Gli autori sono grati per i contributi artistici di Hyewon Cooper.

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tension+receptors+on+the+closer+muscle+apodeme+in+the+walking+legs+of+the+blue+crab+Callinectes+sapidus.">Tension receptors on the closer muscle apodeme in the walking legs of the blue crab Callinectes sapidus.</a> J. Comp. Physiol. A. 157 (3), 355-362 (1985).</li><li>Holsinger, R. <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 effect of regional phenotypic differences of Procambarus clarkii opener muscle on sarcomere length, fiber diameter, and force development MSc thesis [dissertation]. , (2013).</li><li>Parsons, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphology+and+ultrastructure+of+tension+receptors+in+the+walking+legs+of+the+crab,+Carcinus+maenas.">The morphology and ultrastructure of tension receptors in the walking legs of the crab, Carcinus maenas.</a> Cell Tissue Res. 211 (1), 139-149 (1980).</li><li>Parsons, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+and+central+interactions+of+tension+receptors+in+the+leg+muscle+of+Carcinus.">Responses and central interactions of tension receptors in the leg muscle of Carcinus.</a> Comp. Biochem. Physiol. A Physiol. 72 (2), 391-399 (1982).</li><li>Tryba, A. K., Hartman, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+responses+of+series+force+receptors+innervating+the+opener+muscle+apodeme+in+the+blue+crab,+Callinectes+sapidus.">Dynamic responses of series force receptors innervating the opener muscle apodeme in the blue crab, Callinectes sapidus.</a> J. Comp. Physiol. A. 180 (3), 215-221 (1997).</li><li>Whitear, 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+fine+structure+of+crustacean+proprioceptors.+I.+The+chordotonal+organs+in+the+legs+of+the+shore+crab,+Carcinus+meanas.">The fine structure of crustacean proprioceptors. I. The chordotonal organs in the legs of the shore crab, Carcinus meanas.</a> Philos. Trans. R. Soc. Lond. B Biol. Sci. 245 (725), 291-325 (1962).</li><li>Whitear, 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+fine+structure+of+crustacean+proprioceptors.+II.+The+thoracico-coxal+organs+in+Carcinus,+Pagurus+and+Astacus.">The fine structure of crustacean proprioceptors. II. The thoracico-coxal organs in Carcinus, Pagurus and Astacus.</a> Philos. Trans. R. Soc. Lond. B Biol. Sci. 248 (752), 437-456 (1965).</li><li>Leksrisawat, B., Cooper, A. S., Gilberts, A. B., 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=Muscle+Receptor+Organs+in+the+Crayfish+Abdomen:+A+Student+Laboratory+Exercise+in+Proprioception.">Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception.</a> J. Vis. Exp. (45), e2323 (2010).</li><li>Baierlein, B., Thurow, A. L., Atwood, H. L., 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=Membrane+Potentials,+Synaptic+Responses,+Neuronal+Circuitry,+Neuromodulation+and+Muscle+Histology+Using+the+Crayfish:+Student+Laboratory+Exercises.">Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises.</a> J. Vis. Exp. (47), e2322 (2011).</li><li>Delaney, K., Gelperin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+interneurons+controlling+fictive+feeding+in+Limaxmaximus;+I.+Anatomy+and+criteria+for+re-identification.">Cerebral interneurons controlling fictive feeding in Limaxmaximus; I. Anatomy and criteria for re-identification.</a> J. Comp. Physiol. A. 166, 297-310 (1990).</li><li>Hartman, H. B., 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=Regeneration+and+molting+effects+on+a+proprioceptor+organ+in+the+Dungeness+crab,+Cancer+magister.">Regeneration and molting effects on a proprioceptor organ in the Dungeness crab, Cancer magister.</a> J. Neurobiol. 25 (5), 461-471 (1994).</li><li>Hartman, H. B., Austin, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proprioceptor+organs+in+the+antennae+of+Decapoda+Crustacea.">Proprioceptor organs in the antennae of Decapoda Crustacea.</a> J. Comp. Physiol. 81 (2), 187-202 (1972).</li><li>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=Development+of+sensory+processes+during+limb+regeneration+in+adult+crayfish.">Development of sensory processes during limb regeneration in adult crayfish.</a> J. Exp. Biol. 201 (Pt 11), 1745-1752 (1998).</li><li>Chung, Y. -. S., Cooper, R. M., Graff, J., 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=The+acute+and+chronic+effect+of+low+temperature+on+survival,+heart+rate+and+neural+function+in+crayfish+(Procambarus+clarkii)+and+prawn+(Macrobrachium+rosenbergii)+species.">The acute and chronic effect of low temperature on survival, heart rate and neural function in crayfish (Procambarus clarkii) and prawn (Macrobrachium rosenbergii) species.</a> Open J. Mol. Int. Physiol. 2 (3), 75-86 (2012).</li><li>Blundon, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+temperature+and+thermal+history+on+neuromuscular+properties+of+two+crustacean+species.">Effects of temperature and thermal history on neuromuscular properties of two crustacean species.</a> J. Comp. Physiol. B. 158 (6), 689-696 (1989).</li><li>Cooper, R. M., Schapker, H., Adami, 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+and+ventilatory+measures+in+crayfish+during+copulation.">Heart and ventilatory measures in crayfish during copulation.</a> Open J. Mol. Int. Physiol. 1 (3), 36-42 (2011).</li><li>Bierbower, S. M., Cooper, R. 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Gli autori dichiarano di non avere interessi finanziari in competizione.

