I thank Dr. Maureen Barr, under whose supervision the dauer IL2 remodeling phenotype was initially characterized. Thanks to Dr. C. Britt Carlson for critical reading of the manuscript. Funding in the Barr lab was from the NIH (5R01DK59418) and postdoctoral fellowships from the USDA (2010-65106-20587) and the New Jersey Commission on Spinal Cord Research (CSCR12FEL004). Current funding for this work is from a University of Illinois Urbana-Champaign College of ACES FIRE grant.

1. Ham Dauer Feromon Üretimi <ol><li> OP50 E. büyütün Tek bir koloni, O / N 37 ° C &#39;de E. coli 200 rpm&#39;de LB suyu 100 ml çalkalandı. NOT: Bu göre O / N kültür önceden yapılmış ve 4 ° C&#39;de saklanabilir. </li><li> N2 C büyütün elegans, 15 - 20 OP50 E: 50 ul NGM ağarma (Tablo 1 &#39;de tarif bak) ile 6 cm Petri kapları coli bakteri kadar neredeyse 19 tükenmiş. </li><li> 1 L Erlenmeyer&#39;de S medya 5x 250 ml (Tablo 1 tarifi görün) Marka 19 şişeleri. NOT: Bu çözüm önceden yapılabilir. </li><li> OP50 E. kültürünü büyütün E. coli &#39;de OP50 1 ml LB suyu aşılayarak O 4 L / K coli E. coli ve 16 saat boyunca 37 ° C&#39;de çalkalanarak. </li><li> , (S bazal çözeltisi (Tablo 1 &#39;de tarif bak) ve Aşama 1.3&#39; de hazırlanan media, 4: 1 L&#39;lik Erlenmeyer kaplanna her birinin içine, 4-5 plakalarından buraya nematod 1 ml levhalar (adım 1.2) için 4- nematodları yıkayın 5 plakalarıHer şişe için) 19,. </li><li> 10 dakika boyunca 650 x g&#39;de aşama 1.4 OP50 santrifüj ve supernatant çıkarın. G taban çözeltisi 10 ml her bir bakteri pelletini. Nematodların her şişeye yeniden süspansiyon haline getirildi bakteri eşit miktarda aktarın. </li><li> Çözelti, yiyecek tükenmesi gösteren temizlemek için başlayana kadar 5 gün - (- 25 ° C&#39;de 20) ve 100 - 4 150 rpm&#39;de oda sıcaklığında bir orbital çalkalayıcı üzerinde şişeler yerleştirin. </li><li> Gıda bitince, OP50 E. başka bir O / N kültür büyümek LB suyu 4x 1 L E. coli. 10 dakika boyunca 650 x g&#39;de santrifüj ve bakteri, nematodlar ile media şişelere her bir şişeye bir pelet ekleyin. Gıda daha bitene kadar ilave 3-4 gün boyunca, Aşama 1.7 gibi orbital çalkalayıcıya S ortam şişeler dönün. </li><li> Her bir şişeden bir çözelti ~ 1 ml lik bir alın ve her biri 1 ml&#39;lik bir 1 ul alt-numûne alma ve nematodların sayısını sayarak nematod konsantrasyonu tahmin ediyoruz. NOT:75,000 nematodlar / mL - nematod popülasyonu 50,000 olmalıdır. Bu konsantrasyonda nematod çoğunluğu dauers bulunmaktadır. </li><li> Kirlenmiş şişeleri atın. NOT: Kirlilik sık adımı 1.9 sırasında görülen hyphe&#39;nin olarak tezahür eder. </li><li> 4 ° C&#39;de 10 dakika boyunca 4000 x g&#39;de santrifüje nematodlar. Nematod pelet atın ve süpernatant saklayın. Alternatif olarak, düşük potens feromon yapmak için bu hayvanları kullanarak tartışma bakın. </li><li> # 1 Whatman filtre kağıdı ile süpernatantı Filtresi. NOT: Bir termos bu süreci hızlandırmak olacaktır. </li><li> Ayrıca 0.45 mikron steril vakumlu filtrasyon üniteleri ile süpernatant süzün. Not: Filtrat, 4 ° C&#39;de O / N ya da muhafaza 1,14 devam edilebilir. </li><li> Bir karıştırma çubuğu ile bir 2 L&#39;lik bir kap için filtrat ekleyin. Bir ısıtma karıştırma plakası üzerinde bir çeker ocak içinde yerleştirin. Karıştırılarak bir taşım kaynatın ve yaklaşık 200 ml buharlaşır. 