Name: assessment of dictyostelium discoideum response to acute mechanical stimulation
Uploaded: 2017-11-09T12:30:00.000+00:00
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	<li>Swaney, K. F., Huang, C. H., Devreotes, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eukaryotic+chemotaxis:+a+network+of+signaling+pathways+controls+motility,+directional+sensing,+and+polarity.">Eukaryotic chemotaxis: a network of signaling pathways controls motility, directional sensing, and polarity.</a> Annu Rev Biophys. 39, 265-289 (2010).</li><li>Sasaki, A. T., Chun, C., Takeda, K., Firtel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+Ras+signaling+at+the+leading+edge+regulates+PI3K,+cell+polarity,+and+directional+cell+movement.">Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement.</a> J Cell Biol. 167 (3), 505-518 (2004).</li><li>Jeon, T. J., Lee, D. -. J., Merlot, S., Weeks, G., Firtel, R. 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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moving+towards+a+paradigm:+common+mechanisms+of+chemotactic+signaling+in+Dictyostelium+and+mammalian+leukocytes.">Moving towards a paradigm: common mechanisms of chemotactic signaling in Dictyostelium and mammalian leukocytes.</a> Cell Mol Life Sci. 71 (19), 3711-3747 (2014).</li><li>Dalous, J., 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=Reversal+of+cell+polarity+and+actin-myosin+cytoskeleton+reorganization+under+mechanical+and+chemical+stimulation.">Reversal of cell polarity and actin-myosin cytoskeleton reorganization under mechanical and chemical stimulation.</a> Biophys J. 94 (3), 1063-1074 (2008).</li><li>Sato, M. J., 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=Switching+direction+in+electric-signal-induced+cell+migration+by+cyclic+guanosine+monophosphate+and+phosphatidylinositol+signaling.">Switching direction in electric-signal-induced cell migration by cyclic guanosine monophosphate and phosphatidylinositol signaling.</a> Proc Natl Acad Sci U S A. 106 (16), 6667-6672 (2009).</li><li>Zhao, M., Pu, J., Forrester, J. V., McCaig, C. 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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+and+mechanical+stimuli+act+on+common+signal+transduction+and+cytoskeletal+networks.">Chemical and mechanical stimuli act on common signal transduction and cytoskeletal networks.</a> Proc Natl Acad Sci U S A. 113 (47), E7500-E7509 (2016).</li><li>Artemenko, Y., Swaney, K. F., Devreotes, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+development+and+chemotaxis+in+Dictyostelium+discoideum+mutants.">Assessment of development and chemotaxis in Dictyostelium discoideum mutants.</a> Methods Mol Biol. 769, 287-309 (2011).</li><li>Gaudet, P., Pilcher, K. E., Fey, P., Chisholm, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+Dictyostelium+discoideum+with+plasmid+DNA.">Transformation of Dictyostelium discoideum with plasmid DNA.</a> Nat. Protocols. 2 (6), 1317-1324 (2007).</li><li>Veltman, D. M., Lemieux, M. G., Knecht, D. A., Insall, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PIP3-dependent+macropinocytosis+is+incompatible+with+chemotaxis.">PIP3-dependent macropinocytosis is incompatible with chemotaxis.</a> J Cell Biol. 204 (4), 497-505 (2014).</li><li>Cai, H., Huang, C. H., Devreotes, P. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caffeine+blocks+activation+of+cyclic+AMP+synthesis+in+Dictyostelium+discoideum.">Caffeine blocks activation of cyclic AMP synthesis in Dictyostelium discoideum.</a> Dev Biol. 101 (1), 136-146 (1984).</li><li>Bretschneider, T., 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=Dynamic+Actin+Patterns+and+Arp2/3+Assembly+at+the+Substrate-Attached+Surface+of+Motile+Cells.">Dynamic Actin Patterns and Arp2/3 Assembly at the Substrate-Attached Surface of Motile Cells.</a> Curr Biol. 14 (1), 1-10 (2004).</li><li>Swaney, K. F., Borleis, J., Iglesias, P. A., Devreotes, P. 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Yöntem tanımlamak burada nüfus ve tek hücre davranış yanıt akışı yamultmak için değerlendirmek için kullanışlı bir yol sunar. Önceki çalışmalarda lider ve kenar işaretleri geçiş sırasında geri kalmış yerelleştirme analiz ederken, önemlisi, güncel yaklaşımlar ilk etkisi soruşturma akut kesme akışı uygulayarak sağlar. Bu yöntemi kullanarak aslında, ilk yanıt akışı yamultmak için hücre hücre geçiş gerektirmez, gösterdi. Bunun yerine, ilk kesme akışı uyarana hızlı ve geç...

