Introduzione Gli organismi devono presentare una vasta gamma di comportamenti di rispondere agli stimoli minacciosi e cambiamenti nell&#39;ambiente. La risposta autonomica è ben studiata nei vertebrati come la risposta di lotta o fuga in cui il sistema nervoso simpatico prepara fisiologicamente l&#39;ambiente interno (Carpenter, 1976;. Nicholls et al, 2001). È attraverso i cambiamenti fisiologici che un organismo in grado di regolare la risposta allo stress e alterare il comportamento di reagire a un attacco imminente o cambiamenti ambientali. I dati dimostrano che il sistema cardio-vascolare è strettamente collegato con il sistema respiratorio consentendo in tal modo l&#39;ambiente interno di reagire e di attuare una risposta. E &#39;noto che il controllo autonomo del sistema respiratorio e cardiovascolare in grado di regolare la disponibilità di ossigeno e nutrienti ai tessuti target specifici necessari per una risposta imminente comportamentale. Nei vertebrati, la risposta autonomica e fisiologico è così ben evoluto, sembrerebbe probabile che invertebrati complesso sarebbe allo stesso modo in possesso di un sistema di risposta sviluppato (Schapker et al, 2002;. Zavarzin, 1941). Anche se vertebrati e invertebrati sono molto diversi sistematicamente; invertebrati altamente sviluppato richiedono la rapida risposta cardiovascolare e respiratorio per rispondere in maniera lotta o fuga. Per molti invertebrati, un simpatico-come risposta fisiologica può essere quantificato. Risalente al 1927, molti degli studi molto presto sono stati condotti negli invertebrati, in particolare attraverso i artropodi in generale (Alexandrowicz, 1932; Orlov, 1927; Zavarzin, 1941). Gamberi sono noti per esporre una vasta gamma di comportamenti rapido così come la capacità di valutare e rispondere agli stimoli ambientali. Studi molto presto hanno notato un simpatico-come la risposta di una risposta immediata e rapida di atteggiamenti di difesa (Bethe, 1897, Huxley, 1880; Shuranova et al, 2006;. Wiersma, 1961). Il cuore gamberi adulto neurogena da quando il battito e il ritmo sono controllati dal sistema nervoso centrale (Alexandrowicz, 1932; Yamagishi e Hirose, 1997;. Yamagishi et al, 1997; Wilkens, 1999). Di routine, la misurazione della frequenza cardiaca di un animale (HR) fornisce una misura diretta di eccitabilità e disponibilità dell&#39;ambiente interno. In gamberi, un aumento delle risorse umane è notato durante la postura di difesa quando si presenta con uno stimolo percepito minaccioso (Listerman et al., 2000). Questo tipo di risposta del sistema nervoso è stato anche mostrato in aragoste (Yazawa e Katsuyama, 2001). Il sistema di ventilazione è anche neuronally controllata con un generatore di ventilatorio modello centrale (VPG) responsabile per consumo di ossigeno attraverso le branchie con l&#39;azione di pompaggio di due appendici specializzate scaphognathites definito (Mendelson, 1971). Un scaphognathite singolo risiede in ogni camera branchiale alle estremità anteriore e estrae l&#39;acqua attraverso le branchie con un movimento ritmico (Pasztor, 1968). Il VPG, come con il cuore, può essere modulato da molti fattori. Lavori precedentemente svolti in granchio, Cancro magister mostra il ritmo cardiaco e respiratorio può essere alterata dal controllo centrale attraverso i neuroni di comando (Wilkens et al., 1974). Inoltre, l&#39;attività del VPG è noto a cambiare con i cambiamenti nella risposta interna e con le interazioni sociali o cambiamenti ambientali in crostacei (Burmistrov e Shuranova, 1996; Cuadras, 1979, 1980; Li et al, 2000;. Listerman et al. , 2000; McMahon e Wilkens, 1983; Schapker et al, 2002;. Shuranova et al, 2002;. Wilkens, 1976). Come si vede nella gamberi, attività ventilatoria (VR) può variare a seconda dello stato interno e le modifiche in VR possono essere registrati durante inaspettati stimoli esterni (Shuranova et al, 1993;. Shuranova et al, 2002).. A causa della affidabilità e la fattibilità delle risorse umane e le misure di VR, le domande futuro da porsi sono infinite. Come notato in precedenza, il