Lo scopo di questa serie di esperimenti è 1) insegnare ed esporre i principi fondamentali di registrazioni extracellulari da un organo propriocettiva identificabile e la tensione nervosa e 2) per sottolineare l&#39;importanza della mappatura anatomica in relazione alla funzione fisiologica di particolari sistemi sensoriali. Questo approccio sperimentale e modelli animali utilizzati sono poco costoso e relativamente facile da condurre in laboratori didattici neurofisiologia. I neuroni di organi cordotonali sono di due tipi funzionali specifici, quelli che rispondono al movimento e coloro che rispondono a posizioni statiche. Registrazioni di cellule singole a partire da una varietà di organi cordotonali, non importa quale viene esaminato congiunta, hanno dimostrato che questo sia il caso di 3,5. Infatti, gli organi cordotonali associati alle articolazioni antennali di aragoste rivelano gli stessi due tipi sensoriali e anatomia di base 27. Oltre a ci siano due tipi di neuroni (movimento e position), i neuroni condividono la stessa disposizione anatomica sui rispettivi fili elastici. Il grande somata trova prossimale sul filamento tendono ad appartenere ai neuroni sensibili movimento dinamico. I neuroni che segnalano posizioni statiche hanno piccole somata e si trovano distale. Queste cellule sono tonico attivo. Il giunto PD contiene solo un singolo organo cordotonali mentre ci sono due organi cordotonali nel carpo-propodus (CP) e merus-carpo (MC) articolazioni. La dissezione di esporre le strutture propriocettivi in granchi blu (C. sapidus) per la registrazione elettrofisiologica richiede una strategia che permette movimenti articolari che si terrà nelle posizioni naturali, mentre la registrazione da neuroni sensoriali. Il nervo tensione per il muscolo opener nella gamba a piedi è un bellissimo nervo composto da diversi neuroni. A meno che non si fa attenzione, il nervo tensione nonché il nervo motore che innervano il muscolo da stimolare, possono essere danneggiati durante questa dissezione. Perregistrazioni ottimali gli elettrodi di aspirazione devono essere adattati alle dimensioni del nervo. Le registrazioni sono facilmente accessibili in un laboratorio studente utilizzando un microscopio ingrandimento di dissezione 30-40X e micromanipolatori di fascia bassa. Futuri esperimenti che sarebbe interessante proseguire con gli organi cordotonali comune sarebbe quello di esaminare i profili strutturali e fisiologiche durante la rigenerazione gamba in varie specie nelle diverse fasi del ciclo di vita come un follow-up a un primo studio che ha utilizzato Cancer magister 19,26 . Domande ancora da affrontare sono: 1) non effettua la distribuzione e l&#39;organizzazione dei neuroni artificiali dipendono dall&#39;età dell&#39;animale quando rigenerare un arto, 2) sono le proiezioni assonale al CNS (cordone nervoso ventrale) in un arto rigenerante funzionale o lo fa prendere tempo e l&#39;uso congiunto di stabilire connessioni funzionali, e 3) che cosa accade agli assoni mozzate prossimale al piano autotomy quando l&#39;arto è autotomized? 