inci geçişantibiyotik olmadan 0.1x MMR içine E embriyolar bağırsak bakteri gelişimini sağlamak için. </li><li> Onlar besleme sahne (evre 45) ulaşıncaya kadar tetarlar büyür ve 0.1x MMR ile dolu 100 mm Petri kapları içine taşıyabilirsiniz. Aradan gününde 0.1x MMR değiştirerek, bir hafta boyunca her gün onlara ısırgan tozu süpernatant besleyin. </li><li> 100 mm Petri kabındaki Bir ​​hafta sonra, kurbağa su dolu yarım galon tankları için hayvanlar arasında hareket [0.5 g / L anlık okyanus ve 2 mM sodyum fosfat dibazik (Na 2 HPO 4: 141,96 mw) pH 6.8]. Adım 2.4 gibi onları beslemek, ancak su bulutlu olduğunda suya haftada sadece 2-3 kez değiştirin. Işıklar 06:00 de dönüm ile 12 saat aydınlık/12 karanlık ışık döngüsü içinde hayvanları korumak. </li><li> Bir laboratuar tezgah underpad, en az 12 saat O / N, testten önce gibi beyaz bir yüzeye test konularla yarım galon tankı yerleştirin. </li><li> Aşamaları 45-50 Test tadpoles aşağıdaki aşama 3&#39;te tarif edildiği. </li></ol> . 2. Ön davranış Kurulum: Equipment <ol><li> Davranış tahlil için laboratuara bir alanı kenara. Bu standart floresan aydınlatma ile sessiz, düşük trafik alanda olmalıdır. </li><li> Hayvanlar için test tankları hazırlayın. Iç içe, yarım galon test tankları iki kısımdan oluşur: su ve kurbağa yavrularını yapacak bir iç tank; ve görsel uyaran ile bir dış tankı. <ol><li> İç tank için, bir sharpie, alttan 5 cm ile dış köşelerinde küçük bir işareti yapmak; Bu su hattı (Şekil 1A, yeşil noktalı çizgi). </li><li> Aynı zamanda, iç depo için, Sylgard elastomer gibi eylemsiz bir bileşik ile (köşelerinde ve merkezinde) tank içinde divets doldurun. NOT: içeri dolu değilse tadpoles bu alanlarda oyalanmak </li><li> Dış tank için, beyaz kağıt mendil Şekil 1B siyah elektrik bandı ve diğer yarısı ile bir tankın dışında tam yarısını kapsamaktadır. </li></ol></li><li> Üzerine tankları yerleştirinpikap aşılamak. </li><li> Kur kamerası / tripod bu test tankların üstünde olduğunu böylece. Şekil 1C &#39;de gösterildiği gibi, test alanı görüntülenmesine olanak vermek üzere kamera ayarlayın. </li><li> 5 yüklü QuickTime Player yazılımı ile bir bilgisayara kameranızı bağlayın. Kamerayı açın. QuickTime Player, File altında, &#39;Yeni Film Recording&#39; seçeneğini seçin. Davranış tahlil kurulum hemen bilgisayarda görünür olacaktır. </li><li> Larva davranışını etkileyen dış ipuçları azaltmak, hem de, suyun yüzeyinde yansıyan ışığı azaltmak için tüm kurulum boyunca hafif bir pamuklu bez Örtü. </li><li> Emin olun 35-50 cd / m 2 arasında bez önlemleri altında parlaklık. </li></ol> 3.. Davranış Assay <ol><li> Kurbağa su ile adım 1.2.1 yapılan 5 cm su işareti için iç test deposunu doldurun. </li><li> Bir küçük net kullanarak, yavaşça iç test tankına hayvan taşımak. </li><li> Açık Sbilgisayar ve bir rehber olarak dış tankı kullanarak uickTime yazılım, kamera test alanı görselleştirmek ve kaydetmek mümkün olduğundan emin olun. </li><li> Kamera ile kağıt ve kaydın parçası üzerinde hayvan adı, tarih ve saati yazın. </li><li> Doğru Şekil 2&#39;ye tankın