500 ml&#39;lik bir behere çözeltisini. Yaklaşık 50 ml le kadar kaynar Devamft. NOT: çözüm taşmak için mümkün olduğu gibi Bakım bu adım sırasında alınmalıdır. Son 50 ml&#39;lik rengi koyu kahverengi. Pişirme işlemi sırasında herhangi bir zamanda, ısı kapatılabilir ve süzülen madde, oda sıcaklığında ya da 4 ° C &#39;de O / N saklanır. </li><li> G soğuması ve x 1000 de 5 dakika süreyle santrifüj. 100 ml&#39;lik bir beher içine dökün ve süpernatant pelet atılır. </li><li> Kahverengi bir kabuk yüzer aşağı kaynatın. Ateşten alın ve bir spatula ile kabuk kırmaya. Kabuk karşılamak için yeterli 200 derece etanol ekleyin. Parafilm ile beher örtün ve oda sıcaklığında O / N bekletin. Temiz bir beher içine dökün ve etanol tarafına yerleştirin. Kabuk kapağı taze etanol ekleyin. O / N Extractions 3 kez tekrarlayın. Ekstraktları havuzda toplayın. </li><li> Etanol ekstraktları için bir vakum pompası kullanılır. Bir vakum pompası mevcut değilse, bir çeker ocak içinde, bir taraftan karıştırılırken 50 ° C&#39;ye kadar temiz bir cam deney ve sıcak etanol ile özü ekleyin. NOT: etanol l yapışkan bir kalıntı buharlaştırılarak sonraIght kahverengi kalmalıdır. Isıtma kullanıldığı takdirde, çok uzun süre bakım ısıtılarak çözelti yakmak için özen gösterilmesi gerekmektedir. </li><li> Damıtılmış steril su içinde 10 ml bir tortu çözülür. Filtre 1 ml alikotlar içinde -20 ° C&#39;de bir 0.22 um şırınga filtre ve mağaza bu solüsyon sterilize edin. Not: 1 ml su esas sıvı nematod kültürünün her 100 ml için en eritilmesi için kullanılır. Sıvı kültür şişelerinden bir kirlenme nedeniyle göz ardı edilir, örneğin, daha sonra bir tortu çözmek için kullanılan su miktarını ayarlayın. </li></ol> Feromon Doz Tepki 2. belirlenmesi <ol><li> Peptonu olmadan Noble agar NGM ortamı 6x 5 ml yaparak Dauer feromon hazırlanmakta ve 50 ug / ml streptomisin sülfat ug ve dayanıklı feromon bir dizi ile takviye edilmiştir. 4, 19 (Tablo 1 &#39;de tarife bakınız), bir 13x100 mm Karışım bileşenleri cam kültürü tüp, bir Bunsen beki taşmak, ve daha sonra 3 cm Petri kaplarına dökün. </li><li> Am tartılıricrocentrifuge tüpü. E. göre O / N kültürün 1 ml ekleyin mikrosantrifüj tüp içinde ~ 17.000 x g&#39;de tüp ve santrifüj coli OP50. Yüzer sökün. Yine tüp tartılır ve bakteryel tanelerin ağırlığını belirler. % 4&#39;lük bir konsantrasyon için M9 tampon (Tablo 1 &#39;de tarif bakınız) pelletini (a / h). Her bir kısım feromon plaka 4 merkezinin üzerine bakteri süspansiyonundan 10 ul. Mağaza plakaları O / N, oda sıcaklığında. </li><li> Feromon plakalar 4 üzerine yetişkinliğe molt sonra 10-15 gebe N2 vahşi tip çift cinsiyetli bir gün seçin. 4 saat için 3 - 25 ° C&#39;de plakalar saklayın. </li><li> Yetişkin çift cinsiyetli tüm koparmak. Her plaka için bıraktıkları yumurta sayısını kaydedin. Parafilm ile sızdırmaz şekilde kapatılır ve plakaları, 3 gün 17 boyunca 25 ° C&#39;de geri döner. </li><li> Dauers yüzdesini Sayısı (Şekil 2). Not: her iki yöntem için, dikkat edilmelidir ki dauers (ve daha az bir exÇadır olmayan dauers) duvarlara tırmanmaya ve Petri yemekler kapağın üzerine olacaktır. Bu nematod için yemeğin tüm yüzeyini incelemek