28 Crostacei conformi alle condizioni ambientali e la loro temperatura circostante, ma non è chiaro come si mantengono coordinamento all&#39;interno di un circuito neurale come neuroni alterano la loro attività in risposta alle variazioni di temperatura. Un basso tasso di cambiamento potrebbe permettere all&#39;animale po &#39;di tempo per acclimatarsi, mentre un rapido cambiamento può not29, 30. Cambiamenti fisiologici di pH o di osmolarità a causa di metabolismo, il comportamento 31, o l&#39;impatto ambientale, possono presentare problemi simili ai circuiti neurali coinvolti nella propriocezione. Queste preparazioni crostacei sono ideali per affrontare questi tipi di problemi perché la loro funzione è ben caratterizzata a livello di singola cellula. In questo protocollo abbiamo dimostrato l&#39;importanza fisiologica dei neuroni tensione in forza generata dal muscolo apri monitoraggio. Questi recettori di tensione possono essere ricondotte alla loro posizione all&#39;interno della apodeme utilizzando procedure di colorazione. Questeneuroni, come nei mammiferi, rilevano forza a vari livelli e reclutare neuroni addizionali come la forza aumenta. La frequenza di attività è correlata alla frequenza di stimolazione del motoneurone fino a saturazione in ricezione. Utilizzando un protocollo sgancio rapido con il giunto dactylus flessa, attività tensione sparisce rapidamente, ma poi torna sui riconquistare la tensione in un giunto completamente esteso. Questa è una procedura sperimentale classico per illustrare la forza misurata dai recettori di tensione. Vari neuromodulatori possono essere applicati alla preparazione di vedere come effettua lo sviluppo della forza e della risposta neuronale. Uno degli aspetti importanti è come le risposte neurali vengono elaborati e integrati nel sistema nervoso centrale e loro impatto sull&#39;attività dei neuroni motori. Le tecniche che abbiamo mostrato permettono di iniziare ad affrontare ulteriori informazioni sulla (sensoriale) Funzione circuito neurone nervo motore tensione, cioè il segnale in una gamba intatta al ganglio e ritornoal muscolo. Le procedure di colorazione sono dimostrati chiave per comprendere la fisiologia dei neuroni sensoriali che innervano organi propriocettivi. Disposizione anatomica dei neuroni in base alla funzione e la dimensione del soma sono simili nei vari organi cordotonali all&#39;interno delle gambe di granchio. Non è noto se accordi neuronali simili si trovano anche in altre specie di insetti o crostacei. Combinando le registrazioni fisiologiche da singole cellule e la mappatura della posizione permette relazioni funzionali struttura diretta. La conservazione a lungo termine del regime anatomica con CoCl 2 colorazione e dal fissaggio permette di fare ripetutamente le misure e valutare la disposizione strutturale. Propriocezione e ricezione tensione dei muscoli scheletrici sono modalità sensoriali che consentono comportamenti coordinati e risposte ambiente esterno ed interno per animali articolati in una varietà di configurazioni di muscolo scheletricos. L&#39;organo recettore muscolare nell&#39;addome del gambero è un altro preparato ben documentata (vedi il Progetto Crawdad, <a href="http://www.crawdad.cornell.edu/">http://www.crawdad.cornell.edu/</a> ) a fini didattici di propriocezione con soli due neuroni per addominali emi-segmento 23 . Essere in grado di registrare da singoli neuroni a fasci nervosi sensoriali fornisce ulteriori dettagli che aiuti a comprendere i principi fondamentali della percezione sensoriale. Questi relativamente semplici preparazioni di crostacei permettono di affrontare gli aspetti fondamentali della propriocezione e controllo tensione, con il potenziale per determinare i circuiti neurali che consentono l&#39;integrazione centrale di ingressi propriocettivi e altri sensoriali 9-12, 32, 33.

