Giriş Organizmalar, tehdit edici uyaranlara ve çevre değişiklikleri cevap vermek için geniş bir yelpazede davranışları sergilemek zorundadır. Otonom yanıt iyi mücadele veya uçuş yanıt olarak sempatik sinir sisteminin fizyolojik (Nicholls ve ark 2001 Carpenter, 1976) iç ortam hazırlıyor hangi omurgalıların okudu. Bu, bir organizmanın stres yanıtı düzenleyen ve yaklaşan bir saldırı ya da çevresel değişim tepki davranışı değiştirebilir fizyolojik değişiklikler geçer. Kanıtlar, kardiyo-vasküler sistem, böylece bir yanıt yanıt ve uygulamak için iç ortam sağlayan solunum sistemi ile sıkı sıkıya bağlı olduğunu gösterir. Biliyorsunuz, solunum ve kardiyovasküler sistem otonom kontrol oksijen kullanılabilirliği ve yaklaşan bir davranış yanıt için gerekli olan belirli bir hedef dokulara besin düzenleyen olduğu bilinmektedir. Omurgalılar, otonom ve fizyolojik tepki, o kadar iyi gelişti; kompleks omurgasızlar, benzer şekilde gelişmiş bir yanıt sistemine sahip olacağını olası görünüyor (Schapker ve ark, 2002; Zavarzin, 1941) . Omurgalılar ve omurgasızlar sistematik olarak çok farklı olsa da, son derece gelişmiş omurgasızlar bir mücadele veya uçuş şekilde cevap vermek için hızlı kalp-damar ve solunum yanıt gerektirir. Birçok omurgasızlar için, sempatik gibi fizyolojik yanıt belirlenebilir. 1927 yılına Arkadaş, çok erken çalışmaların çoğu, özellikle (; Orlov, 1927; Zavarzin, 1941 Alexandrowicz, 1932) genel olarak eklembacaklılar kullanarak, omurgasızlar yapılmıştır var. Kerevit hızlı davranışların geniş bir yelpazede, değerlendirmek ve çevresel uyaranlara yanıt yeteneği yanı sıra sergilemek için bilinmektedir. Çok erken çalışmaları, anında ve hızlı bir savunma tavır yanıt (; Huxley, 1880; Shuranova ve ark, 2006; Wiersma, 1961 Bethe, 1897) gibi sempatik bir yanıt kaydetti var . Yetişkin kerevit kalp, santral sinir sistemi (Yamagishi ve Hirose, 1997; Yamagishi ve ark, 1997; Wilkens, 1999 Alexandrowicz, 1932) yendi ve ritm tarafından kontrol edilir nörojenik . Rutin, bir hayvanın kalp hızı (HR) ölçümü eksitabilite ve iç ortam hazır doğrudan bir ölçümü sağlar. Algılanan tehdit edici bir uyarıcı (Listerman ve ark, 2000), kerevit, HR bir artış savunma tavır içinde olduğunu kaydetti . Bu tip sinir sistemi tepki de dikenli ıstakozlar (Yazawa ve Katsuyama, 2001) gösterildi. Ventilasyon sistemi de neuronally solungaçları arasında oksijen alımı için iki özel ekleri olarak adlandırılan scaphognathites pompalama eylem (Mendelson, 1971) tarafından, sorumlu bir ventilasyon merkezi desen üreteci (VPG) ile kontrol edilir. Bir tek scaphognathite ön ucunda her brankial odasında bulunduğu ve ritmik bir hareket (Pasztor, 1968) ile solungaçları arasında su çekiyor. VPG, kalp gibi birçok faktör tarafından modüle edilebilir. Yengeç Önceki çalışmalar, Kanser magister komut nöron (Wilkens ve ark, 1974) ile merkezi kontrol ile ventilasyon ve kalp ritmi değişmiş olabilir gösterir. Cuadras, 1979, 1980; Li ve ark, 2000; Listerman ark Buna ek olarak, VPG faaliyet içsel tepkiyi ve sosyal etkileşim ya da kabuklular çevresel değişiklikler (Burmistrov ve Shuranova, 1996 değişiklikleri ile değiştirmek için bilinmektedir. 