Geçiş ökaryotik hücre ortamında gradyanlar çözünür veya yüzey bağlı chemoattractants, değişken sertliği yüzeylerde, elektrik alanları veya yamultma akışı dahil olmak üzere çeşitli kimyasal ve fiziksel ipuçları tarafından önyargılı. Moleküler mekanizmalar kemotaksisi sürüş bizim anlamada birçok gelişmeler rağmen daha az yönlendirilmiş göç ve nasıl bu farklı sinyaller Birleşik bir üretmek için hücresel düzeyde entegre edilmiştir diğer türleri hakkında bilinen göçmen yanıt.
Geçiş doğru bir chemoattractant artan bir konsantrasyon içerir üç davranış bileşenleri Yönetmen: hareketliliği, yönlü algılama ve polarite1. Motilite pseudopod çıkıntı tarafından elde hücrelerin rastgele hareket anlamına gelir. Yönlü bir hücre bir chemoattractant kaynağını tespit yeteneği algılama, hangi hatta oluşabilir hücrelerin immobilize. Polarite hücre içi bileşenler arasında önde gelen ve artan sebat hareketine yol açan bir hücrenin, ahşap kaplama kenarı daha istikrarlı asimetrik dağılımı gösterir.
Bir chemoattractant hücresel yanıt dört kavramsal olarak tanımlanmış düzenleyici ağları faaliyete bağlıdır: reseptör / G protein, sinyal iletimi, aktin sitoiskeleti ve polarite1. G protein birleştiğinde reseptör Chemoattractant bağlama heterotrimeric G protein α ve βγ yönlü sinyali güçlendirir aşağı akım sinyal iletim ağ üzerinden sinyal iletir. Birden çok sinyal iletim ağı içinde paralel olarak hareket ve önyargı aktin polimerizasyon ve bunun sonucunda pseudopod çıkıntı, degradenin yönünü aktin sitoiskeleti ağa beslenir. Ras GTPazlar, TorC2, fosfoinositid 3-kinaz (PI3K), fosfataz ve tensin homolog (PTEN) ve guanylyl cyclase kemotaksisi önemli düzenleyiciler arasında vardır. Geri bildirim mekanizmaları sinyal iletim ağı içinde ve arasında sinyal iletimi ve aktin sitoiskeleti ağları daha fazla yanıt yükseltmek. Son olarak, kötü tanımlanmış polarite ağ giriş aktin sitoiskeleti alır ve daha fazla kalıcı degradenin yönünü geçişinde yaymak için sinyal iletim şebeke önyargıları.
Mekanik anlayışımızı kemotaksisi çoğunu fluorescently öğesini biyosensörler düzenleyici ağların çeşitli bileşenleri için gelişimi nedeniyle mümkün yapıldı. Birçok kemotaksisi düzenleyiciler asimetrik bir dağıtım düzenleyici molekül veya faaliyet var. Örneğin, tanımak biyosensörler harekete geçirmek yorum-in küçük GTPases Ras ve Rap1-Raf1 (RBD burada anılacaktır) Ras bağlayıcı etki ve RalGDS, sırasıyla-öncü bir chemotaxing hücre2,3yerelleştirilmesine. Benzer şekilde, PI3K ve onun ürün phosphatidylinositol (3,4,5)-trisphosphate (PIP3), pleckstrin homoloji (PH) etki alanı tarafından tanınan da göstermek yerelleştirme ön cep4,5. Buna ek olarak, bir 3-fosfataz PTEN hangi dönüştürür PIP3 geri phosphatidylinositol (4,5)-bisphosphate, hücre6ahşap kaplama kenarı yerelleştirir. Önemlisi, bu biyosensörler onların yerelleştirme yanıt küresel stimülasyon ile bir chemoattractant olarak değiştirin. Sitozolik ya da çıkıntılar resting hücredeki ipucu, kortikal Yerelleştirme ve bulunmadığına kenar işaretleri geri kalmış ise korteks için relocalize öncü işaretleri çıkıntılar resting hücrede ipuçları, sonra sitozolik haline uyarım. Biyoalgılayıcı dağıtım yanıt olarak bir chemoattractant ile küresel stimülasyon analizi katkı kez gözlemleri yıkmak hareketliliği ve polarite, en aza indirir. Bir chemoattractant ile bir hücre süspansiyon genel veya üniforma uyarılması da bir araç olarak protein harekete geçirmek, genellikle protein fosforilasyon7,8,9 tarafından algılanan nüfusu genelindeki değişiklikleri değerlendirmek için kullanılan . Mikroskopi düzenleyici ağların çeşitli bileşenlerinin davranışı hakkında zamansal ve mekansal bilgi toplamak için kullanılan, ancak bu biyokimyasal tahlil öncelikle geçici bilgi, elde etmek için kullanılır.
Sinyal iletim ağ bir telaşlı sistem10,11özellikleri içerir. Supra-eşik kemotaktik uyaranlara yanıt "yaparak" ve ateşe dayanıklı dönemleri görüntülemek. Yanıt-e doğru da stochastically tetiklenir ve salınım davranış gösterebilir. Sinyal iletim olaylar dalgalar12,13,14,15yaymak bölgelere korteksin lokalizedir. Ön ya da geri, işaretçileri için işe veya aktif bölgelerinden yayılıyor dalgaların, ayırmak. Onlar karşılamak üzere active bölge izleyen ateşe dayanıklı bölge nedeniyle karşılıklı yönlendirilmiş dalgalar yok edin. Yayılıyor sinyal iletim dalgalar altında yatan hücre göç10arabuluculuk hücresel çıkıntılar.