için bu nedenle önemlidir. </li><li> Bir doz-tepki eğrisi için verilerin Fit ve Şekil 3&#39;te gösterildiği gibi (dauers meydana getirmek üzere hayvanların% 90 uyarılması için yeterli bir feromon) feromonun EC90 konsantrasyonu tahmin Not:. Normal olarak, AK 90 tahmin etmek için bir lojistik regresyon modeli kullanmak ama farklı istatistiksel yazılım paketi benzer tahminler üretmek alternatif yöntemleri olabilir. </li></ol> Dauer IL2 Nöronlar 3. Canlı Görüntüleme <ol><li> Adım 2.6 belirlenen AT 90 değerini kullanarak protokolü 2 gibi Dauer feromon tabak hazırlayın. </li><li> Entegre IL2 muhabiri transgenini taşıyan bir suş (myIs13 [P klp-6 :: GFP], myIs14 [P klp-6 :: GFP], myIs16 [P protokol 2&#39;de yöntemleri kullanarak dauers Uyarmaktadır <em&gt; KLP-6 :: tdTomato] veya qIs56 [P ara-2 :: GFP]) 12, 20, 21 Not: eq. Is56 kullanılırsa, GFP ifade içine birkaç saat kadar IL2Qs görünür olmayacak Dauer dökücü ve ilk biçimlenme bazı olaylar görünür olmayacaktır. P ara-2 :: GFP transgenden flüoresan sinyal P KLP-6 floresan muhabir daha Dauer sırasında IL2s sonuçta parlak iken, ayrıca, p-Gecikmeli 2 :: GFP Transgen kası zayıf biçimde ifade eden ince IL2 dalları maskeleyebilir. </li><li> Yumurtlama izlenerek, ergin hermafrodit bireyler çıkarmak Parafilm ile sızdırmaz plakalar 25 ° C de 17 34 saat boyunca inkübe edilir. NOT: Dauer molt için zaman uzunluğu bireyler arasında biraz değişir. Dauer molt başlangıcından itibaren 5 hr (39 - - dendrit arborization en hızlı dönemdir 4 başlıyor 44saat sonrası yumurtlama 25 ° C) 12,. </li><li> Görüntüleme bir gün önce, EC90 konsantrasyonunda Dauer feromonu ile M9 tampon% 10 ihtiva eden bir çözelti (ağ / hac) agaroz oluşturur. Agaroz kısım yaklaşık 100 ul bir mikroskop lamı üzerine ve hızlı bir şekilde, ancak, hafifçe düz bir yüzey oluşturmak için agaroz üzerine başka bir slayt yerleştirin. Agaroz katılaşmış sonra bir nem odasına (ıslak kağıt havlu ile pipet kutusu), saran sargısına ve mağaza slaytlar sarın. </li><li> 34 saat kuluçka döneminden sonra, yutak pompalamayı durdurdu nematod için feromon plakaları inceleyin. Ayrı slaytlar bir sürgünün sadece bir yüzü üzerinde agaroz sahip bileşimleri içerir. % 10 agaroz pad ve 0.1 mikron polistiren boncuklar 22 1 μ l üzerine bir veya daha fazla nematod seçin. Not: iyi senkronize nüfusu ile plaka üzerinde nematodların çoğunluğu L2D gelen Dauer için molt giren edilecektir. Ancak, i arasında değişimi olabilirndividuals. Bu hayvanlar Dauer molt içinde olmuştur süre (örneğin temsili sonuçlar bakınız) miktarını tahmin etmek için ek özellikler (stoma tıkanıklığı, boğaz yeniden şekillenme) dikkat etmek önemlidir. </li><li> Nematod yönünü (sol / sağ, dorsal / ventral) belirleyin. Mümkün olan en düşük floresan ile 100x büyütmede her 15 dakika bir IL2Q dendritin Z-yığın görüntüleri yakalayabilir veya belirli bir deney için gerektiği gibi. (Yutak pompalama başlar) nematod L2D manikür ayrılmış kadar görüntü yakalamayı devam edin ve hayvan Dauer kurtarır eğer hareketsizlik imkansız ya. Aktif görüntüleme zamanlarda, bir nemlilik bölmesi içinde slayt yerleştirin. NOT: Bir motorlu sahne ve diferansiyel girişim kontrast (DIC) optik bir epifluorescent mikroskop kullanıyoruz. </li></ol>