2000; McMahon ve Wilkens, 1983; Schapker ve ark, 2002; Shuranova ve ark, 2002; Wilkens, 1976). Kerevit de görüldüğü gibi, solunum aktivitesi (VR) beklenmedik dış uyaranlara (Shuranova ve ark, 2002. Shuranova ve ark, 1993) sırasında kaydedilebilir iç durumu ve VR değişikliklere bağlı olarak değişebilir. İK ve VR önlemler güvenilirlik ve fizibilite nedeniyle, gelecek sorular sorulmasını sonsuzdur. Daha önce de belirtildiği gibi, iş, sosyal etkileşimler ve çevresel bozuklukları sırasında otonom tepkisini incelemek için yapılmıştır. İlginçtir ki, pek çok alanda doğal hormonal değişiklikler ve / veya organizmada meydana gelen fizyolojik süreçleri sırasında otonom tepki incelenmesi gibi keşfedilmeyi bırakılır. Gelecekteki muhtemel yönleri çiftleşme ve koku kimyasal resepsiyon sırasında sempatik gibi cevaplar da inceleyebilir. En önemlisi, fizyolojik yanıtlar dahili kendilerini readying zaman hayvanlar her zaman davranışsal yanıt vermiyor bu davranış gözlemlerine göre, çevreye bir organizmanın tepki içine daha fazla bilgi verebilir. Böylece, bir organizmanın genel değişiklikleri açıklayabilecek bir organizmanın iç durumu değerlendirmek için fizyolojik ölçüm biyolojik bir dizin sağlar. Yöntemleri Kerevit tel başlamadan önce, tellerin bir hazırlama gerekli değildir. Kesme yeterince uzun kerevit takın ve yeterli bir empedans dedektör ulaşmak (UFI, modeli 2991); iki yalıtımlı paslanmaz çelik teller (AM Sistemleri, Carlsburg, WA çapı 0.005 inç ve kaplama ile 0,008 inç) Karapaksla eklenecektir hem tellerin ucunda yangın yalıtımı (~ 0.5 mm) çıkarın. Hayvan dışında çıplak tel olarak çok fazla izolasyon kaldırmak dikkatli olun kısa devre empedansı kayıtları önlemek kaplanabilir olmalıdır. Daha sonra, forseps kullanarak karapaksla içine yerleştirme için 90 açı tellerin yanmış biter bükün. Bu iç organlara zarar çünkü tel eklenen kısmı çok uzun olmadığını emin olun. Teller, plastik bir tüp (telleri korur) içine yerleştirmeden fizibilite için, gevşek teller birlikte bükmek için en iyisidir. Kayıt kalp ve havalandırma oranları, yukarıdaki işlemi tekrarlayarak ikinci kez adım ve tellerin iki takım plastik tüp içine yerleştirilmesinden önce birlikte gevşek örgülü olabilir. Teller kayıt için hangi empedans dedektörü eklemek karıştırmayın şekilde kalp ve havalandırma oranı teller tayin emin olun. Teller etiket ya da başında, diğer bir çift daha kısa kesmek en iyisidir. Kerevit fizibil ve güvenli bir şekilde tel için, kişi ve / veya kerevit (Şekil 1) yaralanmasını ortadan kaldırmak için, ıslak bez veya kağıt havlu pençeleri ve bacaklar (arka bırakarak ve bir tarafı maruz) sarmak için en iyisidir. <img src="/files/ftp_upload/1594/1594fig1.jpg" style="float:left;" alt="Şekil 1" /> Şekil 1. Kerevit chelipeds ve bacak Sarma. Islak bir kağıt havlu kerevit tamamlayan kişi ve kerevit yaralanmasını önlemek. Sarma chelipeds ve bacaklarını kerevit yeteneği engel olacaktır. Karapaksla kalınlığı her kerevit ve aşırı güç ile değişir kablolama işlemine başlamak için, solungaç odasında başlamak için en iyi solungaç odası başında, böylece iç organlara zarar verebilir, aşırı basınç kerevit öldürmek için büyük olasılıkla daha az torasik odasına göre. Ilk delik rostral alanında solungaç kamara (; Schapker ve ark, 2002 yani ventilatör hızı monitörü prebranchial odasına) manikür altına yerleştirilen bir tel için yeterli neşter ve sadece büyük bir ince noktası kullanılarak yapılır. Büyük bir delik aşırı hemolimf kaybı olacak ve düzgün bir mühür ve bu delik kalan tellerin olasılığı artacaktır. Tel yerleştirildikten sonra üzerine küçük bir miktar yapıştırıcı (siyanoakrilat ester) ve hızlandırıcı (HobbyTown ABD, Lexington, KY), tel bırakın. Hızlı kuruyan tutkal kullanılan hayvanların taşıma stresi azaltır. Bu kerevit için toksik ve ölüme yol