Benzer düzenleyici mekanizmaları da nötrofil ve diğer memeli hücre türleri16 uygulanabilir olmasına karşın kemotaksisi olarak yukarıda belirtilen bilgilerin çoğunu sosyal amip Dictyostelium discoideum, iyice düşünülmüş--dan geldi . Dictyostelium tek hücreleri binlerce sonunda sporları içeren çok hücreli bir meyve vücut şekillendirme bir toplama merkezine doğru geçirirken, sırasında şiddetli açlık, güçlü kemotaktik tepki süresine sahip bir iyi kurulmuş model organizmadır. Bakteriyel gıda kaynakları bulmak için bu organizmanın tek hücre büyüme aşamasında kemotaksisi esastır. Önemlisi, geçiş tek Dictyostelium hücre memeli nötrofiller veya metastatik kanser hücrelerinin, Bütün bunlar çok hızlı protozlar türü göç geçmesi geçiş oldukça benzer. Aslında, düzenleyici ağların genel topoloji, hem çok bireysel sinyal iletim yollarının kemotaksisi dahil korunmuş Dictyostelium ve memeli lökosit17arasında. Ayrıca, fibroblastlar gibi diğer hücre reseptör tirozin kinaz (RTK) yerine GPCRs kullanın; Ancak, RTKs-ebilmek beslemek benzer ağlar.
Kemotaksis aksine, yönlendirilmiş göç çeşitli modları sürücü sinyal mekanizmaları konusunda eksiksiz bilgi bulunmuyor. Benzer şekilde chemoattractant Gradyanda geçiş hücreleri için çeşitli çalışmalarda harekete geçirmek ve/veya yerelleştirme tipik öncü işaretleri, aktin polimerizasyonu, PIP3 ve/veya hücre dışı sinyal düzenlenir kinaz (ERK) 1/2, dahil olmak üzere, bildirdin Açık geçiren hücre göç akışı veya elektrik alanları18,19,20,21değişimler yamultmak için yanıt olarak yönetti. Ancak, bu çalışmalarda uyarıcı sürekli maruz Ayrıca hücre göç, örneğin, bir uyarana tepki olarak özellikle öncü işaretleri yerelleştirilmesine, veya sadece öncü yerelleştirmek soru açık bırakarak sonuçlandı pseudopods ön geçirme hücre sayısının artmasına yüzünden.
Hücreleri cevaben kesme akışı hem düzeyinde nüfus ve tek tek hücreler22olarak teslim akut mekanik pertürbasyon gözlemlemek için bize izin deneyleri geliştirdik. Benzer bir chemoattractant, akut yaptığı ile küresel stimülasyonTion kesme akışı ile hareketliliği veya polarite mekanik bir uyarana karıştırıcı katkısı olmadan bir hücresel yanıt çalışma sağlar. Bu biyokimyasal ve mikroskobik deneyleri genetik veya farmakolojik tedirginlikler ile birleştirerek hakkında nasıl mekanik uyaranlara algılandığını öğrenmek için bize izin verir ve iletilen. Ayrıca, bu yaklaşım da yokluğunda böylece sinyal iletim ve aktin sitoiskeleti ağları reseptör/g izole eden bir chemoattractant, chemoattractant reseptör sistemi aşağı faydalanarak için yeni bir yöntem sağlar protein ağ.
Son zamanlarda biz açıklanan teknikleri kullanarak akut kesme stres kemotaktik sinyal iletim ve aktin sitoiskeleti ağlar22birden çok bileşeni etkinleştirme için yol gösterdi. Akut mekanik uyarıcı değişen aralıklarla uygulayarak, biz aynı şekilde chemoattractants için mekanik bizi canlı tutan da altında yanıt yaparak davranışını da dahil olmak üzere bir telaşlı sisteminin özelliklerini sergileyen, gösterdi koşullar ve ateşe dayanıklı bir süre varlığı doyurarak. Son olarak, mekanik ve kimyasal uyarımı birleştirerek iki uyaranlara büyük olasılıkla birden çok uyaranlara karşı önyargı hücre göç entegrasyon için izin sinyal iletim ve aktin sitoiskeleti ağlar, paylaşmak gösterdi.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dextrose</td><td>Fisher</td><td>D16-1</td><td>Use to prepare HL-5 media and SM plates (steps 1.1, 1.2)</td></tr><tr><td>Proteose peptone</td><td>Fisher</td><td>DF0120-17-6</td><td>Use to prepare HL-5 media (step 1.1)</td></tr><tr><td>Yeast extract</td><td>Fisher</td><td>50-550-445</td><td>Use to prepare HL-5 media and SM plates (steps 1.1, 1.2)</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher</td><td>S373-500</td><td>Use to prepare HL-5 media, and 10X Phosphate Buffer (steps 1.1, 1.3)</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>P386-500</td><td>Use to prepare HL-5 media, SM plates, and 10X Phosphate Buffer (steps 1.1-1.3)</td></tr><tr><td>Streptomycin (dihydrostreptomycin sulfate)</td><td>Fisher</td><td>ICN10040525</td><td>Use to prepare HL-5 media (step 1.1)</td></tr><tr><td>Peptone (Bacto)</td><td>Fisher</td><td>DF0118-17-0</td><td>Use to prepare SM plates (step 1.2)</td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>P288-100</td><td>Use to prepare SM plates (step 1.2)</td></tr><tr><td>Agar</td><td>Fisher</td><td>BP1423-500</td><td>Use to prepare SM plates (step 1.2)</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>M65-500</td><td>Use to prepare DB Buffer (step 1.4)</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher</td><td>C79-500</td><td>Use to prepare DB Buffer (step 1.4)</td></tr><tr><td>Caffeine</td><td>Fisher</td><td>O1728-500</td><td>Use to prepare caffeine (step 1.5)</td></tr><tr><td>cAMP (EMD Millipore Calbiochem Adenosine 3 ft.,5 ft.-cyclic Monophosphate, Sodium Salt)</td><td>Fisher</td><td>11-680-1100MG</td><td>Use to prepare cAMP (step 1.6)</td></tr><tr><td>Folic acid</td><td>Sigma</td><td>F7876</td><td>Use to prepare folic acid (step 1.7)</td></tr><tr><td>Tris base</td><td>Fisher</td><td>BP152-500</td><td>Use to prepare 3X sample buffer (step 1.8)</td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>Fisher</td><td>BP166-500</td><td>Use to prepare 3X sample buffer (step 1.8)</td></tr><tr><td>Glycerol</td><td>Fisher</td><td>G33-500</td><td>Use to prepare 3X sample buffer (step 1.8)</td></tr><tr><td>Dithiothreitol (DTT)</td><td>Bio-Rad</td><td>1610610</td><td>Use to prepare 3X sample buffer (step 1.8)</td></tr><tr><td>Bromophenol blue</td><td>Bio-Rad</td><td>1610404</td><td>Use to prepare 3X sample buffer (step 1.8)</td></tr><tr><td>NaF</td><td>Fisher</td><td>S299-100</td><td>Use to prepare sample buffer with protease and phosphatase inhibitors (step 1.8)</td></tr><tr><td>Na&lt;sub&gt;3&lt;/sub&gt;VO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>50-994-911</td><td>Use to prepare sample buffer with protease and phosphatase inhibitors (step 1.8)</td></tr><tr><td>Sodium pyrophosphate decahydrate</td><td>Fisher</td><td>S390-500</td><td>Use to prepare sample buffer with protease and phosphatase inhibitors (step 1.8)</td></tr><tr><td>Complete EDTA-free protease inhibitor cocktail (Roche)</td><td>Sigma</td><td>11873580001</td><td>Use to prepare sample buffer with protease and phosphatase inhibitors (step 1.8)</td></tr><tr><td>Latrunculin A</td><td>Enzo Life Sciences</td><td>BML-T119-0100</td><td>Use to prepare Latrunculin A (step 1.9)</td></tr><tr><td>4-15% Tris-HCl polyacrylamide gel</td><td>Bio-Rad</td><td>3450029</td><td>Use for immunoblotting (step 3.3)</td></tr><tr><td>Polyvinylidene fluoride membrane</td><td>Bio-Rad</td><td>1620177</td><td>Use for immunoblotting (step 3.3)</td></tr><tr><td>Phospho-PKC (pan) (zeta Thr410) (190D10) Rabbit antibody</td><td>Cell Signaling</td><td>2060</td><td>Primary antibody for use in immunoblotting (step 3.3)</td></tr><tr><td>Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP antibody</td><td>Cell Signaling</td><td>4370</td><td>Primary antibody for use in immunoblotting (step 3.3)</td></tr><tr><td>Rabbit IgG HRP Linked Whole Ab (from Donkey)</td><td>GE Healthcare</td><td>NA934-100UL</td><td>Secondary antibody for use in immunoblotting (step 3.3)</td></tr><tr><td>Enhanced chemiluminescence substrate (Clarity Western ECL Substrate)</td><td>Bio-Rad</td><td>1705060</td><td>Use for immunoblotting (step 3.3)</td></tr><tr><td>Stripping buffer (Restore Plus Western blot stripping buffer)</td><td>Pierce</td><td>PI46430</td><td>Use for immunoblotting (step 3.3)</td></tr><tr><td>Coomassie Brilliant Blue</td><td>Fisher</td><td>PI20278</td><td>Use for immunoblotting (step 3.3)</td></tr><tr><td>K. aerogenes strain (non-pathogenic)</td><td>Dicty Stock Center (dictybase.org)</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Materials and Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Orbital shaker (model G-33, or a comparable alternative)</td><td>New Brunswick Scientific</td><td></td><td>Use to grow &lt;em&gt;K. aerogenes&lt;/em&gt; (step 2.1.1), and to basalate and stimulate &lt;em&gt;D. discoideum&lt;/em&gt; cells (step 3). The radius of gyration for this shaker is 9.5 mm.