Propriocezione è la sensazione di posizione dell&#39;arto e movimento che consente comportamento coordinato motore. Propriocettori consistono in posizione (statico) e il movimento recettori (cinestetica). In insetti e crostacei, gli organi cordotonali sono le strutture che forniscono tali informazioni al sistema nervoso centrale 1. Non tutti gli organi cordotonali abbracciano una comune, ma possono ancora controllare i movimenti articolari a causa del loro attaccamento alle apodemes (come le strutture tendinee), che coprono il giunto e si muovono in associazione con il muscolo scheletrico e l&#39;articolazione congiunta. Granchio gambe hanno sei giunti, ciascuno con uno o due organi cordotonali 2. Tipicamente un organo cordotonali ha 60-100 o più neuroni sensoriali incorporati all&#39;interno di un filo elastico, i neuroni che segnalano la posizione comune statica, direzione e velocità di movimento 3-6. L&#39;ingresso da organi cordotonali ad ogni articolazione e la gamba viene poi elaborato centralmente permettendo movimenti coordinati da parte degli animali. <p class="jove_content"&gt; Le forze che muscoli delle gambe producono durante contrazioni isometriche e isotoniche vengono rilevati dai recettori di tensione associati alle fibre muscolari e dei loro allegati ai apodemes7-9. Nei protocolli gambe piedi crostacei che seguono vi presentiamo metodologia per registrazioni da neuroni sensoriali primari che controllano la propriocezione e neuroni che rispondono alle forze generate da fibre muscolari. Una tecnica per l&#39;attivazione movimenti delle gambe e quantificare generazione della forza è presentato anche, così come le tecniche anatomiche che possono essere utilizzati per caratterizzare la disposizione di queste strutture del sistema nervoso periferico. Le procedure illustrate in dettaglio successivamente consentire l&#39;analisi strutturale e funzionale dei neuroni che innervano entrambi i tipi di recettori relativi alla loro posizione su un filo elastico cordotonali e apodeme. Per illustrare, usiamo il propodite-dactylopodite (PD) organo cordotonali, l&#39;organo che attraversa il segmento più distale della gamba granchio 3.Anche se studi elettrofisiologici dettagliati iniziarono nel 1930 e sono ancora in corso oggi, alcuni aspetti sono diventati noti per le connessioni segmentale dei propriocettori nelle varie articolazioni e il loro ruolo nel controllo coordinato di muscles10-16. Stabilire il rapporto struttura-funzione tra gli organi propriocettivi, i muscoli e il sistema nervoso ulteriormente aiutare a definire questi ruoli. Ad esempio, l&#39;etichettatura del somata e terminazioni distali dei neuroni tensione inserite nel apodeme rivelerà la loro posizione rispetto alle fibre muscolari 8,17-21. Presentiamo tre tecniche di colorazione per le gambe dei crostacei che possono essere utilizzati nella ricerca o laboratori accademici. Metilene colorazione blu offre un contrasto adeguato per muscoli e nervi ed è raccomandato come una semplice tecnica per gli studenti di imparare l&#39;anatomia. Labs che hanno configurazioni microscopia a fluorescenza possono realizzare più selettivo colorazione neuronale esponendo brevemente il nervos al colorante vitale a 4-di-2-ASP. La terza alternativa è CoCl 2 di recupero, che colora e fissa i neuroni, e non necessita di imaging di fluorescenza. Anche se è lavoro e tempo intenso, questo processo di colorazione conferisce un elevato contrasto e specificità per i nervi che sono pieni. Insieme, queste tecniche possono essere utilizzate per la comparazione dei vari organi cordotonali, non solo all&#39;interno di un arto o tra gli arti, ma anche tra le altre specie di crostacei e insetti 20-22. Granchi blu (C. sapidus) utilizzati nelle registrazioni fisiologiche e anatomiche per la colorazione sono facilmente disponibili in tutto il confine meridionale e sud-est degli Stati Uniti. Questa specie funge da rappresentante del regime nervose cordotonali e la tensione si trovano nella maggior parte dei granchi. Laboratori sulla costa occidentale preferiscono utilizzare il molto più grande granchio Dungeness (Cancer magister) per questi esperimenti.