siyah tarafı ile dış tanka test tankı yerleştirin. </li><li> Depoyu düzenlenmesi hemen sonra film kaydı başlatın ve 2 dakika için zamanlayıcı ayarlayın. </li><li> Zamanlayıcı çalar, iç deney tankı kaldırdığınızda, dış tankı 180 ° döndürmek ve dış tankın içindeki iç deney tankı yerleştirin. Sayacı başlatmak. Not: siyah tarafı şimdi diğer tarafta olmalıdır. </li><li> On denemenin toplamı için adım 3.7, sekiz kez daha tekrarlayın. Her deneme işaretleyerek, bir rehber olarak Şekil 2 kullanın. </li><li> Iki ayrı günlerde davranış testi tekrarlayın. </li></ol> 4. Retina Axotomy <ol><li> Onlar tepkisiz kadar% 0.02 tricaine hayvan yerleştirinBir # 3 forseps ile bir kuyruk tutam. </li><li> 0.1x MMR içinde% 1 agaroz eritilir ve daha sonra bir 60 mm Petri kabı ekleyin. Hayvan kısmen sıvı içine daldırılmış, böylece sonra soğutuldu, agaroz küçük dikdörtgen çimen yapmak ve biraz% 0.02 tricaine ile çimen için iribaş ekleyin. </li><li> Pierce 25 G iğne ile, gözün dorsal bölgesi arkasında bir 45 derecelik bir açı ile, deri, karşı tarafta forseps karşı hayvan takviye ise. </li><li> Optik sinir yanındadır ven, makasla kesme özen, dikkatli, # 5 forseps ile deliğe ulaşmak optik sinir kesiyorlar ve yolumdan çevirmek. Şiddetli kanama nedeniyle kaza arteri kırpma var ise, o hızla euthanize% 2 tricaine hayvan. DİKKAT:% 2 tricaine çözüm insanlarda uyuşma neden olabilir. Bu çözüm eldiven ele alınmalıdır. </li><li> Hayvan kurtarmak için ameliyat sonrası, 0.7X MMR ile 100 mm Petri tabağına ve 20 dakika boyunca 50 ug / ml gentamisin içinde hayvan. Next, kurbağa su ile kurtarma tankına transfer iribaş. Hayvan dinlenme O / incelemeleri edelim </li><li> Ertesi sabah, (3,1-3,9 adım) yukarıda tarif edildiği gibi görsel fonksiyonunu test edin. </li></ol> 5.. Sonuçları Analiz <ol><li> Çalışmalar bittikten sonra, iribaş tankın siyah tarafında kalır süreyi ölçmek. <ol><li> Elle zaman görüntüler ve sık sık duraklamasına izin verir yazılımını kullanarak videoları görüntülemek. Video görüntü zaman bakmak. Tank iç başarıyla dış tankın içinde yuva yapar ve kameranın görünümünde karesi alındığında bir iki dakikalık deneme, başlangıç ​​yazın. </li><li> Hayvanların yüzme tankın hangi tarafı tanımlamak için iribaş gözlerinin konumunu kullanın. Iki gözü siyah / beyaz çizgiyi geçti yalnızca diğer tankın bir tarafında üzerinde geçiş tanımlayın. </li></ol></li><li> Hayvan siyah tarafta harcıyor (sn) her bir aralığın başlangıcını ve sonunu yazmak. Pause ve yenidendoğruluğunu sağlamak için videoyu rüzgar. Her deneme içinde sn toplam. </li><li> Ekli Davranış çalışma kitabına girdi bu numaraları. Sayfası, hayvan saniyede toplam zaman, toplam 120 sn deneme ve uçurumdan tankın siyah tarafında harcanan zamanı çıkarılarak beyaz tarafta harcanan zamanın oranını hesaplamak [(120 - toplam saniye harcanan Siyah tarafı) / 120 sn]. 10 çalışmalarda üzerinde çalışma ortalamaları bu oran. </li><li> Gün arasında denemeler (P ≤ 0.05) önemli olup olmadığını belirlemek için bir öğrencinin T-testi, eşleştirilmiş iki kuyruklu dağılımı kullanın. </li></ol>