</td></tr><tr><td>10 cm Petri dish</td><td>Fisher</td><td>FB0875712</td><td>Use to prepare SM plates (step 1.2)</td></tr><tr><td>Hemocytometer</td><td>Fisher</td><td>02-671-51B</td><td>Use to count &lt;em&gt;D. discoideum&lt;/em&gt; cells (step 2.1.2)</td></tr><tr><td>35 mm dish (Corning Falcon Easy-Grip Tissue Culture Dish)</td><td>Fisher</td><td>08-772A</td><td>Use to plate &lt;em&gt;D. discoideum&lt;/em&gt;cells for mechanical stimulation followed by cell lysis (step 3.1.1)</td></tr><tr><td>Polystyrene cup (5 mL)</td><td>VWR</td><td>13915-985</td><td>Use to stimulate &lt;em&gt;D. discoideum&lt;/em&gt; cell suspension with a chemoattractant (step 3.2.2)</td></tr><tr><td>Fluidic unit with pump (ibidi Pump System)</td><td>Ibidi</td><td>10902</td><td>Use to deliver mechanical stimulation to &lt;em&gt;D. discoideum&lt;/em&gt; cells in a channel (steps 4.1, 4.2, and 5)</td></tr><tr><td>Slide with a channel (&amp;mu;-Slide III 3in1 ibiTreat: #1.5 polymer coverslip, tissue culture treated, sterilized)</td><td>Ibidi</td><td>80316</td><td>Use to plate &lt;em&gt;D. discoideum&lt;/em&gt; cells for mechanical stimulation delivered by a fluidic device (steps 4.1, 4.2, and 5)</td></tr><tr><td>50 mL reservoirs (Ibidi Reservoir Holder for Fluidic Unit)</td><td>Ibidi</td><td>10978</td><td>Use to setup the fluidic device for delivering mechanical stimulation to &lt;em&gt;D. discoideum&lt;/em&gt; cells in a channel (steps 4.1, 4.2, and 5)</td></tr><tr><td>Lines for the fluidic unit (Perfusion Set YELLOW-and-GREEN)</td><td>Ibidi</td><td>10964</td><td>Use to setup the fluidic device for delivering mechanical stimulation to &lt;em&gt;D. discoideum&lt;/em&gt; cells in a channel (steps 4.1, 4.2, and 5). Note: the two lines can also be cut from the 1.6 mm tubing (catalog number 10842).</td></tr><tr><td>Line for the drain (1.6 mm ID tubing)</td><td>Ibidi</td><td>10842</td><td>Use to setup the fluidic device for delivering mechanical stimulation to &lt;em&gt;D. discoideum&lt;/em&gt; cells in a channel (steps 4.1, 4.2, and 5).</td></tr><tr><td>Zeiss Observer.Z1 inverted microscope equipped with a 40X/1.3 oil objective and a 20X/0.3 air objective (or a comparable alternative)</td><td>Zeiss</td><td></td><td>Used to image &lt;em&gt;D. discoideum&lt;/em&gt; cells with phase-contrast or fluorescence illumination (steps 4 and 5)</td></tr><tr><td>UltraView spinning disk confocal microscope equipped with a 40&amp;times;/1.25&amp;ndash;0.75 oil objective (or a comparable alternative)</td><td>Perkin-Elmer</td><td>DM 16000</td><td>An alternative used to image &lt;em&gt;D. discoideum&lt;/em&gt; cells (step 4.1.4)</td></tr><tr><td>FemtoJet Microinjector (Eppendorf; model 5247, or a comparable alternative) with Eppendorf InjectMan</td><td>Eppendorf</td><td>5252000021D and 5192000027</td><td>Use to deliver mechanical stimulation to &lt;em&gt;D. discoideum&lt;/em&gt; cells by a micropipette (step 4.3). Note: catalog number are for the newest version of the equipment (4i).</td></tr><tr><td>Micropipette (Femtotip; 1 &amp;mu;m outside diameter, 0.5 &amp;mu;m inside diameter )</td><td>Eppendorf</td><td>930000035</td><td>Use to deliver mechanical stimulation to &lt;em&gt;D. discoideum&lt;/em&gt; cells by a micropipette (step 4.3)</td></tr><tr><td>Microloader tips (Eppendorf Femtotips Microloader Tips for Femtojet Microinjector)</td><td>Eppendorf</td><td>5242956.003</td><td>Use to fill the micropipette (femtotip) with buffer (step 4.3.1)</td></tr><tr><td>1-well chamber (Thermo Scientific Nunc Lab-Tek Chambered Coverglass)</td><td>Fisher</td><td>12-565-472</td><td>Use to plate &lt;em&gt;D. discoideum&lt;/em&gt; cells for mechanical stimulation delivered by a micropipette (step 4.3.2)</td></tr><tr><td>8-well chamber (Thermo Scientific Nunc Lab-Tek II Chambered Coverglass)</td><td>Fisher</td><td>12-565-338</td><td>Use to plate &lt;em&gt;D. discoideum&lt;/em&gt; cells for mechanical stimulation delivered by bulk buffer addition (step 4.4.1)</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Image Analysis Software (Fiji)</td><td>NIH</td><td>https://fiji.sc/</td><td>Use to quantify response to mechanical stimulation (step 4.5)</td></tr><tr><td>Migration analysis software (Tracking Tool PRO)</td><td>Gradientech</td><td>http://gradientech.se/tracking-tool-pro/</td><td>Use to quantify cell speed and persistence (step 5.4)</td></tr></tbody></table>

