Yazarlar bu çalışma Doğa Bilimleri ve Kanada&#39;nın (NSERC) hibe Mühendislik Araştırma Konseyi tarafından desteklenen olduğunu kabul etmek istiyorum.

<ol>
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WormBook, The C. elegans Research Community. , 1-61 (2006).</li><li>Maddox, A. S., Habermann, B., Desai, A., Oegema, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+roles+for+two+C.+elegans+anillins+in+the+gonad+and+early+embryo.">Distinct roles for two C. elegans anillins in the gonad and early embryo.</a> Development. 132, 2837-2848 (2005).</li><li>Sulston, J. E., Schierenberg, E., White, J. G., Thomson, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+embryonic+cell+lineage+of+the+nematode+Caenorhabditis+elegans.">The embryonic cell lineage of the nematode Caenorhabditis elegans.</a> Dev. Biol. 100, 64-119 (1983).</li><li>Chin-Sang, I. D., Chisholm, A. 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Cell Sci. 125, 3039-3049 (2012).</li><li>Guenther, C., Garriga, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+distribution+of+the+C.+elegans+HAM-1+protein+in+neuroblasts+enables+daughter+cells+to+adopt+distinct+fates.">Asymmetric distribution of the C. elegans HAM-1 protein in neuroblasts enables daughter cells to adopt distinct fates.</a> Development. 122, 3509-3518 (1996).</li><li>Audhya, A., 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=A+complex+containing+the+Sm+protein+CAR-1+and+the+RNA+helicase+CGH-1+is+required+for+embryonic+cytokinesis+in+Caenorhabditis+elegans.">A complex containing the Sm protein CAR-1 and the RNA helicase CGH-1 is required for embryonic cytokinesis in Caenorhabditis elegans.</a> J. Cell Biol. 171, 267-279 (2005).</li><li>Maddox, A. 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Yazarlar ifşa hiçbir şey yok.

Bu protokol, orta embriyojenez sırasında görüntü hücre bölünmesi için mikroskopla çeşitli kullanımını tarif eder. Özellikle, bu protokol nasıl epidermal morfogenetiğine kolaylaştırabilir neuroblasts bölünme hücreleri görüntü vurguluyor. Hücre-hücre iletişimi metazoan gelişimi ve C. sırasında doku oluşumu için önemlidir elegans in vivo doku oluşumunu incelemek için mükemmel bir model. Güzel dokuların etkileşimi canlandıran bir olay epitel hücrelerinin bir tabaka...