<table border="0" cellpadding="0" cellspacing="0" width="686">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sylgard</td>
			<td style="width:184px;">Dow Corning</td>
			<td style="width:119px;">182 silicone kit</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">S7653</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">P9333</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CaCl2</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">C5670</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES acid</td>
			<td style="width:184px;">Sigma</td>
			<td style="width:119px;">3375</td>
			<td style="width:167px;">Free acid, crystalline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES base</td>
			<td style="width:184px;">Sigma</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">D-Glucose</td>
			<td style="width:184px;">Sigma</td>
			<td style="width:119px;">G7021</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">MgSO4&#8226;7H2O</td>
			<td style="width:184px;">Sigma</td>
			<td style="width:119px;">M2643</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Na2SO4&#8226;10H2O</td>
			<td style="width:184px;">Sigma</td>
			<td style="width:119px;">246980</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Bouin&#8217;s solution fixative</td>
			<td style="width:184px;">Sigma</td>
			<td style="width:119px;">HT10-1-32</td>
			<td style="width:167px;">Caution: Hazardous material (Special shipping cost required)</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">CoCl2</td>
			<td style="width:184px;">Sigma</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Caution: Hazardous material. Please follow proper disposal according to local and federal regulations.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Methylene blue chloride</td>
			<td style="width:184px;">Matheson Co., Inc</td>
			<td style="width:119px;">Basic Blue 9, C.I. 52015</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">4-Di-2-ASP</td>
			<td style="width:184px;">Molecular Probes</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">4-(4-diethylaminostyryl)-N-methylpyridinium iodide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bleach</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">To chloride silver wire</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaOH</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">221465</td>
			<td style="width:167px;">To adjust pH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HCl</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">H1758</td>
			<td style="width:167px;">To adjust pH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Materials</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissecting tools</td>
			<td style="width:184px;">World Precision Instruments</td>
			<td style="width:119px;">assortment</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Intracellular electrode probe</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Faraday cage</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Insect Pins</td>
			<td style="width:184px;">Fine Science Tools, Inc</td>
			<td style="width:119px;">26001-60</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissecting microscope (100X)</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fiber optic lamp</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Small adjustable mirror</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">To direct light beam toward the preparation.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass electrodes</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">CLS7095B5X</td>
			<td style="width:167px;">Box of 200, suction electrodes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micromanipulator</td>
			<td style="width:184px;">World Precision Instruments</td>
			<td style="width:119px;">MD4-M3-R</td>
			<td style="width:167px;">Can fix for base or on a metal rod</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Raised preparation stand</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Silver wire (10/1,000 inch)</td>
			<td style="width:184px;">A-M Systems</td>
			<td style="width:119px;">782500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Computer</td>
			<td style="width:184px;">Any company</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AC/DC differential amplifier</td>
			<td style="width:184px;">A-M Systems</td>
			<td style="width:119px;">Model 3000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PowerLab 26T</td>
			<td style="width:184px;">AD Instruments</td>
			<td style="width:119px;">27T</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Force transducer</td>
			<td style="width:184px;">AD Instruments</td>
			<td style="width:119px;">0-50g</td>
			<td style="width:167px;">MLTF050/ST</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Head stage</td>
			<td style="width:184px;">AD Instruments</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Comes with AC/DC amplifier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LabChart7</td>
			<td style="width:184px;">AD Instruments</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electrical leads</td>
			<td style="width:184px;">Any company</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass tools</td>
			<td style="width:184px;">Make yourself</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">For manipulating nerves</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cable and connectors</td>
			<td style="width:184px;">Any company</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipettes with bulbs</td>
			<td style="width:184px;">Fisher Scientific</td>
			<td style="width:119px;">13-711-7</td>
			<td style="width:167px;">Box of 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Beakers</td>
			<td style="width:184px;">Any company</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Wax or modeling clay</td>
			<td style="width:184px;">Any company or local stores</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stimulator</td>
			<td style="width:184px;">Grass Instruments</td>
			<td style="width:119px;">SD9 or S88</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="84" rowspan="2" style="height:84px;width:216px;">Plastic tip for suction electrode</td>
			<td rowspan="2" style="width:184px;">Local hardware store (Watt&#8217;s brand)</td>
			<td rowspan="2" style="width:119px;">&#188; inch OD x 0.170 inch ID</td>
			<td style="width:167px;">Cut in small pieces. Pull out over a flame and cut back the tip to the correct size</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:167px;"></td>
		</tr>
	</tbody>
</table>