assessment of dictyostelium discoideum response to acute mechanical stimulation

Kemotaksis, ya da bir chemoattractant kadar Degrade geçiş en iyi anlaşılmış, yönlendirilmiş geçiş modudur. Sosyal amip kullanarak çalışmalar ortaya Dictyostelium discoideum paralel yollar karmaşık sinyal iletim ağı chemoattractants cevaben güçlendirir ve önyargılı aktin polimerizasyon ve çıkıntı bir pseudopod yol açar degradenin yönünü. Buna ek olarak, diğer türleri, örneğin, yönlendirilmiş göç akışı veya elektrik alanları, yamultmak için maruz kalma nedeniyle sürüş moleküler mekanizmaları bilinmemektedir. Kemotaksis birçok düzenleyiciler, yerelleştirme ya da harekete geçirmek bir chemoattractant ile küresel stimülasyon sonrasında geçici değişiklikler gösterir yanı sıra, yerelleştirme önde gelen veya geçirme bir hücrenin ahşap kaplama kenarı sergilemek. Moleküler mekanizmalar yönlendirilmiş göç diğer türleri anlamak için hücresel yanıt-e doğru akış yamultmak için kısa (2-5 s) pozlama dayalı akut mekanik stimülasyon incelenmesi sağlayan bir yöntem geliştirdi. Bu uyarımı hücreleri tek hücre davranışı incelemek için biyosensörler fluorescently etiketli ifade görüntüleme sırasında bir kanal olarak teslim edilebilir. Ayrıca, hücre nüfus bir tabak, lysed ve immunoblotted ilgi proteinlerin etkin versiyonlar tanımak antikorları kullanarak teşvik. Her iki deneyleri birleştirerek, bir moleküller hücre altı Yerelleştirme ve/veya fosforilasyon değişikliklerden aktif geniş bir dizi inceleyebilirsiniz. Bu yöntemle akut mekanik stimülasyon kemotaktik sinyal iletim ve aktin sitoiskeleti ağları aktivasyonu tetikler belirledi. Başlatan olaylar kesme akış kaynaklı hareketliliği için gerekli anlamak için önemli hücresel yanıt akut mekanik uyarılması için incelemek için yeteneğidir. Bu yaklaşım da kemotaktik sinyal iletim ağ chemoattractant reseptör karıştırıcı etkisi olmadan çalışmak için bir aracı sağlar.

Zamansal kemotaksisi düzenleyiciler cevaben akut mekanik stimülasyon
D. discoideum hücre mekanik stimülasyon yanıtı değerlendirmek için yapisan toplama yetkili hücreleri için kesme akımı nabız kısa maruz bırakıldı. Toplama yetkili D. discoideum hücreler hücreleri komşu tarafından hissedilen kampı salgılar. Hücre-hücre sinyallemesi katkı üstesinden gelmek için hücreleri gozlemler cyclas...

Watch this Scientific Journal Video about Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation at JoVE.com

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Dictyostelium discoideum yanıt-e doğru akut mekanik stimülasyon değerlendirilmesi

Burada akut mekanik stimülasyon hücresel yanıt değerlendirmek için yöntemleri açıklanmaktadır. Mikroskopi tabanlı tahlil fluorescently etiketli biyosensörler kesme akışı ile kısa stimülasyon takip yerelleştirilmesini inceleyin. Ayrıca akut mekanik stimülasyon karşılık olarak ilgi çeşitli proteinlerin aktivasyonu biyokimyasal olarak test ediyoruz.

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

assessment-dictyostelium-discoideum-response-to-acute-mechanical

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

6.9K Görüntüleme.  SUNY Oswego. Burada akut mekanik stimülasyon hücresel yanıt değerlendirmek için yöntemleri açıklanmaktadır. Mikroskopi tabanlı tahlil fluorescently etiketli biyosensörler kesme akışı ile kısa stimülasyon takip yerelleştirilmesini inceleyin. Ayrıca akut mekanik stimülasyon karşılık olarak ilgi çeşitli proteinlerin aktivasyonu biyokimyasal olarak test ediyoruz.

Video: Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

Sıçanlarda Spinal Kord Yaralanma sonrası Akut ve Kronik Dokunsal Duyu Testi

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

Tıp

Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of the population1,2. Symptoms include spontaneous (tingling, burning, electric-shock like) pain, dysaesthesia, paraesthesia, allodynia (pain resulting from normally non-painful stimuli) and hyperalgesia (an increased response to painful stimuli). The sensory symptoms are co-morbid with behavioural disabilities, such as insomnia and depression. To study chronic neuropathic pain several animal models mimicking peripheral nerve injury have been developed, one of the most widely used is Bennett and Xie's (1988) unilateral sciatic nerve chronic constriction injury (CCI)3 (Figure 1). Here we present a method for performing CCI and testing pain hypersensitivity.
CCI is performed under anaesthesia, with the sciatic nerve on one side exposed by making a skin incision, and cutting through the connective tissue between the gluteus superficialis and biceps femoris muscles. Four chromic gut ligatures are tied loosely around the sciatic nerve at 1 mm intervals, to just occlude but not arrest epineural blood flow. The wound is closed with sutures in the muscle and staples in the skin. The animal is then allowed to recover from surgery for 24 hrs before pain hypersensitivity testing begins.
For behavioural testing, rats are placed into the testing apparatus and are allowed to habituate to the testing procedure. The area tested is the mid-plantar surface of the hindpaw (Figure 2), which falls within the sciatic nerve distribution. Mechanical withdrawal threshold is assessed by mechanically stimulating both injured and uninjured hindpaws using an electronic dynamic plantar von Frey aesthesiometer or manual von Frey hairs4. The mechanical withdrawal threshold is the maximum pressure exerted (in grams) that triggers paw withdrawal. For measurement of thermal withdrawal latency, first described by Hargreaves et al (1988), the hindpaw is exposed to a beam of radiant heat through a transparent glass surface using a plantar analgesia meter5,6. The withdrawal latency to the heat stimulus is recorded as the time for paw withdrawal in both injured and uninjured hindpaws. Following CCI, mechanical withdrawal threshold, as well as thermal withdrawal latency in the injured paw are both significantly reduced, compared to baseline measurements and the uninjured paw (Figure 3). The CCI model of peripheral nerve injury combined with pain hypersensitivity testing provides a model system to investigate the effectiveness of potential therapeutic agents to modify chronic neuropathic pain. In our laboratory, we utilise CCI alongside thermal and mechanical sensitivity of the hindpaws to investigate the role of neuro-immune interactions in the pathogenesis and treatment of neuropathic pain.