lavoro è stato fatto per esaminare la risposta autonomica durante le interazioni sociali e disturbi ambientali. È interessante notare che molte zone sono lasciati da esplorare come l&#39;esame della risposta autonomica durante naturali cambiamenti ormonali e / o altri processi fisiologici che avvengono nell&#39;organismo. Possibili direzioni future possono anche esaminare simpatico-come le risposte durante l&#39;accoppiamento e la ricezione chimici olfattivi. Ma soprattutto, le risposte fisiologiche possono fornire una conoscenza più approfondita in una risposta dell&#39;organismo con l&#39;ambiente rispetto alle osservazioni comportamentali poiché gli animali non sempre rispondono comportamentale quando si stanno preparando internamente. Così, la misura fisiologica fornisce un indice biologico per valutare lo stato interno di un organismo che può spiegare i cambiamenti globali in un organismo. Metodi Prima di iniziare a filo di un gambero, la preparazione di fili è richiesto. Tagliate due fili isolati in acciaio inox (0,005 pollici di diametro e con il rivestimento 0,008 pollici; Sistemi AM, Carlsburg, WA), abbastanza a lungo da inserire nel gamberi e adeguatamente raggiungere un rilevatore di impedenza (UFI, modello 2991). Rimuovere l&#39;isolamento (~ 0,5 mm) da un incendio alle estremità dei fili che sarà inserito nel carapace. Fare attenzione a non rimuovere l&#39;isolamento troppo come un filo scoperto sulla parte esterna del l&#39;animale deve essere ricoperto per evitare corto circuito registrazioni impedenza. Quindi, utilizzando pinze piegare le estremità dei fili bruciati in un angolo di 90 per il posizionamento nel carapace. Assicurarsi che la parte inserita del filo non sia troppo lungo perché questo potrebbe fare danni agli organi interni. Per la fattibilità di inserire i cavi in ​​un tubo di plastica (che protegge i fili), è meglio girare liberamente i fili insieme. Se il cuore di registrazione e tassi di ventilazione, ripetere i passi sopra una seconda volta e le due serie di fili possono essere liberamente intrecciati tra loro prima collocazione nel tubo di plastica. Assicurati di designare i fili per il cuore e la velocità di ventilazione in modo da non confondere i fili che attribuiscono alla quale rivelatore impedenza per la registrazione. E &#39;meglio di etichettare i cavi o tagliare un paio più corta dell&#39;altra all&#39;inizio. Per plausibilmente e sicuro filo il gambero di fiume, è meglio per avvolgere gli artigli e le gambe (lasciando la parte posteriore e un lato a vista) in panno umido o tovaglioli di carta per eliminare il pregiudizio alla persona e / o gamberi (Figura 1). <img src="/files/ftp_upload/1594/1594fig1.jpg" style="float:left;" alt="Figura 1" /> Figura 1. Avvolgimento del chelipeds e le gambe del gambero. Avvolgere i gamberi in un tovagliolo di carta bagnato per prevenire lesioni alla persona e il gambero di fiume. La confezione sarà ostacolare la capacità del gambero per spostare il chelipeds e le gambe. Per iniziare il processo di cablaggio, è meglio iniziare nella camera branchiale poiché lo spessore del carapace varia con ogni gamberi e forza in eccesso possono causare danni agli organi interni, così da partire nella camera branchiale, eccessiva pressione è meno probabile che uccidere come i gamberi rispetto alla camera del torace. La prima buca è fatto usando una punta sottile bisturi e appena abbastanza grande per un filo da inserire sotto la cuticola in area rostrale della camera branchiale (cioè, camera prebranchial di monitoraggio della frequenza respiratoria;. Schapker et al, 2002). Un grande buco avrà eccessiva perdita di emolinfa e non tenuta bene e questo aumenterà la probabilità che i fili non rimanere nel foro designato. Una volta che il filo è a posto, cadere una piccola quantità di colla (cianoacrilato estere) e acceleratore (HobbyTown USA, Lexington, KY) sul filo. L&#39;uso della colla a rapida essiccazione riduce lo stress trattamento degli animali. Fare attenzione a posto solo una piccola quantità di acceleratore sulla cuticola poiché questo è tossico per i