açacağından hızlandırıcı sadece küçük bir miktar manikür üzerine yerleştirmek için dikkatli olun. En iyisi hemen, kağıt havlu ile herhangi bir aşırı gaz pedalından silin. Solungaç odasında ikinci bir tel için bu adımları tekrarlayın. Bu iki teller her scaphognathite hareketi sırasında (Şekil 2) güçlü bir doğru empedans önlem sigortalamak için scaphognathite (solunum organı) span yerleştirilir. Doğru bir kayıt almak için, ikinci tele solungaç odası dışında ve göğüs odasına yerleştirilen olmadığından emin olun. <img src="/files/ftp_upload/1594/1594fig2.jpg" style="float:left;" alt="Şekil 2" /> Şekil 2. Solungaç odası paslanmaz çelik tel yerleştirilmesi. Karapaksta beyaz noktalar, manikür altına yerleştirilen paslanmaz çelik teller için noktalarını temsil eder. Teller, dinamik empedans üzerinden scaphognathite ve ölçü hareketi kapsar. Torasik odasında kablo kalp hızı adımları teller doğrudan bir rostral-kaudal düzenleme kalp ve kalp a (Şekil 3) üzerinde dorsal karapaksla altına yerleştirilen dışında yukarıdaki gibi aynıdır. Bu yerleşim her kalp kasılması (Listerman ve ark, 2001) sırasında güçlü bir doğru empedansı ölçmek sağlayacaktır. <img src="/files/ftp_upload/1594/1594fig3.jpg" style="float:left;" alt="Şekil 3" /> Şekil 3. Torasik odasında Yerleştirme paslanmaz çelik teller. Karapaksta beyaz noktalar, manikür altına yerleştirilen paslanmaz çelik teller için noktalarını temsil eder. Teller rostral-kaudal düzenleme kalp kapsar. Bu dinamik empedans ile kalp kasılma önlemler için izin verecektir. Dört tel yerleştirilmesi tamamlandıktan sonra, (Eastman, 5 dakika kurutma e bir anlık yapıştırıcı karışımıPoxy) ve ceket her konum yanı sıra tel için delikler. Teller ve delikler yeterince kaplıdır sonra kalan epoksi plastik boru, kerevit arkasına saklamak için kullanın. Bu teller gelecek handlings delikleri çıkardı olacağını garanti eder. Epoksi kuruması için birkaç dakika sürer, epoksi artık dokunmatik yapışkan kadar tutun ya da bir kap su olmadan kerevit. Epoksi kuruduktan sonra, fazla kimyasal maddelerin kaldırmak ve onları bir su ortamına geri yerine su kerevit yıkayın. Kerevit sağlığını korumak için, daha önce çıkarılmış herhangi bir kimyasal madde kaldırmak için yaklaşık bir saat içinde su değiştirmek için en iyisidir. , Bu nedenle, hayvanlar 2 ila 3 gün bırakılmalıdır herhangi bir deney öncesi, önceki araştırmacılar belirtildiği gibi, bu uzun süreli kullanım nedeniyle stres, 3 gün (. Wilkens ve ark, 1985) 24 saatlik bir süre fizyolojik ölçümler değiştirir bulundu. . EKG&#39;lerinin kaydetmek için, kerevit empedans dedektörleri telleri bağlamak. Kalp (ölçü İK) kapsayan iki tel scaphognathite (ölçü VR) kapsayan iki tel için aynı şekilde tek bir empedans detektörü bağlı ve olmalıdır. Empedans dedektörü, iki paslanmaz çelik teller arasındaki dinamik direnç ölçer. Bu sinyaller, bir PowerLab/4SP arayüz (AD Aletleri) ile bir PowerLab dedektör on-line bağlayan kaydedilir. Tüm olaylar ölçülür ve PowerLab Grafik yazılım sürümü 5.5.6 (AD Instruments, Avustralya) ile kalibre edilir. Başlamak için bir kazanım oranı, 4 kHz ayarlayın. Büyük deplasmanlar, kalp atışı ve havalandırma oranı temsil edecek. Yumuşatma fonksiyonu veri seti uygulanan her İK / VR, sayma kolaylığı için. 100 puan düzeltti olsaydı (yani ortalama) ortaya çıkan eser, sadece kalp atışları tasvir. Kalp hızı 10-s aralıklarla üzerindeki her beat direk sayımı tarafından belirlenir ve daha sonra dk (BPM) başına atışlarda dönüştürülür olabilir.