Due to the simplicity of surgery and the robust behavioural outcome, chronic constriction of the sciatic nerve is one of the pre-eminent animal models of neuropathic pain. Within 24 hrs following surgery, pain hypersensitivity is established and can be quantitatively measured using a von Frey aesthesiometer (mechanical test) and plantar analgesia meter (thermal test).

Siyatik Sinir Sıçanlarda Ağrı aşırı duyarlılık Test Kronik daraltma

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of ...

Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently shown that UPEC have an important role in the initiation of chronic pelvic pain2, a feature of Chronic prostatitis/Chronic pelvic pain syndrome (CP/CPPS)3,4. Infection of the prostate by clinically relevant UPEC can initiate and establish chronic pain through mechanisms that may involve tissue damage and the initiation of mechanisms of autoimmunity5.
A challenge to understanding the pathogenesis of UPEC in the prostate is the relative inaccessibility of the prostate gland to manipulation. We utilized a previously described intraurethral infection method6 to deliver a clinical strain of UPEC into male mice thereby establishing an ascending infection of the prostate. Here, we describe our protocols for standardizing the bacterial inoculum7 as well as the procedure for catheterizing anesthetized male mice for instillation of bacteria.
CP/CPPS is primarily characterized by the presence of tactile allodynia4. Behavior testing was based on the concept of cutaneous hyperalgesia resulting from referred visceral pain8-10. An irritable focus in visceral tissues reduces cutaneous pain thresholds allowing for an exaggerated response to normally non-painful stimuli (allodynia). Application of normal force to the skin result in abnormal responses that tend to increase with the intensity of the underlying visceral pain. We describe methodology in NOD/ShiLtJ mice that utilize von Frey fibers to quantify tactile allodynia over time in response to a single infection with UPEC bacteria.

Infection of the prostate may be a contributing factor in mediating pelvic pain in chronic prostatitis. We describe the procedure for preparation of standardized bacterial inoculum, instillation of bacteria into the urethra of male mice and methodology for measuring tactile allodynia in mice over time.

Bakteriyel Prostatit bir fare modelinde Dokunsal allodini Ölçümü

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently ...

İmmünoloji ve Enfeksiyon

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.
The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species1,2. The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology4-6 In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus8. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9 in addition to their recently discovered role as photoreceptors10. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells11,12. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11 where they may connect to second-order neurons that project to the brain.
Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates.

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.

Termal nosisepsiyon değerlendirilmesi Yerel ve Küresel Yöntemleri<em&gt; Drosophila</em&gt; Larva

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of ...

Nörobilim

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain perception in rodents. Withdrawal thresholds can be measured first at baseline and then following drug, venom, injury, allergen,&#160;or otherwise evoked inflammation by applying an accurate force on very specific areas of the skin. An electronic von Frey apparatus allows precise assessment of mouse hind paw withdrawal thresholds that are not limited by the available filament sizes in contrast to classical von Frey measurements. The ease and rapidity of measurements allow for incorporation of assessment of tactile sensitivity outcomes in diverse models of rapid-onset inflammatory and neuropathic pain as multiple measurements can be taken within a short time period. Experimental measurements for individual rodent subjects can be internally controlled against individual baseline responses and exclusion criteria easily established to standardize baseline responses within and across experimental groups. Thus, measurements using an electronic von Frey apparatus represent a useful modification of the well-established classical von Frey filament-based assays for rodent mechanical allodynia that may also be applied to other nonhuman mammalian models.

Electronic von Frey measurements are an effective and improved alternative to classical von Frey filaments, as they allow rapid and precise measurement of changes in rodent mechanical withdrawal thresholds to continuously applied pressure stimuli before and after an inflammatory event.

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain ...

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila larvae, customized metal nickel-titanium alloy (nitinol) filaments. These mechanical probes are similar to the von Frey filaments used in vertebrates to measure mechanical nociception. Here, we demonstrate how to make and calibrate these mechanical probes and how to generate a full behavioral dose-response from subthreshold (innocuous or non-noxious range) to suprathreshold (low to high noxious range) stimuli. To demonstrate the utility of the probes, we investigated tissue damage-induced hypersensitivity in Drosophila larvae. Mechanical allodynia (hypersensitivity to a normally innocuous mechanical stimulus) and hyperalgesia (exaggerated responsiveness to a noxious mechanical stimulus) have not yet been established in Drosophila larvae. Using mechanical probes that are normally innocuous or probes that typically elicit an aversive behavior, we found that Drosophila larvae develop mechanical hypersensitization (both allodynia and hyperalgesia) after tissue damage. Thus, the mechanical probes and assay that we illustrate here will likely be important tools to dissect the fundamental molecular/genetic mechanisms of mechanical hypersensitivity.

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

The goal of this protocol is to show how to perform an improved assay for mechanical nociception in Drosophila larvae. We use the assay here to demonstrate that mechanical hypersensitivity (allodynia and hyperalgesia) exists in Drosophila larvae.