proprioception and tension receptors in crab limbs: student laboratory exercises

Lo scopo principale di queste procedure è di dimostrare per fini didattici e di ricerca come registrare l&#39;attività dei neuroni sensoriali primari vivere responsabili della propriocezione come sono Rilevazione della posizione comune e il movimento, e la tensione muscolare. Attività elettrica da propriocettori crostacei e recettori di tensione viene registrata dalla strumentazione neurofisiologico di base, e un trasduttore viene utilizzato per misurare simultaneamente forza generata stimolando un nervo motore. Inoltre, dimostriamo come macchiare i neuroni per una rapida valutazione della loro organizzazione anatomica o per il fissaggio permanente. La colorazione rivela organizzazione anatomica che è rappresentativo di organi cordotonali nella maggior parte dei crostacei. Confrontando le risposte tensione nervose verso le risposte propriocettive è uno strumento didattico efficace nel determinare come questi neuroni sensoriali sono definiti funzionalmente e come l&#39;anatomia è correlata alla funzione. Tre tecniche di colorazionesono presentati consentendo ai ricercatori e istruttori di scegliere un metodo che è ideale per il loro laboratorio.

Quando l&#39;organo PD è allungato estendendo completamente il giunto, attività nel nervo PD è robusto durante il movimento come illustrato per il primo secondo in Figura 10. Alcune attività rimane mentre è tenuto fermo nella posizione di apertura. Questa attività è dai neuroni sensibili statici posizione (seconda metà di registrazione mostrato nella Figura 10). Movimento evoca una risposta durante lo spostamento, e la cottura è presente soprattutto durante l&#39;allungamento del filamento cordotonali (Figura 11). Ulteriori analisi delle punte può essere facilmente avvicinato di classificare le relative ampiezze. Questo è un approccio per dimostrare le diverse popolazioni di neuroni sensoriali essere reclutati per posizioni o tipi di movimenti 5. Ampiezze tipiche vanno 0,25-1,5 mV, ma questi valori dipendono dalla resistenza (cioè tenuta) della tenuta elettrodo di aspirazione. Frequenza dei picchi di tha varie gamme di dimensioni può anche essere rappresentato graficamente per l&#39;analisi. Forze generate dal muscolo apri rispetto alla frequenza di stimolazione possono essere confrontati sovrapponendo le rispettive tracce tensione-tempo sopra l&#39;altra (Figure 13A e B). Questo può anche essere eseguita per ogni posizione collettiva a ogni data frequenza di stimolazione. Attività del nervo tensione può poi essere correlato con la quantità di forza relativa generato per ciascuna frequenza di stimolazione, e per ogni posizione comune. Come nel nervo PD, una varietà di ampiezze picco sono considerati in risposta alla contrazione del muscolo apri (Figura 13C). Disposizione anatomica dei neuroni nella gamba a piedi è chiaramente osservata con colorazione blu di metilene (Figura 14). Notare il cordotonali filo e la tensione neurone elastico che si trova vicino al apodeme. Diversi somata con differenti diametri did località specifiche sono visibili anche in questa figura. L&#39;intero corso del nervo tensione e barella nervo motore sono mostrati in Figura 15. Neuroni individuali del nervo PD sono mostrati con maggiore contrasto utilizzando 4-Di-2-ASP (Figura 16) e CoCl 2 (Figura 17) di recupero informazioni tecniche. Ad alto ingrandimento le terminazioni sensoriali possono essere visti all&#39;interno delle scolopales di supporto (Figure 16B) 21,22,26. <img alt="Figura 10" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig10.jpg" width="500px" /> Figura 10. Spostare e tenere a 0 °. I neuroni dinamici sono robusti in cottura durante il movimento e picchi di neuroni sensibili all&#39;elettricità statica sono presenti mentre il comune è tenuto aperto. <a href="https://www.jove.com/files/ftp_upload/51050/51050fig10highres.jpg" target="_blank">Clicca qui per vedere l&#39;immagine ingrandita &lt;</a>/ A&gt;. <img alt="Figura 11" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig11.jpg" width="500px" /> Figura 11. Rapid aperto e chiusura da completamente flessa in posizione estesa (90-0 °). <a href="https://www.jove.com/files/ftp_upload/51050/51050fig11highres.jpg" target="_blank">Clicca