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The authors have nothing to disclose

Dauer özgü nöroplastik Dauer ve diğer ilişkili morfolojik değişikliklerin bir inceleme kontrol edilebilir ve her iki genetik mutasyon (örneğin, DAF,-C mutantları) vasıtasıyla veya feromon vahşi tip hayvanların maruz bırakılarak dauers güvenilir oluşturulmasını gerektirir. Dauers ikna etmek için bir genetik mutasyon kullanımı uygun olsa da, bu sonuçlar yanıltabilecek. Bu nedenle Daf-c mutasyonlar ile toplanan veriler daha sonra Dauer feromon maruz Dauer sahne girmek için uyarı...

Gelişimsel ve davranışsal plastisite fenatipik plastisite, olumsuz çevre koşullarına adaptasyonları için önemlidir ve evrim 1 itici bir güç olarak kabul edilir. C. elegans sık geliştirme ve nörobiyoloji çalışmalar için kullanılan bir model organizmadır. İdeal koşullar altında, C. elegans yetişkin üreme aşamasına başlamadan önce dört larva aşamalardan geçerek gelişir. Ancak, düşük gıda kullanılabilirlik ve yüksek nüfus yoğunluğu, C koşulları altında elegans gelişimsel anahtarı geçmesi ve uzun ömürlü ve stres dayanıklı Dauer evre 2 içine girebilirsiniz. Dauers olasılıkla bu stres direnci aracılık olmayan dauers birkaç morfolojik ve davranışsal farklılıklar gösterecektir. Dauer girişi düzenleyen çevre kuyrukları ve genetik yollar yoğun oldu, 5, 4, 3 6, 7 nitelendirdiler. Ancak, bireysel fenotipik değişiklikleri kontrol eden moleküler mekanizmalar d meydana olduğunurada Dauer oluşumu daha az iyi anlaşılmaktadır. Dauer özgü fenotiplerinin incelenmesi Dauer hayvanların üretimini gerektirir. Bu üç şekilde yapılabilir: 1) C bir açlıktan kültüründen Çekme elegans, 2) Dauer oluşum bünye (Daf-c) mutantlar, arıtılmış feromon sayesinde Dauer oluşumu 3) ndüksiyonunun kullanımı. Bir C ile standart bir NGM plaka ikinci dauers almak için nispeten basit onun bakteriyel gıda arzını tükenmiş elegans nüfus. Elimizde olanlar, standart bir NGM plaka orjinal 3 gün büyümeye bırakıldı ve daha sonra 3 ile aşılanmış Escherichia coli OP50, 50 ul ile tohumlanmış - 4 ergin hermafrodit bireyler bir hafta içinde gıda kaynağı dışarı atmasını sağlar ve birkaç bin üretecek 25 ° C&#39;de tutuldu İlave bir 3 gün içinde dauers (çok sayıda açlıktan olmayan dauers ek olarak). Bununla birlikte, bu yöntem, d içine molt sırasında meydana gelen incelenmesi işlemleri için tatmin edici değildirauer. Bu Dauer girmektedir tipik bir açlıktan plaka üzerinde bir kaç bin hayvan belirlemek zordur. Ayrıca, hasret plaka kullanımı senkronizasyonu için hiçbir imkanı sunuyor. DAF-C mutantlarının kullanımı senkronizasyon ve dauers güvenilir oluşturulmasına izin verir. Ancak, belirli bir Daf-c mutasyonu tek başına veya ek mutasyonlar ile birlikte diğer Dauer özgü fenotipleri etkilemez garantisi yoktur. Bir Dauer feromon varlığı ilk Cassada ve Russell tarafından ima ve daha sonra Altın ve Riddle tarafından gösterilmiştir. Dauer feromon yana kimyasal olarak 9, 10 8, 2 karakterize edilmiştir. Arıtılmış feromon farklı şekillerde 9, bir ham özü Dauer feromon 11. Kullanımlar sağlar hem hareketsel hem de gelişim süreçleri düzenleyen ascarosides karmaşık bir harmanından oluşur doğal tipte bir arka senkronize dauers güvenilir kontrol indüksiyonu. </p&gt; Sinir sistemi ve ilişkili glial hücreler Dauer larva oluşumu 12, 13, 14 sırasında biçimlenme geçmesi. Bu değişikliklerin bazıları, Dauer 13, 14 sırasında glia hücrelerinin yeniden olarak, geri döndürülemez. Ancak, başka biçimlenme olayları olumlu bir dönüş üzerine geri dönüşümlüdür çevre koşulları. Dendrit arborization, multidendritic nöronlar ve akson 12 biçimlenme tek dendritik bir anahtarı: Örneğin, dört IL2Q nöronların bir dizi dahil Dauer oluşumu sırasında hızlı ve geri dönüşümlü nöronal biçimlenme tabi. Buradaki yöntemler, bir model organizmada strese bağlı nöroplastisitesi güvenilir bir in vivo görüntüleme için izin verir. Üretim ve ham Dauer feromon testleri için sunulan yöntemler, daha önce çeşitli kaynaklardan 4, 8, 15, 16, 17, 18 tarif edilen ve derlenmektedir.