gamberi e causerà la morte. La cosa migliore è di cancellare immediatamente qualsiasi acceleratore eccesso con un tovagliolo di carta. Ripetere questi passaggi per il secondo filo nella camera branchiale. Questi due fili sono in grado di coprire l&#39;scaphognathite (organo respiratorio) per assicurare una misura forte impedenza accurata durante ogni movimento scaphognathite (Figura 2). Per ottenere una registrazione accurata, assicurarsi che il secondo filo non è posto all&#39;esterno della camera di branchie e nella camera toracica. <img src="/files/ftp_upload/1594/1594fig2.jpg" style="float:left;" alt="Figura 2" /> Figura 2. Il posizionamento di fili di acciaio inossidabile nella camera branchiale. I puntini bianchi sul carapace rappresentano i punti per i fili di acciaio inox da posizionare sotto la cuticola. I fili coprono l&#39;scaphognathite e del movimento attraverso la misura di impedenza dinamica. La procedura per la frequenza cardiaca cablaggio nella camera toracica è la stessa di sopra eccetto i fili sono posti sotto il carapace dorsale direttamente sul cuore e attraversano il cuore in un rostrale-caudale disposizione (Figura 3). Questa posizione garantisce una misura accurata forte impedenza durante ogni contrazione cardiaca (Listerman et al., 2001). <img src="/files/ftp_upload/1594/1594fig3.jpg" style="float:left;" alt="Figura 3" /> Figura 3. Il posizionamento di fili di acciaio inossidabile nella camera del torace. I puntini bianchi sul carapace rappresentano i punti per i fili di acciaio inox da posizionare sotto la cuticola. I fili attraversano il cuore in un rostrale-caudale accordo. Ciò consentirà di misure di contrazione del cuore attraverso l&#39;impedenza dinamica. Una volta che il posizionamento di tutte e quattro fili è completa, un mix adesivo istantaneo (Eastman, a 5 min e asciugaturaPOXY) e coprire i buchi per ogni posizione così come i fili. Una volta che i fili e fori siano adeguatamente coperti, utilizzare la resina epossidica rimanenti per tenere il tubo di plastica sul retro del gambero. Questo farà sì che i fili non sarà tirato fuori i fori maneggi futuro. Dal momento che la resina epossidica richiede pochi minuti per asciugare, detenere o posto i gamberi in un contenitore senza acqua fino a quando la resina epossidica non è più appiccicoso al tatto. Una volta che la resina epossidica è asciutta, lavare i gamberi in acqua per rimuovere le sostanze chimiche in eccesso e riporle in un ambiente acquatico. Per garantire la salute del gambero, è meglio cambiare l&#39;acqua in circa un&#39;ora per rimuovere tutti i prodotti chimici non precedentemente rimosso. Come rilevato da ricercatori precedenti, abbiamo trovato che una manipolazione prolungata altera le misure fisiologiche dello stress dovuto per la durata di 24 ore a 3 giorni (Wilkens et al, 1985.), Pertanto, gli animali dovrebbero essere lasciati per 2 a 3 giorni prima di ogni sperimentazione . Per registrare ECG, collegare i fili dal gambero di rilevatori di impedenza. I due fili che attraversa il cuore (HR misura) deve essere collegato ad un rilevatore di impedenza singolo e allo stesso modo per i due fili che attraversa il scaphognathite (misura VR). Il rilevatore impedenza misure resistenza dinamica tra i due fili di acciaio inossidabile. Questi segnali vengono registrati collegando il rilevatore on-line ad un PowerLab tramite un&#39;interfaccia PowerLab/4SP (Strumenti dC). Tutti gli eventi sono misurati e calibrati con la versione software 5.5.6 Grafico PowerLab (AD Instruments, Australia). Per iniziare, impostare una velocità di acquisizione a 4 kHz. Le deviazioni di grandi dimensioni rappresenta il battito cardiaco e la frequenza di ventilazione. Per una maggiore facilità nel conteggio ogni HR / VR, può applicare una funzione di smoothing per il set di dati. Se 100 punti sono stati attenuato (cioè la media) la traccia risultante sarà raffigurano solo i battiti cardiaci. La frequenza cardiaca può essere determinato mediante una conta diretta di ogni battito più di 10-s intervalli e poi convertito in battiti per minuto (BPM).