<ol>
	<li>Alexandrowicz, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+innervation+of+the+heart+of+Crustacea.">The innervation of the heart of Crustacea.</a> I. Decapoda. Wuart. J. Microsc. Sci. 75, 181-249 (1932).</li><li>Bethe, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vergleichende+Untersuchungen+uber+die+Funtionen+des+Zentrainervensystems+der+Arthropoden.">Vergleichende Untersuchungen uber die Funtionen des Zentrainervensystems der Arthropoden.</a> Pfluger s Arch. Ges. Physiol. 68, 449-545 (1897).</li><li>Burmistrov, Y. M., Shuranova, Z. P., Abramson, C. I., Shuranova, Z. P., Burmistrov, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Russian Contributions to Invertebrate Behavior. , 111-144 (1996).</li><li>Carpenter, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+autonomic+nervous+system.">The autonomic nervous system.</a> Human Neuroanatomy. , 191-212 (1976).</li><li>Cuadras, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+rate+and+agonistic+behavior+in+unrestrained+crabs.">Heart rate and agonistic behavior in unrestrained crabs.</a> Mar. Behav. Physiol. 6, 189-196 (1979).</li><li>Cuadras, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+responses+to+visual+detection+of+movement,+mechanostimulation+and+cheliped+improsed+movement+in+hermit+crabs.">Cardiac responses to visual detection of movement, mechanostimulation and cheliped improsed movement in hermit crabs.</a> Comp. Biochem. Physiol. A. 66, 113-1171 (1980).</li><li>Huxley, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Crayfish: An introduction to the study of zoology. , (1977).</li><li>Li, H., Listerman, L. R., Doshi, D., Cooper, 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=Use+of+heart+rate+to+measure+intrinsic+state+of+blind+cave+crayfish+during+social+interactions.">Use of heart rate to measure intrinsic state of blind cave crayfish during social interactions.</a> Comp. Bichem. Physiol. 127A, 55-70 (2000).</li><li>Listerman, L. R., Deskins, J., Bradacs, H., Cooper, 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=Heart+rate+within+male+crayfish:+social+interactions+and+effects+of+5-HT.">Heart rate within male crayfish: social interactions and effects of 5-HT.</a> Comp. Biochem. Physiol. 125, 251-263 (2000).</li><li>McMahon, B. R., Wilkens, J. L., Mantel, L., Bliss, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventilation,+perfusion+and+oxygen+uptake.">Ventilation, perfusion and oxygen uptake.</a> Biology of Crustacea. 6, 289-372 (1983).</li><li>Mendelson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillator+neurons+in+crustacean+ganglia.">Oscillator neurons in crustacean ganglia.</a> Science. 171, 1170-1173 (1971).</li><li>Nicholls, J. G., Martin, A. R., Wallace, B. G., Fuchs, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> From Neuron to Brain. , 315-317 (2001).</li><li>Orlov, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Das+Magenganglion+des+Flu&#946;krebses,+Ein+Beitrag+zur+vergleichenden+Histologis+des+sympathischen+Nervensystem.">Das Magenganglion des Flu&#946;krebses, Ein Beitrag zur vergleichenden Histologis des sympathischen Nervensystem.</a> Z. Mikrosk. Anat. Forschung. 8, 67-102 (1927).</li><li>Pasztor, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurophysiology+of+respiration+in+decapod+Crustacea:+The+motor+system.">The neurophysiology of respiration in decapod Crustacea: The motor system.