<table border="0" cellpadding="0" cellspacing="0" width="873">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:259px;">N2 C. elegans Bristol strain</td>
			<td style="width:152px;">Caenorhabditis Genetics Center</td>
			<td style="width:212px;"></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PT2660 myIs13[Pklp-6::gfp] III</td>
			<td style="width:152px;">Upon request from Schroeder lab</td>
			<td></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PT2762 myIs14[Pklp-6::gfp] V</td>
			<td style="width:152px;">Upon request from Schroeder lab</td>
			<td></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">JK2868 qIs56[Plag-2::gfp] V</td>
			<td style="width:152px;">Caenorhabditis Genetics Center</td>
			<td></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">OP50 E. coli</td>
			<td style="width:152px;">Caenorhabditis Genetics Center</td>
			<td></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dishes</td>
			<td style="width:152px;">Tritech Research</td>
			<td>60 mm T3308, 35 mm T3501</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="132">
			<td height="132" style="height:132px;">NGM agar</td>
			<td style="width:152px;">Various</td>
			<td></td>
			<td style="width:251px;">3 g NaCl, 17 g agar, 2.5 g peptone in 975 mL H2O autoclaved cooled to 55&#176;C followed by 1 mL of 5 mg/ml cholesterol in ethanol, 25 mL of 1 M KPO4 buffer (pH 6.0), 1 mL of 1 M CaCl2, 1 mL of 1 M MgSO4</td>
		</tr>
		<tr height="356">
			<td height="356" style="height:356px;">S Media</td>
			<td style="width:152px;">Various</td>
			<td></td>
			<td style="width:251px;">S media consists of S basal solution (1.46 g NaCl, 0.25g K2HPO4, 1.5 g KH2PO4, 0.25 ml of 5 mg/ml cholesterol in ethanol and H2O to 250 mL. Autoclaved.) To the S basal solution add 2.5 mL of potassium citrate pH 6.0 (2 g citric acid monohydrate, 29.4 g tri-potassium citrate monohydrate, H2O to 100 mL, autoclaved), 2.5 mL of Trace Metals Solution (1.86 g disodium EDTA, 0.69 g FeSO4&#160;&#8226;7 H2O, 0.2 g MnCl2&#8226;4 H2O, 0.29 g ZnSO4&#160;&#8226;7 H2O, 0.025 g CuSO4&#160;&#8226;5 H2O, H2O to 1 liter. Autoclaved and stored in the dark), 0.75 mL of 1 M CaCl2, and 0.75 mL of 1 M MgSO4.</td>
		</tr>
		<tr height="392">
			<td height="392" style="height:392px;width:259px;">Dauer pheromone plates for dose-response assay</td>
			<td style="width:152px;">Various</td>
			<td></td>
			<td style="width:251px;">Mix 0.085 g Noble agar, 0.5 mL of 0.513 M NaCl and 5 &#181;L of 5 mg/ml cholesterol dissolved in ethanol in each of 6x 15 mL culture tubes. Add sufficient crude pheromone to each tube to create a range of concentrations. Add H2O to bring the volume to 4.725 mL.&#160; Boil over a Bunsen burner until the agar is dissolved and then cool to 55 &#176;C. Add 125 &#181;L 1 M KPO4 buffer (pH 6.0), 50 &#181;l 0.1 M CaCl2, 50 &#181;L 0.1 M MgSO4 and 50 &#181;l of 5 mg/ml streptomycin sulfate to each tube.&#160; Pour the contents of each tube into a 35 mm diameter Petri dish. Let the agar solidify at room temperature overnight (if longer, store in enclosed container to prevent excessive drying).</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microsphere beads 0.1 micron</td>
			<td style="width:152px;">Polysciences Inc.</td>
			<td>00876-15</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;">M9 buffer</td>
			<td style="width:152px;">Various</td>
			<td></td>
			<td style="width:251px;">Mix 3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 liter. Sterilize by autoclaving</td>
		</tr>
	</tbody>
</table>