qui per vedere l&#39;immagine ingrandita</a> . <img alt="Figura 12" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig12.jpg" width="500px" /> Figura 12. Extracellulari picchi registrati dal nervo PD. Il giunto è completamente estesa e poi rapidamente spostato in una ½ posizione flessa e poi tenendola ferma. Si noti l&#39;attività durante il movimento e la diminuzione dell&#39;attività quando statica. <a href="https://www.jove.com/files/ftp_upload/51050/51050fig12highres.jpg" target="_blank">Clicca qui per ingrandire ima</a>ge. <img alt="Figura 13" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig13.jpg" width="500px" /> Figura 13. Le forze relative che si sviluppano con il giunto completamente flesse e stimolati alle varie frequenze. (A) tracce Voltage-time dal trasduttore di forza sono indicati con ogni frequenza di stimolazione. (B) Le tracce nel pannello A si sovrappongono in diversi colori per la facilità di confronto. (C) Tensione in tempo le tracce di attività elettrica registrate dal nervo tensione quando il nervo motore è stato stimolato a 80 Hz. Si noti la struttura regolare di manufatti come stimolo rispetto all&#39;attività neurale. Inoltre, notare le diverse ampiezze delle risposte neurali. <a href="https://www.jove.com/files/ftp_upload/51050/51050fig13highres.jpg" target="_blank">Clicca qui per vedere l&#39;immagine ingrandita</a> . Figura 14. Blu di metilene macchia della preparazione gamba a piedi. Somas individuali vengono mostrati con le loro terminazioni sensoriali proiettando nel filo elastico. Vicino alla apodeme un neurone tensione è mostrato. <a href="https://www.jove.com/files/ftp_upload/51050/51050fig14highres.jpg" target="_blank">Clicca qui per vedere l&#39;immagine ingrandita</a> . <img alt="Figura 15" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig15.jpg" width="500px" /> Figura 15. Il nervo tensione derivante dalla estremità distale (frecce rosse) e unire il nervo motore (freccia verde). <a href="https://www.jove.com/files/ftp_upload/51050/51050fig15highres.jpg" target="_blank">Clicca qui per vedere l&#39;immagine ingrandita</a> . Figura 16. (A) Un back-riempimento del nervo PD in Cancer magister con 4-Di-2-ASP. (B) Maggiore ingrandimento delle terminazioni sensitive. <a href="https://www.jove.com/files/ftp_upload/51050/51050fig16highres.jpg" target="_blank">Clicca qui per vedere l&#39;immagine ingrandita</a> . <img alt="Figura 17" fo:content-width="5in" src="/files/ftp_upload/51050/51050fig17.jpg" width="500px" /> Figura 17. I neuroni che sono stati riempiti con CoCl 2 e trattati (A). Contorno Tracciato della preparazione macchiato mostrato (B). <a href="https://www.jove.com/files/ftp_upload/51050/51050fig17highres.jpg" target="_blank">Clicca qui per vedere l&#39;immagine ingrandita</a> . <table border="0" cellpadding="0" cellspacing = &quot;0&quot; width = &quot;392&quot;&gt; <colgroup><col /><col /></colgroup><tbody><tr height="21"><td height="21" style="height:21px;width:235px;"> Salino </td><td style="width:157px;"> g / L </td></tr><tr height="20"><td height="20" style="height:20px;width:235px;"> NaCl </td><td style="width:157px;"> 27.29 </td></tr><tr height="20"><td height="20" style="height:20px;width:235px;"> KCl </td><td style="width:157px;"> 0.81 </td></tr><tr height="26"><td height="26" style="height:26px;width:235px;"> MgSO 4 • 7H 2 O </td><td style="width:157px;"> 4.81 </td></tr><tr height="26"><td height="26" style="height:26px;width:235px;"> CaCl 2 • 2H 2 O </td><td style="width:157px;"> 1.85 </td></tr><tr height="26"><td height="26" style="height:26px;width:235px;"> Na 2 SO 4 • 10H 2 O </td><td style="width:157px;"> 0.97 </td></tr><tr height=&quot;20&quot;&gt; <td height="20" style="height:20px;width:235px;"> Destrosio (D-glucosio) </td><td style="width:157px;"> 1.982 </td></tr><tr height="20"><td height="20" style="height:20px;width:235px;"> L&#39;acido HEPES </td><td style="width:157px;"> 0,476 </td></tr><tr height="20"><td height="20" style="height:20px;width:235px;"> HEPES sale </td><td style="width:157px;"> 2.08 </td></tr><tr height="40"><td height="40" style="height:40px;width:235px;"> Regolare a pH 8.1 con NaOH o HCl </td><td style="width:157px;"></td></tr></tbody></table> Tabella 1. Ricette per granchi salina.