<ol>
	<li>Maurer, C. M., Huang, Y. Y., Neuhauss, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+zebrafish+oculomotor+behavior+to+model+human+disorders.">Application of zebrafish oculomotor behavior to model human disorders.</a> Rev Neurosci. 22 (1), 5-16 (2011).</li><li>Solessio, E., Scheraga, D., Engbretson, G. A., Knox, B. E., Barlow, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+modulation+of+temporal+properties+of+the+rod+pathway+in+larval+Xenopus.">Circadian modulation of temporal properties of the rod pathway in larval Xenopus.</a> J Neurophysiol. 92 (5), 2672-2684 (2004).</li><li>Moriya, T., Kito, K., Miyashita, Y., Asami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preference+for+background+color+of+the+Xenopus+laevis+tadpole.">Preference for background color of the Xenopus laevis tadpole.</a> J Exp Zool. 276 (5), 335-344 (1996).</li><li>Viczian, A. S., Solessio, E. C., Lyou, Y., Zuber, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+functional+eyes+from+pluripotent+cells.">Generation of functional eyes from pluripotent cells.</a> PLoS Biol. 7 (8), (2009).</li><li>Choi, R. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cone+degeneration+following+rod+ablation+in+a+reversible+model+of+retinal+degeneration.">Cone degeneration following rod ablation in a reversible model of retinal degeneration.</a> Invest Ophthalmol Vis Sci. 52 (1), 364-373 (2011).</li><li>Viczian, A. S., Zuber, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+determination+using+the+animal+cap+transplant+(ACT)+assay+in+Xenopus+laevis.">Tissue determination using the animal cap transplant (ACT) assay in Xenopus laevis.</a> J Vis Exp. 16 (39), (2010).</li><li>Nieuwkoop, P. D., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal table of Xenopus laevis (Daudin) : a systematical and chronological survey of the development from the fertilized egg till the end of metamorphosis. , (1994).</li><li>Blackiston, D., Shomrat, T., Nicolas, C. L., Granata, C., Levin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+second-generation+device+for+automated+training+and+quantitative+behavior+analyses+of+molecularly-tractable+model+organisms.">A second-generation device for automated training and quantitative behavior analyses of molecularly-tractable model organisms.</a> PLoS One. 5 (12), (2010).</li><li>Rosemberg, D. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+spatio-temporal+behavior+of+zebrafish+in+the+open+tank+paradigm+after+a+short-period+confinement+into+dark+and+bright+environments.">Differences in spatio-temporal behavior of zebrafish in the open tank paradigm after a short-period confinement into dark and bright environments.</a> PLoS One. 6 (5), (2011).</li><li>Dong, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+Avoidance+in+Xenopus+Tadpoles+is+Correlated+With+the+Maturation+of+Visual+Responses+in+the+Optic+Tectum.">Visual Avoidance in Xenopus Tadpoles is Correlated With the Maturation of Visual Responses in the Optic Tectum.</a> J Neurophysiol. 101 (2), 803-815 (2009).</li><li>Lan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noggin+Elicits+Retinal+Fate+In+Xenopus+Animal+Cap+Embryonic+Stem+Cells.">Noggin Elicits Retinal Fate In Xenopus Animal Cap Embryonic Stem Cells.</a> Stem Cells. 27 (9), 2146-2152 (2009).</li><li>De Robertis, E. M., Kuroda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal-ventral+patterning+and+neural+induction+in+Xenopus+embryos.">Dorsal-ventral patterning and neural induction in Xenopus embryos.</a> Annu Rev Cell Dev Biol. 20, 285-308 (2004).</li></ol>