</a> Can. J. Zool. 46, 585-596 (1968).</li><li>Schapker, H., Breithaupt, T., Shuranova, Z., Burmistrov, Y., Cooper, 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=Heart+rate+and+ventilatory+correlative+measures+in+crayfish+during+environmental+disturbances+and+social+interactions.">Heart rate and ventilatory correlative measures in crayfish during environmental disturbances and social interactions.</a> Comp. Biochem. Physiol., A. 131, 397-407 (2002).</li><li>Shuranova, Z. P., Vekhov, A. V., Burmistrov, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+behavioral+reactions+of+fresh-water+crayfish+to+sensory+exposures:+the+autonomic+components.">The behavioral reactions of fresh-water crayfish to sensory exposures: the autonomic components.</a> Zh. Vyssh Nerv. Deiat Im I P Pavlova. 43, 1159-1169 (1993).</li><li>Shuranova, Z. P., Burmistrov, Y. M., Wiese, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventilatory+activity+in+free+moving+crayfish+is+indicative+of+its+functional+state+and+perceiving+external+stimuli.">Ventilatory activity in free moving crayfish is indicative of its functional state and perceiving external stimuli.</a> The Crustacean Nervous System. , 526-535 (2002).</li><li>Shuranova, Z. P., Burmistrov, Y. M., Strawn, J. R., Cooper, 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=Evidence+for+an+autonomic+nervous+system+in+decapod+crustaceans.">Evidence for an autonomic nervous system in decapod crustaceans.</a> Inter. J. Zool. Res. 2, 242-283 (2006).</li><li>Wiersma, C. A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reflexes+and+the+central+nervous+system.">Reflexes and the central nervous system.</a> The physiology of Crustacea, vol II, Sense organs, integration, and behavior. , 241-279 (1961).</li><li>Wilkens, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+control+of+respiration+in+decapod+Crustacea.+Fed.">Neuronal control of respiration in decapod Crustacea. Fed.</a> Fed. Proc. 35, 2000-2006 (1976).</li><li>Wilkens, J. L., Wilkens, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+control+of+cardiac+and+scaphognathite+pacemakers+in+the+crab+Cancer+magister.">Central control of cardiac and scaphognathite pacemakers in the crab Cancer magister.</a> J. Comp. Physiol. 90, 89-104 (1974).</li><li>Wilkens, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+control+of+cardiac+rhythmicity+and+of+blood+distribution+in+crustaceans.">The control of cardiac rhythmicity and of blood distribution in crustaceans.</a> Comp. Biochem. Physiol. A. 124, 531-538 (1999).</li><li>Yamagishi, H., Ando, H., Makioka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myogenic+heartbeat+in+the+primitive+crustacean+Triops+longicaudatus.">Myogenic heartbeat in the primitive crustacean Triops longicaudatus.</a> Biol. Bull. 193, 350-358 (1997).</li><li>Yazawa, T., Katsuyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+and+repetitive+cardiac+slowdown+in+the+freely+moving+spiny+lobster,+Panulirus+japonicas.">Spontaneous and repetitive cardiac slowdown in the freely moving spiny lobster, Panulirus japonicas.</a> J. Comp. Physiol. 187, 817-824 (2001).</li><li>Zavarzin, A. A., Zavarzin, A. A., Izbrannye, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ocherki po evol utsionnoj gistologii nervnoj sistemy (Essays on the evolutionary histology of the nervous system). III, (1950).</li></ol>