<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

La risposta alla paura, fuga o lotta serve come base fisiologica per l&#39;esame fondamentale la consapevolezza di un organismo del suo ambiente in un attacco imminente predatore. Anche se non è noto se gli invertebrati possiedono un sistema nervoso autonomo identica a quella dei vertebrati, le prove dimostrano gli invertebrati hanno una simpatica reazione simile per regolare l&#39;ambiente interno e pronto l&#39;organismo ad agire comportamentale a determinati stimoli. Inoltre, questa risposta fisiologica può essere misurata e tenerne agisce come un indice biologico per lo stato interno dell&#39;animale. Misure della risposta fisiologica può essere direttamente correlati ai fattori di stress interni ed esterni attraverso i cambiamenti nel coordinamento del sistema nervoso centrale controllato dei sistemi cardio-vascolari e respiratorie. Più in particolare, il monitoraggio cardiaco e dei tassi di ventilazione forniscono misure quantificabili della risposta allo stress comportamentale non sempre osservate. Gamberi sono organismi buon modello per la misurazione della frequenza cardiaca e respiratoria a causa della possibilità di registrazione, così come la ricca storia conosciuta della morfologia del gambero, risalente al Huxley nel 1888, ed i comportamenti tipici ben studiato.

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

Invertebrati mostrano un autonomo simpatico, come risposta simile a quella descritta per i vertebrati. Il coordinamento dei sistemi cardio-vascolare e respiratorio permette per la misurazione di un indice biologico in cui per valutare uno stato interno dell&#39;organismo.

Misure di Cuore e Tariffe ventilatoria a muoversi liberamente gamberi di fiume

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 Views.  University of Kentucky. Invertebrati mostrano un autonomo simpatico, come risposta simile a quella descritta per i vertebrati. Il coordinamento dei sistemi cardio-vascolare e respiratorio permette per la misurazione di un indice biologico in cui per valutare uno stato interno dell'organismo.

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.

L&#39;impianto di ingegneria tessutale nel Cuore Rat

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

Ricerca

Biologia

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.

Le registrazioni di attivazione circuito neurale in Liberamente Comportarsi Animali

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.

Vedi storici e dimostrazione di Fisiologia presso la NMJ del Muscolo Opener gamberi di fiume

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.

Preparazione di embrioni per la microscopia elettronica del Drosophila Tubo cuore embrionale

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.

Quantificazione dei γH2AX Foci in risposta alle radiazioni ionizzanti

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.

ΓH2AX quantificazione dei Foci, a campioni di tessuto

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.

Valutazione della distribuzione spaziale delle γH2AX seguenti radiazioni ionizzanti

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.

Clonogenica saggio: cellule aderenti

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.

Valutazione del calcio Sparks in intatte fibre muscolari scheletriche

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.

Misura di mRNA Decay Prezzi in<em&gt; Saccharomyces cerevisiae</em&gt; Utilizzo<em&gt; Rpb1-1</em&gt; Ceppi

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