The authors have nothing to disclose.

Biz burada kolayca az eğitimli laboratuvar personeli tarafından bir hafta altında yapılabilir basit bir vizyon eşliğinde davranış deneyi rapor. Diğer deneyler hayvan davranışları, özel ekipman ve uzmanlık gerektiren, bu tahlil hızlı bir test görme fonksiyonunu belirlemenize olanak sağlar. Başka bir davranışsal deney, Görsel Kaçınma tahlil, TECTUM Xenopus&#39;ta 10 görsel algı nasıl katkıda belirlemek için geliştirilmiştir. Istikrarlı bir arka plan karşı hareketli, ve...

Xenopus laevis göz oluşumunu çalışmak için bir model organizma olarak kullanılmaktadır. Gözleri görme gelişimi ve fonksiyonu üzerinde bir etkiye sahip test genlerin ya da yollar için en az bir hafta içinde vadeye kadar büyüyen, hızla gelişir. Görme fonksiyonunu test etmek için, optokinetik ve optomotor yanıtı sırasıyla 1,2, Zebra balığı ve Xenopus iribaşlara kullanılmaktadır. Xenopus iribaşların gözleri zebrabalıkları nispeten daha küçük olduğundan, bu deney Xenopus&#39;ta ince kuyruk-Flip ve göz hareketleri davranışları tespit için özel ekipman kullanımı ve eğitimli personel gerektirir. Xenopus&#39;ta daha sağlam bir davranış, burada 3 tanımlanan bir beyaz arka plan ile bir tank içinde yüzme için tercihtir. Bir buçuk siyah / yarı-beyaz tankı bir iribaş yerleştirirken, ön-metamorfik iribaş hızla tankın beyaz tarafına yüzer. Daha önce pluripotent hücre-türevli olmadığını belirlemek için bu tahlil kullanılmıştırGözleri 4 fonksiyonel vardı. Burada, premetamorphic Xenopus iribaşların görme fonksiyonunu test etmek için kullanılabilir, bu tahlilde ayrıntılı bir versiyonu, rapor etmektedir. Sadece en Ksenopus laboratuvarlarda bulunan bir monte dijital video kamera, dijital kamera yazılımı ve standart ekipman gerektirir çünkü bu deney, optomotor tepki deneyi daha basittir. Buna ek olarak, kaydedilen tepki taksitli sonuçlarına özel bir eğitim gerektirir. Bizim temsilcisi sonuçları çift retina axotomy geçirmiş iribaşların aynı grup, tankın etrafına rastgele yüzmek olduğunu göstermektedir. Ayrıca bir göz görsel tepki açısından test edilebilir gösteren temsili bir tadpole arasındaki davranış deney sonuçları dahil ettik. Deneyde sırasında elde edilen sayı eklenir ve analiz edilebilir, böylece bir çalışma dahil edilmiştir. Bu çalışma, test edilen tadpoles görsel bir tepki var olup olmadığını belirlemek için kullanılabilir.

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	<tbody>
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			<td height="21" style="height:21px;width:216px;">1/2-gal. Flex-Tank with Cover</td>
			<td style="width:95px;">eNasco</td>
			<td style="width:119px;">SB19271M</td>
			<td style="width:225px;">size: 5-3/8&#34; x 7&#34; x 3-3/4&#34;</td>
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			<td height="21" style="height:21px;width:216px;">Black electrical tape</td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">White tissue paper</td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Large Inoculating Turntable</td>
			<td style="width:95px;">VWR</td>
			<td style="width:119px;">50809-022</td>
			<td>size: Dia 114.3 x&#160; H 76.2 mm (4 1/2&#160; x&#160; 3&#34;)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Durasorb Underpad</td>
			<td style="width:95px;">VWR</td>
			<td style="width:119px;">82004-836</td>
			<td>size: 43.2 x 60.1 cm (17 x 24&#34;)</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Kim wipe</td>
			<td style="width:95px;">Krackeler Scientific, Inc.</td>
			<td style="width:119px;">1945-34155-CS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Standard Tripod</td>
			<td style="width:95px;"></td>
			<td style="width:119px;">various</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">iSight camera or webcam</td>
			<td style="width:95px;">Apple</td>
			<td style="width:119px;">M8817LL/A</td>
			<td>Good for larger tadpoles but small ones are difficult to see</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Portable computer</td>
			<td style="width:95px;">Apple/PC</td>
			<td style="width:119px;">various</td>
			<td>We used a 13&#34; MacBook, 2 GHz Intel Core 2 Duo running MacOSX Lion 10.7.5</td>
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		<tr height="21">
			<td height="21" style="height:21px;">MiniDV Handycam Camcorder</td>
			<td style="width:95px;">SONY</td>
			<td style="width:119px;">DCR-HC42</td>
			<td>Connected by firewire to the computer with a 6-conductor and 4-conductor alpha FireWire 400&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Handycam Station</td>
			<td style="width:95px;">SONY</td>
			<td style="width:119px;">DCRA-C121</td>
			<td>This can be used for connecting firewire to camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QuickTime Player software</td>
			<td style="width:95px;">Quicktime</td>
			<td style="width:119px;">Version 10.1</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">26 Gauge Needle (5/8&#34; length)</td>
			<td style="width:95px;">VWR</td>
			<td style="width:119px;">BD305115</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Dumont #5 Forceps Biology</td>
			<td style="width:95px;">Fine Science Tools</td>
			<td style="width:119px;">11295-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td style="width:225px;"></td>
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		<tr height="24">
			<td height="24" style="height:24px;width:216px;">Disposables</td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td style="width:225px;"></td>
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		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Gentamicin Sulfate [50 mg/ml]</td>
			<td style="width:95px;">Fisher Scientific</td>
			<td style="width:119px;">17-528Z</td>
			<td>Stored at room temperature</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Sylgard 184 Silicone Elastomer</td>
			<td style="width:95px;">Fisher Scientific</td>
			<td style="width:119px;">NC9644388</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Instant Ocean</td>
			<td style="width:95px;">Doctors Foster and Smith</td>
			<td style="width:119px;">CD-116528</td>
			<td>Stock solution = 100 g/L stored at room temperature</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Sodium phosphate dibasic</td>
			<td style="width:95px;">Sigma Aldrich</td>
			<td style="width:119px;">S0876</td>
			<td>Stock solution = 0.4 M stored at room temperature</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Tricaine (Ethyl-3-aminobenzoate methanesulfonate salt)</td>
			<td style="width:95px;">Sigma Aldrich</td>
			<td style="width:119px;">A5040</td>
			<td>Stock solution = 10% stored at -20&#730;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">In vitro fertilized embryos</td>
			<td style="width:95px;">eNasco</td>
			<td style="width:119px;">LM00490MX</td>
			<td>100 embryos/unit</td>
		</tr>
	</tbody>
</table>