Nasıl görüntü hücre bölünmesi için erken C. anlatan protokoller var olsa elegans embriyo, bu protokol nasıl görüntü hücre bölünmesi orta Embriyogenez sırasında bir doku içinde açıklanır. Gelişimi sırasında organizmaların görüntülenmesinde önemli zorluklardan biri fototoksisite onların duyarlılık olmuştur. Ancak, dönen bir disk konfokal mikroskoplar veya süpürüldü alan mikroskoplar artan erişilebilirlik daha yaygın görüntüleme uygulamaları izin verdi. Her iki sistem de organizmalar maruz kaldığı UV sınırlandırarak, katı hal lazerleri ve dağınık ışık kullanır. Ancak, Widefield standları hala yüksek duyarlılık (örn. EMCCD), diyafram denetimi ve ışık kontrolü (örneğin, LED&#39;ler veya ayarlanabilir cıva ampul) ile kameralar ile donatılmış olan, özellikle in vivo görüntüleme için kullanılabilir. Bu protokol, C, gelişmekte olan görüntü hücre bölünmesi için bir konfokal tabanlı bir sistem veya bir widefield sistemi ya nasıl kullanılacağını açıklar elegans &lt;/ Em&gt; embriyolar. Örnek olarak, biz nasıl resim neuroblast hücre bölünmesini açıklayınız. Nöro-blastlar örten epidermal hücrelere, kimyasal ya da mekanik ipuçları sağlayarak epidermal morfolojilerinden kolaylaştırmak ve dokuların oluşumunda arası iletişimin önemi mükemmel bir örnek sağlayabilir. Caenorhabditis elegans şeffaflığıyla ve basit doku organizasyonu 1 nedeniyle mikroskopi tabanlı çalışmalar için ideal bir model organizmadır. Ayrıca, C. elegans genetik yöntemler ve RNAi için uygun olan, ve genlerin çoğu insan homologlarını beri, bu doku oluşumu için 2-5 korunmuş mekanizmalarını tanımlamak için kullanılabilir. C. In Embriyo&gt; 300 hücreleri olduğunda elegans, epidermis oluşumu, orta embriyojenez sırasında oluşur. Epidermal morfogenez embriyo büzülür epidermal hücrelerin bir tabaka içine ve embr dönüştürmek için uzanan bir süre boyunca birkaç büyük aşamadan oluşurBir solucan 6&#39;nın uzun oval bir şekil formundan yo. Ventral epidermal hücreler embriyonun ventral yüzeyi (Şekil 1) kapsayacak şekilde ventral orta hat doğru geçiş yaparken, ventral muhafaza, bu morfogenetik olaylardan birini açıklamaktadır. İlk olarak, anterior bulunan öncü hücrelerin iki çift kontralateral komşularıyla 6 ile ventral onlar uygun orta hat ve sigorta doğru göç. Bu, ventral cep 6-7 oluşturma şekiller gibi kama oluşturan merkezi bir arka cep hücrelerinin taşınması takip eder. Cebi kapatır mekanizması iyi anlaşılmamıştır. Bir olasılık, bir supracellular aktin miyozin kasılma yapısı 8 yara iyileştirici benzer, moda gibi bir çanta dize birlikte cep hücreleri bağlar olduğunu. İlginç bir şekilde, cep hücrelerin bazı göç epidermi altında bulunan temel neuroblasts 9 (nöronal ön-maddeleri arasında, belirli alt-aracılık eders, Şekil 1 B). Önceki çalışmalar nöro. VAB-1 (efrin reseptörü) ve VAB-2 (efrin ligand) yüksek neuroblasts olarak ifade edilmiştir ve birbirinden anterior ve posterior neuroblasts sıralama kolaylaştırmak edilir ventral epidermal hücre göçü ve ventral muhafaza düzenleyen gösterdi, ve VAB-1 veya VAB-2 neden ventral muhafaza mutasyonlar 10-13 fenotip. Bununla birlikte, promotör kurtarma deneyler VAB-1, aynı zamanda neuroblasts salgıladığı diğer rehberlik ipuçlarının için üstte ventral epidermal hücreleri ve reseptör olarak gerekli olduğunu göstermiştir ventral epidermal hücrelerinde 9 olarak ifade edilmiştir. Bu reseptörlerin herhangi mutasyonlar ventral muhafaza fenotiplerini neden olsa da kusurları nedeniyle neuroblast konumlandırma veya bağlı rehberlik yanıt ventral epidermal hücrelerin başarısızlık 14 istekalar Sorunlara ortaya çıkması durumunda, bu açık değildir. Nöroblastlar wi bölümü değiştirilmesirehberlik ipuçları salgılama yeteneğini etkileyen thout neuroblasts rolü ve epidermal morfolojilerinden sırasında mekanik girdi sağlama yeteneği ışık tutabilir. Son zamanlarda, bir hücre bölünmesi geni, ani-1 (anillin) yüksek neuroblasts (Şekil 2A) olarak ifade edilmiştir ve tükenme neuroblast bölme kusurlarına neden olduğu bulunmuştur. İlginçtir, bu embriyolar ventral muhafaza fenotipleri (Fotopoulos, Wernike ve Piekny, yayınlanmamış gözlemler) gösterilecek. Anillin hücre bölünmesi için gerekli olan ve özellikle de, bir ana hücre fiziksel olarak iki yavru hücrelere bölündüğü işlemi tarif sitokinez, içindir. Sitokinler sıkıca düzgün kardeş kromatid ayrılması ile birlikte olduğundan emin olmak için mekan ve zaman olarak kontrol edilmesi gereken bir aktomizin kontraktil halka oluşumu ile tahrik edilir. Metazoan sitokinez ana düzenleyicisi RhoA (C. elegans RHO-1), bir küçük bir GTPaz olduğunuonun GTP-bağlı formda yönerge. GEF Ect2/ECT-2 RhoA-GTP kontraktil halka oluşturur ve girişini 15 aracılık eden alt baş efektörlerinin ile etkileşime sonra, RhoA etkinleştirir. Anillin C-terminalinde ile ve N-terminal ile aktin ve miyozin için RhoA bağlanan bir çok alan bir proteindir. Anillin memeli veya Drosophila S2 hücreleri 16 kontraktil halkanın konumunu stabilize etmek için gereklidir. Anillin tükenmesi yanal salınımlar geçmesi kasılma yüzük olur, ve sitokinez sonunda çok çekirdekli hücreler 17-19 oluşturulması başarısız. İlginç bir şekilde, her ne kadar C. elegans ani-1 it sitokinez için gerekli değildir, erken embriyo aktomizin kasılmasını koordine eder. Yukarıda açıklandığı gibi Ancak, ani-1 orta Embriyogenesis (Fotopoulos, Wernike ve Piekny, yayınlanmamış gözlemler) sırasında neuroblast sitokinez için gereklidir. Sitokinez başarısızlık neuroblasts sayısını ve konumunu değiştirecek ve loc etkileyebilecekKimyasal rehberlik ipuçlarının tirme ya da doku mekanik özelliklerini değiştirebilir. Her iki model de ventral muhafaza için neuroblasts otonom olmayan rolünü, ve embriyonik gelişim sırasında doku doku iletişimin önemini vurgulamak. Bu deneysel protokol anlatıyor görüntü hücre bölünmesi sırasında C. elegans floresan mikroskopi kullanılarak orta embriyogenezis. Hücre bölünmesi mekanizmalar üzerinde çalışmaya Deneylerin çoğu hücre sınırlı sayıda (örneğin C. tek hücreli embriyo, Xenopus veya echinoderm elegans ile kültür tabaklarında (örn.. HeLa veya S2 hücreleri) tek hücre ya da erken embriyolar gerçekleştirilmiştir embriyo). Ancak, bu bölünme uçağın zamanlaması ve yerleştirme etkileyebilecek dış ipuçları olduğu gibi, aynı zamanda dokuların içinde hücre bölünmesi çalışma için önemlidir. Ayrıca, hücreler komşu doku gelişimini etkilemek için kimyasal veya mekanik ipuçları verebilirs, ve hücreler arası iletişim gelişimi sırasında meydana dokuların nasıl yardımcı anlamak önemlidir.