Drosophila Larvalarında Mekanik Nosiception Ölçümü için Geliştirilmiş Bir Tahlil ve Araçlar

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila ...

Davranış

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The approach allows unbiased identification and functional characterization of individual colorectal afferents. Extracellular recordings of propagated action potentials (APs) that originate from one or a few afferent (i.e., single-fiber) receptive fields (RFs) in the colorectum are made from teased nerve fiber fascicles. The colorectum is removed with either the pelvic (PN) or lumbar splanchnic (LSN) nerve attached and opened longitudinally. The tissue is placed in a recording chamber, pinned flat and perfused with oxygenated Krebs solution. Focal electrical stimulation is used to locate the colorectal afferent endings, which are further tested by three distinct mechanical stimuli (blunt probing, mucosal stroking and circumferential stretch) to functionally categorize the afferents into five mechanosensitive classes. Endings responding to none of these mechanical stimuli are categorized as mechanically-insensitive afferents (MIAs). Both mechanosensitive and MIAs can be assessed for sensitization (i.e., enhanced response, reduced threshold, and/or acquisition of mechanosensitivity) by localized exposure of RFs to chemicals (e.g., inflammatory soup (IS), capsaicin, adenosine triphosphate (ATP)). We describe the equipment and colorectum&#8211;nerve recording preparation, harvest of colorectum with attached PN or LSN, identification of RFs in the colorectum, single-fiber recording from nerve fascicles, and localized application of chemicals to the RF. In addition, challenges of the preparation and application of standardized mechanical stimulation are also discussed.

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

Fare Kolorektal Afferent Endings in vitro Fonksiyonel Karakterizasyonu içinde

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the age of 50, yet it is the least understood and studied chronic pelvic pain disorder. A significant subset of patients with chronic pelvic pain report having experienced early life stress or abuse, which can markedly affect the functioning and regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Mast cell activation, which has been shown to be increased in both urine and expressed prostatic secretions of CP/CPPS patients, is partially regulated by downstream activation of the HPA axis. Neonatal maternal separation (NMS) has been used for over two decades to study the outcomes of early life stress in rodent models, including changes in the HPA axis and visceral sensitivity. Here we provide a detailed protocol for using NMS as a preclinical model of CP/CPPS in male C57BL/6 mice. We describe the methodology for performing NMS, assessing perigenital mechanical allodynia, and histological evidence of mast cell activation. We also provide evidence that early psychological stress can have long-lasting effects on the male urogenital system in mice.

We are measuring perigenital mechanical sensitivity and mast cell activation in the prostate of male C57BL/6 mice that underwent an early life stress paradigm &#8211; neonatal maternal separation, in order to induce a preclinical model of chronic prostatitis/chronic pelvic pain syndrome.

Yenidoğan Anneden Ayrılma Fare Modelinde perigenital Duyarlılık ve Prostat Mast Hücre aktivasyonu Değerlendirilmesi

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

Soğuk nosisepsiyon için Roman Tahlili<em&gt; Drosophila</em&gt; Larva

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

Implantation of Osmotic Pumps and Induction of Stress to Establish a Symptomatic, Pharmacological Mouse Model for DYT/PARK-ATP1A3 Dystonia

Genetically modified mouse models face limitations, especially when studying movement disorders, where most of the available transgenic rodent models do not present a motor phenotype resembling the clinical aspects of the human disease. Pharmacological mouse models allow for a more direct study of the pathomechanisms and their effect on the behavioral phenotype. Osmotic pumps connected to brain cannulas open up the possibility of creating pharmacological mouse models via local and chronic drug delivery. For the hereditary movement disorder of rapid-onset dystonia-parkinsonism, the loss-of-function mutation in the &#945;3-subunit of the Na+/K+-ATPase can be simulated by a highly specific blockade via the glycoside ouabain. In order to locally block the &#945;3-subunit in the basal ganglia and the cerebellum, which are the two brain structures believed to be heavily involved in the pathogenesis of rapid-onset dystonia-parkinsonism, a bilateral cannula is stereotaxically implanted into the striatum and an additional single cannula is introduced into the cerebellum. The cannulas are connected via vinyl tubing to two osmotic pumps, which are subcutaneously implanted on the back of the animals and allow for the chronic and precise delivery of ouabain. The pharmacological mouse model for rapid-onset dystonia-parkinsonism carries the additional advantage of recapitulating the clinical and pathological features of asymptomatic and symptomatic mutation carriers. Just like mutation carriers of rapid-onset dystonia parkinsonism, the ouabain-perfused mice develop dystonia-like movements only after additional exposure to stress. We demonstrate a mild stress paradigm and introduce two modified scoring systems for the assessment of a motor phenotype.

We provide a protocol to generate a pharmacological DYT/PARK-ATP1A3 dystonia mouse model via implantation of cannulas into basal ganglia and cerebellum connected to osmotic pumps. We describe the induction of dystonia-like movements via application of a motor challenge and the characterization of the phenotype via behavioral scoring systems.

DYT/PARK-ATP1A3 Distonisi Için Semptomatik, Farmakolojik Fare Modeli Kurmak için Osmotik Pompaların İmplantasyonu ve StresIn İndüksiyonu

Genetically modified mouse models face limitations, especially when studying movement disorders, where most of the available transgenic rodent models do ...

Dictyostelium discoideum yanıt-e doğru akut mekanik stimülasyon değerlendirilmesi

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