a simple behavioral assay for testing visual function in xenopus laevis

Kurbağa kurbağa yavruları görsel fonksiyon ölçümü, Xenopus laevis, canlı hayvanlarda körlüğe için tarama sağlar. Optokinetik yanıtı, test edilen bütün omurgalı gözlenmiştir bir görüntü-bazlı, dönüşlü bir davranıştır. Böylece kuyruk kapak tepkisi ince yanıtı kaydetmek için eğitimli bir teknisyen gerektirir alternatif tedbir olarak kullanılan Tadpole gözler küçük. Biz tadpoles siyah ve beyaz her iki taraf ile bir tank içinde yerleştirilen zaman bir tank beyaz tarafında yüzmek için tercih ettiği gerçeğine dayalı alternatif bir davranış testi geliştirdi. Burada sunulan deney, kolaylıkla ölçülür bir yanıt oluşturan bir basit, ucuz bir alternatiftir. Kurulum en Ksenopus laboratuarlarında hazır bir tripod, web kamerası ve iç içe test tanklar, oluşur. Bu makalede önce ve optik sinir kesilmesinin ardından, iribaşların davranışını gösteren bir film içermektedir. Bir gözün işlevini test etmek için, biz de Repre dahilHer göz ardışık günlerde retina axotomy uygulanan hangi bir kurbağa yavrusu bir sentative sonuçları. Gelecek çalışmalar seferde birçok iribaşların vizyonunu test etmek için bu testte bir otomatik versiyonunu gelişebilir.

Önceki raporlar bu testte, Biz iribaşların gözünde ya görme işlevini test etmek için bu tahlil değişti Arkaplan Renk Tercihi Tahlili 3 premetamorphic Ksenopus tadpoles siyah / beyaz tankın beyaz tarafına yüzmeyi tercih ettiğini göstermiştir ve çağrıda Bir haftadan daha az. Bu şekilde, gözleri histolojik inceleme için toplanabilir. Bu tepki görsel işaretlerin kaynaklanmaktadır kadar burada gösterir. Film 1, sol ve sağ panellerind...

Watch this Scientific Journal Video about A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis at JoVE.com

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus tadpoles siyah / beyaz tankın beyaz tarafında yüzme tercih laevis&#39;den. Bu davranış, onların vizyonu tarafından yönlendirilir. Bu davranışa dayanarak, iribaşların görme fonksiyonunu test etmek için basit bir test sunuyoruz.