in vivo imaging of dauer-specific neuronal remodeling in c. elegans

Hayvanlarda stres ile indüklenen fenotipik esneklik kontrol mekanizmaları incelemek için in vivo olarak çoğunlukla kompleks ve zordur. Stres ile indüklenen plastisite klasik bir örneği, C Dauer aşamasıdır elegans. Dauers bakteriyel gıda ve Dauer feromon yüksek konsantrasyonlarının düşük konsantrasyonlarda koşulları altında oluşan alternatif bir gelişim larva bulunmaktadır. Dauers kapsamlı gelişimsel ve davranışsal plastisite gösterir. Örneğin, dört iç dudak kadran bir dizi (IL2Q) nöronları Dauer oluşumu sırasında Reverzibl remodeling tabi tutulur. Büyük ölçekli sıvı nematod kültürlerinden ham Dauer feromonunun üretimi için iyi bilinen çevre yollarını düzenlemeye Dauer girişi, önceden belirlenmiş bir yöntem kullanılarak gösterilmiştir. Bu yöntemle, 50,000 &#39;lik bir konsantrasyon - sıvı kültürü 75,000 nematod / ml bir yüksek ölçüde kuvvetli ham Dauer feromon üretmek için yeterlidir. Ham feromon gücü olan deteBir doz-tepki biyo-tahlil ile rmined. Son olarak, Dauer oluşumu sırasında IL2Qs in vivo zaman atlamalı görüntüleme için kullanılan yöntemler tarif edilmiştir.

Isı, soğuk ve stabil olarak sarı bir sıvı ham Dauer feromon sonuçları (Şekil 1) üretimi. Ham feromon alikotlar aktivitesinde belirgin bir kayıp olmadan, süresiz olarak -20 ° C&#39;de saklanır. Feromonun üretiminin ardından, tekli bir doz-yanıt potens biyolojik tayin, kaba bir tahmin için yeterlidir. 3 kez ve her deneyde bir ortalamasını alın - Bununla birlikte, çoğu deneyleri için bu biyo-analizi tekrar 2 için gereklidir. Iki feromon biyoanalizlere Temsilcisi sonuçları ...

Watch this Scientific Journal Video about In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans at JoVE.com

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 ...

in-vivo-imaging-of-dauer-specific-neuronal-remodeling-in-c-elegans

Research

JoVE Journal

Neuroscience

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

11.2K Görüntüleme.  University of Illinois Urbana-Champaign. Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant ‘dauer’ juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer. 

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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Fare Retina Ganglion Hücreleri Transfeksiyon tarafından In vivo Elektroporasyon

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Nörobilim

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 ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Akut Hipokampal Dilimler içinde Synaptically-uyarılmış astroglial ve Nöronal Cevaplarının İkili Elektrofizyolojik Kayıtlar

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

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, ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Glutamat ve GABA hızlı Micro-iyontoforez: Synaptic Entegrasyon Araştırma yararlı bir araç

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Super-resolution Imaging of Neuronal Dense-core Vesicles