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			<td height="21" style="height:21px;width:291px;">Agar</td>
			<td style="width:247px;">BioShop Canada Inc.</td>
			<td style="width:185px;">#AGR001.1</td>
			<td style="width:335px;">For making C. elegans NGM and RNAi plates</td>
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			<td height="21" style="height:21px;">Agar</td>
			<td>Bio Basic Inc.</td>
			<td>#9002-18-0</td>
			<td>For making bacteria LB agar plates</td>
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			<td height="21" style="height:21px;">Agarose</td>
			<td>BioShop Canada Inc.</td>
			<td>#AGA001.500</td>
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			<td height="21" style="height:21px;">Anti-mouse Alexa 488 antibody</td>
			<td>Life Technologies Corporation (Invitrogen)</td>
			<td>#A11029</td>
			<td></td>
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			<td height="21" style="height:21px;">Anti-mouse anti-GFP antibody</td>
			<td>Roche Applied Science</td>
			<td>#11814460001</td>
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			<td height="21" style="height:21px;">Anti-rabbit Alexa 568 antibody</td>
			<td>Life Technologies Corporation (Invitrogen)</td>
			<td>#A11011</td>
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			<td height="21" style="height:21px;">Ampicillin</td>
			<td>BioShop Canada Inc.</td>
			<td>#AMP201.5</td>
			<td>Store powder at 4 &#176;C and dissolved ampicillin at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bactopetone&#160; (peptone-A)</td>
			<td>Bio Basic Inc.</td>
			<td>#G213</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2 (calcium chloride)</td>
			<td>BioShop Canada Inc.</td>
			<td>#C302.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cholesterol</td>
			<td>BioShop Canada Inc.</td>
			<td>#CHL380.25</td>
			<td>Dissolve in ethanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>#D9542</td>
			<td>Use to stain nucleic acids (DNA)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>BioShop Canada Inc.</td>
			<td>#GLY001.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IPTG (isopropylthio-&#946;-galactoside)</td>
			<td>Bio Basic Inc.</td>
			<td>#367-93-1</td>
			<td>Store powder and dissolved IPTG at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KH2PO4 (potassium phosphate, monobasic)</td>
			<td>BioShop Canada Inc.</td>
			<td>#PPM666.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">K2HPO4 (potassium phosphate, dibasic)</td>
			<td>BioShop Canada Inc.</td>
			<td>#PPD303.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L4440&#160; (feeding vector)</td>
			<td>Addgene</td>
			<td>#1654</td>
			<td>Keep as glycerol stock at -80 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4&#160;&#160; (magnesium sulfate)</td>
			<td>BioShop Canada Inc.</td>
			<td>#MAG511.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl (sodium chloride)</td>
			<td>Bio Basic Inc.</td>
			<td>#7647-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4 (sodium phosphate, dibasic)</td>
			<td>Bio Basic Inc.</td>
			<td>#7558-79-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal Donkey Serum (NDS)</td>
			<td>Wisent Bioproducts</td>
			<td>#035-110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">n-propyl 3.4.5-trihydroxybenzoate (propyl gallate)</td>
			<td>Alfa Aesar</td>
			<td>#A10877</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-lysine</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>#P8920</td>
			<td>For optimal results coat microscope slides three times&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptomycin</td>
			<td>BioShop Canada Inc.</td>
			<td>#STP101.50</td>
			<td>Store powder at 4 &#176;C and dissolved streptomycin at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetracyclin</td>
			<td>BioShop Canada Inc.</td>
			<td>#TET701.10</td>
			<td>Store powder at 4 &#176;C and dissolved tetracycline at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Bio Basic Inc.</td>
			<td>CAS#9005-64-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tryptone</td>
			<td>BioShop Canada Inc.</td>
			<td>#TRP402.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Extract</td>
			<td>Bio Basic Inc.</td>
			<td>#8013-01-2</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing neuroblast cytokinesis during c. elegans embryogenesis