<table border="1">
<tbody>
<tr>
<td>Material Name</td>
<td>Type</td>
<td>Company</td>
<td>Catalogue Number</td>
</tr>	
<tr>
<td>Scalpel (Stainless steel surgical blades No. 11 for No. 3 handle)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Stainless steel wire (diameter 0.005 inches with the coating 0.008 inches; A-M Systems, Carlsburg, WA)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Impedance detector (UFI, model 2991)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Plastic tubing (Tygon Tubing ID .0812, OD .1492, Cole-Parmer Instrument Company)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Fast-drying glue (cyanoacrylate ester, HobbyTown USA, Lexington, KY)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Accelerator (HobbyTown USA, Lexington, KY)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Adhesive (Eastman, 5-min drying epoxy)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/ML866/" target="_blank">PowerLab via a PowerLab/4SP interface (AD Instruments)</a>.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td><a href="http://www.adinstruments.com/products/software/research/LabChart-Software/" target="_blank">PowerLab Chart software version 5.5.6 (AD Instruments, Australia)</a></td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td><a href="http://www.adinstruments.com/products/software/research/LabChart-Software/" target="_blank">Chart software version 3.5.6 (AD Instruments, Australia)</a></td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
</tbody>
		</table>

measures of heart and ventilatory rates in freely moving crayfish

Korku, uçuş ve savaşma tepkisi yaklaşan bir yırtıcı saldırı altında bir organizmanın, çevrenin bilincini incelemek için temel fizyolojik temeli olarak hizmet vermektedir. Omurgasızlar omurgasızlar sahip omurgalıların benzeyen bir otonom sinir sistemi olsun, kanıtlar kesin olarak bilinmemekle birlikte, iç ortam ve hazır organizmanın belli bir uyarana davranışsal hareket düzenleyen bir sempatik gibi tepki var. Ayrıca, bu fizyolojik yanıt fizibil ölçülür ve hayvanın iç devlet için biyolojik bir dizin gibi davranır olabilir. Fizyolojik yanıt ölçümleri, kalp-damar ve solunum sistemleri, merkezi sinir sistemi kontrollü bir koordinasyon içinde değişiklikleri ile doğrudan iç ve dış stresörler ilgili olabilir. Daha spesifik olarak, kalp ve havalandırma oranlarının izlenmesi, her zaman davranışsal gözlenen stres tepkisi ölçülebilir önlem alınmasını sağlamaktadır. Kerevit kayıt fizibilite, yanı sıra kerevit morfolojisi bilinen zengin tarihini, 1888 yılında Huxley kadar uzanan, ve iyi çalışılmış tipik davranışları nedeniyle kalp ve ventilasyon hızı ölçümleri için iyi bir model organizmalar.

Watch this Scientific Journal Video about Measures of Heart and Ventilatory Rates in Freely Moving Crayfish at JoVE.com

Measures of Heart and Ventilatory Rates in Freely Moving Crayfish

Omurgasızlar omurgalıların için açıklanan benzer bir otonom sempatik gibi tepki gösterir. Bir organizmanın iç durumu değerlendirmek için biyolojik bir indeksi ölçümü için kardiyo-vasküler ve ventilasyon sistemlerinin koordinasyonu sağlar.