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and medical applications. Strengths of fluorescence include its sensitivity, specificity, and compatibility with live imaging. Unfortunately, conventional forms of fluorescence microscopy suffer from one major weakness, diffraction-limited resolution in the imaging plane, which hampers studies of structures with dimensions smaller than ~250 nm. Recently, this limitation has been overcome with the introduction of super-resolution fluorescence microscopy techniques, such as photoactivated localization microscopy (PALM). Unlike its conventional counterparts, PALM can produce images with a lateral resolution of tens of nanometers. It is thus now possible to use fluorescence, with its myriad strengths, to elucidate a spectrum of previously inaccessible attributes of cellular structure and organization.
Unfortunately, PALM is not trivial to implement, and successful strategies often must be tailored to the type of system under study. In this article, we show how to implement single-color PALM studies of vesicular structures in fixed, cultured neurons. PALM is ideally suited to the study of vesicles, which have dimensions that typically range from ~50-250 nm. Key steps in our approach include labeling neurons with photoconvertible (green to red) chimeras of vesicle cargo, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a high-resolution PALM image. We also demonstrate the efficacy of our approach by presenting exceptionally well-resolved images of dense-core vesicles (DCVs) in cultured hippocampal neurons, which refute the hypothesis that extrasynaptic trafficking of DCVs is mediated largely by DCV clusters.

We describe how to implement photoactivated localization microscopy (PALM)-based studies of vesicles in fixed, cultured neurons. Key components of our protocol include labeling vesicles with photoconvertible chimeras, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a super-resolution image.

Nöronal Yoğun çekirdekli Keseciklerinde süper çözünürlüklü Görüntüleme

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

Kombine Metodolojileri kullanan Akson Dallanman ve Nöronal Morfogenez sırasında Plazma Membran Teslimat Rolü tanımlayın

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its completely mapped nervous system and transparent anatomy, presents an ideal model for understanding real-time neural dynamics using calcium indicators. In combination with microfluidic technologies and experimental designs, calcium-imaging studies using these indicators are performed in both free-moving and trapped animals. However, most previous studies utilizing trapping devices, such as the olfactory chip described in Chronis et al., have devices designed for use in the more common hermaphrodite, as the less common male is both morphologically and structurally dissimilar. An adapted olfactory chip was designed and fabricated for increased efficiency in male neuronal imaging with using young adult animals. A turn was incorporated into the worm loading port to rotate the animals and to allow for the separation of the individual neurons within a bilateral pair in 2D imaging. Worms are exposed to a controlled flow of odorant within the microfluidic device, as described in previous hermaphrodite studies. Calcium transients are then analyzed using the open-source software ImageJ. The procedure described herein should allow for an increased amount of male-based C. elegans calcium imaging studies, deepening our understanding of the mechanisms of sex-specific neuronal signaling.

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of an adapted &#34;olfactory chip&#34; for the efficient calcium imaging of C. elegans males is described here. Studies of male exposure to glycerol and a pheromone are also shown.

Yanıt olarak erkek C. Elegans baş nöronlar Feromonlar nöronal aktivite görüntüleme için adapte mikrosıvısal koku kozu kullanımı

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its ...

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease across species. Although these neurotoxic challenges have long been considered to be cell-intrinsic, considerable evidence now supports that misfolded human disease proteins originating in one neuron can appear in neighboring cells, a phenomenon proposed to promote pathology spread in human neurodegenerative disease.
C. elegans adult neurons that express aggregating proteins can extrude large (~4 &#181;m) membrane-surrounded vesicles that can include the aggregated protein, mitochondria, and lysosomes. These large vesicles are called &#8220;exophers&#8221; and are distinct from exosomes (which are about 100x smaller and have different biogenesis). Throwing out cellular debris in exophers may occur by a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, relevant to processes by which aggregates spread in human neurodegenerative diseases.
While exophers have been mostly studied in animals that express high copy transgenic mCherry within touch neurons, these protocols are equally useful in the study of exophergenesis using fluorescently tagged organelles or other proteins of interest in various classes of neurons.
Described here are the physical features of C. elegans exophers, strategies for their detection, identification criteria, optimal timing for quantitation, and animal growth protocols that control for stresses that can modulate exopher production levels. Together, details of protocols outlined here should serve to establish a standard for quantitative analysis of exophers across laboratories. This document seeks to serve as a resource in the field for laboratories seeking to elaborate molecular mechanisms by which exophers are produced and by which exophers are reacted to by neighboring and distant cells.

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

This protocol describes approaches for detection and quantitation of large aggregate and/or organelle extrusions (~4 &#181;m) produced by C. elegans cells in the form of membrane-bound exophers. We describe strains, growth conditions, scoring criteria, timing, and microscopy considerations needed to facilitate dissection of this debris expulsion mechanism.