Bu protokol, Caenorhabditis elegans, gelişmekte olan embriyo bölünen hücreleri görüntüye floresan mikroskobu kullanımını tarif eder. Özellikle, bu protokol epidermal hücrelerin altında bulunan ve epidermal morfogenezisi önemli olabilir neuroblasts, bölünmesi nasıl görüntü üzerinde duruluyor. Doku oluşumu metazoan gelişimi için çok önemlidir ve komşu dokuların dış işaretlere dayanmaktadır. C. elegans mikroskobu ile çalışmak için kendi dokuları kolay hale nedeniyle şeffaflık ve basit organizasyon in vivo doku morfogenetiğine incelemek için mükemmel bir model organizmadır. Ventral muhafaza embriyonun ventral yüzey epitel hücrelerinin tek bir tabaka ile kaplanmıştır süreçtir. Bu olay örten epitel hücrelerinin göçüne aracılık kimyasal rehberlik ipuçları sağlamak temel neuroblasts, kolaylaştırılabilir düşünülmektedir. Bununla birlikte, son derece nöro-proliferatif ve aynı zamanda hareket edebilirventral epidermal hücreler için mekanik bir alt-tabaka olarak. Bu deney protokolü kullanılarak yapılan çalışmalar, doku oluşumu sırasında arası iletişimin önemini ortaya çıkarmak olabilir, ve gelişmekte olan dokular içinde hücre bölünmesi ile ilgili genlerin rollerini ortaya çıkarmak için kullanılabilir.