Serbestçe Kerevit Hareketli Kalp ve Ventilatuar Oranları Ölçüleri

The fear, flight or fight response serves as the fundamental physiological basis for examining an organism's awareness of its environment under an ...

measures-of-heart-and-ventilatory-rates-in-freely-moving-crayfish

Research

JoVE Journal

Biology

15.2K Görüntüleme.  University of Kentucky. Omurgasızlar omurgalıların için açıklanan benzer bir otonom sempatik gibi tepki gösterir. Bir organizmanın iç durumu değerlendirmek için biyolojik bir indeksi ölçümü için kardiyo-vasküler ve ventilasyon sistemlerinin koordinasyonu sağlar.

Video: Measures of Heart and Ventilatory Rates in Freely Moving Crayfish

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Sıçan Kalp Engineered Doku implantasyonu

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Biyoloji

Recordings of Neural Circuit Activation in Freely Behaving Animals

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. Traditional non-invasive methods require at least partially restrained research subjects, and they only allow identification of large numbers of simultaneously activated neurons. On the other hand, small ensembles of neurons or individual neurons can only be measured using single-cell recordings obtained from largely reduced preparations. Since the expression of natural behavior is limited in restrained and dissected animals, the underlying neural mechanisms that control such behavior are difficult to identify. 
Here, I present a non-invasive physiological technique that allows measuring neural circuit activation in freely behaving animals. Using a pair of wire electrodes inside a water-filled chamber, the bath electrodes record neural and muscular field potentials generated by juvenile crayfish during natural or experimentally evoked escape responses. The primary escape responses of crayfish are mediated by three different types of tail-flips which move the animals away from the point of stimulation. Each type of tail-flip is controlled by its own neural circuit; the two fastest and most powerful escape responses require activation of different sets of large &#8220;command&#8221; neurons. In combination with behavioral observations, the bath electrode recordings allow unambiguous identification of these neurons and the associated neural circuits. Thus activity of neural circuitry underlying naturally occurring behavior can be measured in unrestrained animals and in different behavioral contexts.

Non-invasive measurements of neural activity patterns in freely behaving animals are obtained by combining neurophysiological recordings with high speed videography.

Serbestçe Hayvanlar Behaving Sinir Devre Etkinleştirme kayıtları

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. ...

Historical View and Physiology Demonstration at the NMJ of the Crayfish Opener Muscle

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is still much to learn from this model preparation. In understanding the history one can appreciate why even today this NMJ still offers a rich playground to address questions regarding pre- and post-synaptic function and plasticity. The viability and ease of access to the terminal for intracellular as well as extracellular electrophysiology and imaging are significant advantages. The mechanisms behind the modulation of vesicular kinetics and fusion within the high- and low-output terminals are begging for investigation. The preparation also offers a testable model system for computational assessments and manipulations to examine key variables in theoretical models of synaptic function, for example calcium dynamics during short-term facilitation. The synaptic complexity of active zone and statistical nature of quantal release is also an open area for future investigation both experimentally and computationally.

The opener muscle of the crayfish leg is presented for its historical importance and experimental versatility in muscle phenotype, synaptic physiology and plasticity.

Kerevit Açıcı Kas NMJ Tarihsel ve Fizyolojisi gösteri

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Elektron Mikroskobu için embriyo Hazırlık Drosophila Embriyonik kalp tüpü

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

İyonlaştırıcı Radyasyon Cevabı γH2AX Foci Kantitasyonu

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

Doku Örnekleri γH2AX Foci kantitasyonu

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

Radyasyondan aşağıdaki γH2AX Mekansal Dağılımı Değerlendirilmesi

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Clonogenic Testi: yapışkan Hücreler

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Kalsiyum değerlendirilmesi Bozulmamış İskelet kas liflerinin içinde Sparks

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell&#8217;s specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.

MRNA Çürüme Oranlarının Ölçümü<em&gt; Saccharomyces cerevisiae</emKullanma&gt;<em&gt; Rpb1-1</em&gt; Suşlar

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...