Test Görsel Fonksiyonu için Basit Bir Davranış Testi<em&gt; Xenopus laevis</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

a-simple-behavioral-assay-for-testing-visual-function-xenopus

Research

JoVE Journal

Neuroscience

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

9.7K Görüntüleme.  Center for Vision Research, SUNY Eye Institute, Upstate Medical University.  Xenopus tadpoles siyah / beyaz tankın beyaz tarafında yüzme tercih laevis'den. Bu davranış, onların vizyonu tarafından yönlendirilir. Bu davranışa dayanarak, iribaşların görme fonksiyonunu test etmek için basit bir test sunuyoruz. 

Video: A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

Şehriye ve Capellini İşleme Testleri: sıçan ve fareler hünerli forepaw fonksiyonu Basit kantitatif tedbirler

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

Nörobilim

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

Sıçanlarda Sakkaroz Craving Kuluçka değerlendirilmesi için Genel Bir Yöntem

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

Dan Sinir Eksplantasyon Kültürleri<em&gt; Xenopus laevis</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

Diseksiyon, Kültür ve Analizi<em&gt; Xenopus laevis</em&gt; Embriyonik Retina Doku

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

Hipokampal İnsülin Mikroenjeksiyon ve<em&gt; In vivo</emMekansal Bellek Test sırasında&gt; mikrodivaliz

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

A Lightweight, Headphones-based System for Manipulating Auditory Feedback in Songbirds

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and sensorimotor plasticity1. Consistent sensory perturbations result in compensatory changes in motor output, reflecting changes in feedforward motor control that reduce the experienced feedback error. By quantifying how different sensory feedback errors affect human behavior, prior studies have explored how visual signals are used to recalibrate arm movements2,3 and auditory feedback is used to modify speech production4-7. The strength of this approach rests on the ability to mimic naturalistic errors in behavior, allowing the experimenter to observe how experienced errors in production are used to recalibrate motor output.
Songbirds provide an excellent animal model for investigating the neural basis of sensorimotor control and plasticity8,9. The songbird brain provides a well-defined circuit in which the areas necessary for song learning are spatially separated from those required for song production, and neural recording and lesion studies have made significant advances in understanding how different brain areas contribute to vocal behavior9-12. However, the lack of a naturalistic error-correction paradigm - in which a known acoustic parameter is perturbed by the experimenter and then corrected by the songbird - has made it difficult to understand the computations underlying vocal learning or how different elements of the neural circuit contribute to the correction of vocal errors13.
The technique described here gives the experimenter precise control over auditory feedback errors in singing birds, allowing the introduction of arbitrary sensory errors that can be used to drive vocal learning. Online sound-processing equipment is used to introduce a known perturbation to the acoustics of song, and a miniaturized headphones apparatus is used to replace a songbird's natural auditory feedback with the perturbed signal in real time. We have used this paradigm to perturb the fundamental frequency (pitch) of auditory feedback in adult songbirds, providing the first demonstration that adult birds maintain vocal performance using error correction14. The present protocol can be used to implement a wide range of sensory feedback perturbations (including but not limited to pitch shifts) to investigate the computational and neurophysiological basis of vocal learning.

We describe the design and assembly of miniaturized headphones suitable for replacing a songbird&#x2019;s natural auditory feedback with a manipulated acoustic signal. Online sound processing hardware is used to manipulate song output, introduce real-time errors in auditory feedback via the headphones, and drive vocal motor learning.

Songbirds İşitsel Geribildirim kurgulama için Hafif, Kulaklıklar tabanlı Sistemi

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Eşzamanlı<em&gt; Ex vivo</emİki retina&gt; Fonksiyonel Test tarafından<em&gt; In vivo</em&gt; Elektroretinogram Sistemi

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

Arasında Mechanosensation için bir davranış tahlilinde klonlarda MARCM tabanlı<em&gt; Drosophila melanogaster</em

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

koku ampul kalsiyum Değişiklikleri İzleme yoluyla Sıcaklık kaynaklı Nöronal Aktivite Kayıt<em&gt; Xenopus laevis</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

Ürünleri ve tüm hücre yama kelepçe kayıt Xenopus laevis Tectal nöronlar için iletişim kuralları

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...