Bu deneysel protokol nasıl C. görüntü hücre bölünmesi açıklanmaktadır elegans ortalarında Embriyogenez sırasında embriyolar. Özellikle, epidermal morfolojilerinden kolaylaştırabilir ki ne resim neuroblasts için tarif etmektedir. Epidermal morfogenez nedeniyle epidermal hücre şekli değişiklikleri, göç ve yapışma, aynı zamanda altında yatan neuroblasts (Şekil 1 B) kimyasal ya da mekanik ipuçları dayanır bir arada oluşur. Nöro-blastlar göç 10,14,25<...

Watch this Scientific Journal Video about Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis at JoVE.com

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

Bu protokol, görüntüye Caenorhabditis elegans embriyolarında bir doku içinde bölünen hücreleri açıklamaktadır. Birkaç protokoller embriyonun erken görüntü hücre bölünmesi için, bu protokol nasıl mid geç embriyonik dönemde gelişmekte olan bir doku içinde hücre bölünmesi görüntü açıklar nasıl tarif ederken.

Sırasında Neuroblast Sitokinez görselleştirme<em&gt; C. elegans</em&gt; Embriyo

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this ...

visualizing-neuroblast-cytokinesis-during-c-elegans-embryogenesis

Research

JoVE Journal

Neuroscience

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

11.7K Görüntüleme.  Concordia University.  Bu protokol, görüntüye Caenorhabditis elegans embriyolarında bir doku içinde bölünen hücreleri açıklamaktadır. Birkaç protokoller embriyonun erken görüntü hücre bölünmesi için, bu protokol nasıl mid geç embriyonik dönemde gelişmekte olan bir doku içinde hücre bölünmesi görüntü açıklar nasıl tarif ederken. 

Video: Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &amp;#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

Transgenik kullanarak Assaying β-amiloid Toksisite C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

Nörobilim

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment through neuron-specific detection of food and chemical odors1, 2, and can associate nutritive states with chemical odors3, temperature4, and the pathogenicity of a food source5.
Here, we describe assays of C. elegans associative learning and short- and long-term associative memory. We modified an aversive olfactory learning paradigm6 to instead produce a positive response; the assay involves starving ~400 worms, then feeding the worms in the presence of the AWC neuron-sensed volatile chemoattractant butanone at a concentration that elicits a low chemotactic index (similar to Toroyama et al.7). A standard population chemotaxis assay1 tests the worms' attraction to the odorant immediately or minutes to hours after conditioning.
After conditioning, wild-type animals' chemotaxis to butanone increases ~0.6 Chemotaxis Index units, its "Learning Index". Associative learning is dependent on the presence of both food and butanone during training. Pairing food and butanone for a single conditioning period ("massed training") produces short-term associative memory that lasts ~2 hours. Multiple conditioning periods with rest periods between ("spaced training") yields long-term associative memory (&lt;40 hours), and is dependent on the cAMP Response Element Binding protein (CREB),6 a transcription factor required for long-term memory across species.8
Our protocol also includes image analysis methods for quick and accurate determination of chemotaxis indices. High-contrast images of animals on chemotaxis assay plates are captured and analyzed by worm counting software in MatLab. The software corrects for uneven background using a morphological tophat transformation.9 Otsu's method is then used to determine a threshold to separate worms from the background.10 Very small particles are removed automatically and larger non-worm regions (plate edges or agar punches) are removed by manual selection. The software then estimates the size of single worm by ignoring regions that are above a specified maximum size and taking the median size of the remaining regions. The number of worms is then estimated by dividing the total area identified as occupied by worms by the estimated size of a single worm.
We have found that learning and short- and long-term memory can be distinguished, and that these processes share similar key molecules with higher organisms.6,8 Our assays can quickly test novel candidate genes or molecules that affect learning and short- or long-term memory in C. elegans that are relevant across species.

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.

C. elegans Pozitif bütanon Öğrenme, Kısa vadeli ve uzun vadeli ilişkilendirilebilir Bellek Tahliller

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment ...

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

C in vivo Lazer Axotomy elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

Içinde Synaptic florasan Otomatik Sayısallaştırma<em&gt; C. elegans</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

C. elegans Tracking and Behavioral Measurement

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the surface of an agar plate. For the study of the genetic, biochemical, and neuronal basis of behavior, C. elegans is an ideal organism because it is genetically tractable, amenable to microscopy, and shows a number of complex behaviors, including taxis, learning, and social interaction1,2. Behavioral analysis based on tracking the movements of worms as they crawl on agar plates have been particularly useful in the study of sensory behavior3, locomotion4, and general mutational phenotyping5. Our system works by moving the camera and illumination system as the worms crawls on a stationary agar plate, which ensures no mechanical stimulus is transmitted to the worm. Our tracking system is easy to use and includes a semi-automatic calibration feature. A challenge of all video tracking systems is that it generates an enormous amount of data that is intrinsically high dimensional. Our image processing and data analysis programs deal with this challenge by reducing the worms shape into a set of independent components, which comprehensively reconstruct the worms behavior as a function of only 3-4 dimensions6,7. As an example of the process we show that the worm enters and exits its reversal state in a phase specific manner.

C. elegans Tracking and Behavioral Measurement

We have developed a video-rate tracking microscope system that can record and quantify C. elegans behavior at high resolution and high speeds. We have also developed computational methods to reduce the dimensionality of the worm images to a fundamental set of measurements that completely describe the shape of the worm.

C. elegans Takip ve Davranış Ölçme

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

Uzun vadeli Olfaktör İntibak A Moleküler Saati<em&gt; C. elegans</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

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 ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

C. vivo Nöronal Kalsiyum Imaging&#39;de elegans

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted area of the brain, neural stem cells proliferate and constantly generate neuroblasts that migrate tangentially in chains along the rostral migratory stream (RMS) to reach the olfactory bulb (OB). Once in the OB, neuroblasts switch to radial migration and then differentiate into mature neurons able to incorporate into the preexisting neuronal network. Proper neuroblast migration is a fundamental step in neurogenesis, ensuring the correct functional maturation of newborn neurons. Given the ability of SVZ-derived neuroblasts to target injured areas in the brain, investigating the intracellular mechanisms underlying their motility will not only enhance the understanding of neurogenesis but may also promote the development of neuroregenerative strategies.
This manuscript describes a detailed protocol for the transfection of primary rodent RMS postnatal neuroblasts and the analysis of their motility using a 3D in vitro migration assay recapitulating their mode of migration observed in vivo. Both rat and mouse neuroblasts can be quickly and efficiently transfected via nucleofection with either plasmid DNA, small hairpin (sh)RNA or short interfering (si)RNA oligos targeting genes of interest. To analyze migration, nucleofected cells are reaggregated in &#39;hanging drops&#39; and subsequently embedded in a three-dimensional matrix. Nucleofection per se does not significantly impair the migration of neuroblasts. Pharmacological treatment of nucleofected and reaggregated neuroblasts can also be performed to study the role of signaling pathways involved in neuroblast migration.

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Neuroblast migration is a crucial step in postnatal neurogenesis. The protocol described here can be used to investigate the role of candidate regulators of neuroblast migration by employing DNA/small hairpin RNA (shRNA) nucleofection and a 3D migration assay with neuroblasts isolated from the rodent postnatal rostral migratory stream.

Kemirgen neuroblasts nucleofection Neuroblast Göç Eğitim için<em&gt; In vitro</em

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted ...

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of stress-induced plasticity is the dauer stage of C. elegans. Dauers are an alternative developmental larval stage formed under conditions of low concentrations of bacterial food and high concentrations of a dauer pheromone. Dauers display extensive developmental and behavioral plasticity. For example, a set of four inner-labial quadrant (IL2Q) neurons undergo extensive reversible remodeling during dauer formation. Utilizing the well-known environmental pathways regulating dauer entry, a previously established method for the production of crude dauer pheromone from large-scale liquid nematode cultures is demonstrated. With this method, a concentration of 50,000 - 75,000 nematodes/ml of liquid culture is sufficient to produce a highly potent crude dauer pheromone. The crude pheromone potency is determined by a dose-response bioassay. Finally, the methods used for in vivo time-lapse imaging of the IL2Qs during dauer formation are described.

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant &#8216;dauer&#8217; juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer.

C. Dauer-spesifik nöronal Yaptırma Vivo Görüntülemede elegans

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of ...