Watch this Scientific Journal Video about Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises at JoVE.com

Proprioception and Tension Receptors in Crab Limbs | Biology | Video

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

Tecniche fisiologiche e anatomiche sono dimostrati per affrontare funzione e la struttura per propriocettori articolari e recettori tensione muscolare degli arti crostacei piedi.

Propriocezione e la tensione recettori in Crab Arti: Esercizi di laboratorio per studenti

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons ...

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25.0K Views.  University of Kentucky.  Tecniche fisiologiche e anatomiche sono dimostrati per affrontare funzione e la struttura per propriocettori articolari e recettori tensione muscolare degli arti crostacei piedi. 

Video: Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

Combinando comportamentali Endocrinologia ed Economia Sperimentale: Decisione testosterone e sociale Fare

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

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A Procedure for Studying the Footshock-Induced Reinstatement of Cocaine Seeking in Laboratory Rats

The most insidious aspect of drug addiction is the high propensity for relapse. Animal models of relapse, known as reinstatement procedures, have been used extensively to study the neurobiology and phenomenology of relapse to drug use. Although procedural variations have emerged over the past several decades, the most conventional reinstatement procedures are based on the drug self-administration (SA) model. In this model, an animal is trained to perform an operant response to obtain drug. Subsequently, the behavior is extinguished by withholding response-contingent reinforcement. Reinstatement of drug seeking is then triggered by a discrete event, such as an injection of the training drug, re-exposure to drug-associated cues, or exposure to a stressor 1.
Reinstatement procedures were originally developed to study the ability of acute non-contingent exposure to the training drug to reinstate drug seeking in rats and monkeys 1, 2. Reinstatement procedures have since been modified to study the role of environmental stimuli, including drug-associated cues and exposure to various forms of stress, in relapse to drug seeking 1, 3, 4.
Over the past 15 years, a major focus of the reinstatement literature has been on the role of stress in drug relapse. One of the most commonly used forms of stress for studying this relationship is acute exposures to mild, intermittent, electric footshocks. The ability of footshock stress to induce reinstatement of drug seeking was originally demonstrated by Shaham and colleagues (1995) in rats with a history of intravenous heroin SA5. Subsequently, the effect was generalized to rats with histories of intravenous cocaine, methamphetamine, and nicotine SA, as well as oral ethanol SA 3, 6.
Although footshock-induced reinstatement of drug seeking can be achieved reliably and robustly, it is an effect that tends to be sensitive to certain parametrical variables. These include the arrangement of extinction and reinstatement test sessions, the intensity and duration of footshock stress, and the presence of drug-associated cues during extinction and testing for reinstatement. Here we present a protocol for footshock-induced reinstatement of cocaine seeking that we have used with consistent success to study the relationship between stress and cocaine seeking.

Animal models of relapse, known as reinstatement procedures, have been used extensively to study the role of stress in relapse to drug seeking. Here, we report on a method for inducing the reinstatement of cocaine seeking in laboratory rats via acute exposures to mild, intermittent electric footshock.

Una procedura per lo studio della Footshock indotta Ripristino di cocaina Cerco nei ratti di laboratorio

The most insidious aspect of drug addiction is the high propensity for relapse.  Animal models of relapse, known as reinstatement procedures, have been ...

Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell's membrane potential and which we address in these experiments are: (1) Ion concentration of K+ on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.

The experiments demonstrate an easy approach for students to gain experience in examining muscle structure, synaptic responses, the effects of ion gradients and permeability on membrane potentials. Also, a sensory-CNS-motor-muscle circuit is presented to show a means to test effects of compounds on a neuronal circuit.

Potenziali di membrana, Risposte Synaptic, circuiti neuronali, neuromodulazione e Istologia muscolare Utilizzando la Gamberi: Esercizi di laboratorio per studenti

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that ...

Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive information for an animal. An additional objective of this experiment is to learn anatomy of the preparation by staining, dissection and viewing of neurons and sensory structures under a dissecting microscope. This is performed by using basic neurophysiological equipment to record the electrical activity from a joint receptor organ and staining techniques. The muscle receptor organ (MRO) system in the crayfish is analogous to the intrafusal muscle spindle in mammals, which aids in serving as a comparative model that is more readily accessible for electrophysiological recordings. In addition, these are identifiable sensory neurons among preparations. The preparation is viable in a minimal saline for hours which is amenable for student laboratory exercises. The MRO is also susceptible to neuromodulation which encourages intriguing questions in the sites of modulatory action and integration of dynamic signals of movements and static position along with a gain that can be changed in the system.

The primary purpose of this experiment is to understand how primary sensory neurons convey information of joint movements and positions as proprioceptive information for an animal. An additional objective of this report is present the anatomy of the preparation by dissection and viewing of neurons under a dissecting microscope.

Receptor Organi muscolare all&#39;addome gamberi: un esercizio di laboratorio per studenti in propriocezione

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive ...

Physiological Experimentation with the Crayfish Hindgut: A Student Laboratory Exercise

The purpose of the report is to describe dissection techniques for preparing the crayfish hindgut and to demonstrate how to make physiological recordings with a force transducer to monitor the strength of contraction. In addition, we demonstrate how to visually monitor peristaltic activity, which can be used as a bioassay for various peptides, biogenic amines and neurotransmitters. This preparation is amenable to student laboratories in physiology and for demonstrating pharmacological concepts to students. This preparation has been in use for over 100 years, and it still offers much as a model for investigating the generation and regulation of peristaltic rhythms and for describing the mechanisms underlying their modulation. The pharmacological assays and receptor sub-typing that were started over 50 years ago on the hindgut still contribute to research today. This robust preparation is well suited to training students in physiology and pharmacology.

In this report we demonstrate techniques that can be used to investigate the biology of the crayfish hindgut. We show how to dissect a crayfish abdomen and study the associated anatomy, physiology and modulation of activity. The peristaltic activity and strength of contractions are measured using a force transducer.

La sperimentazione con la fisiologica hindgut gamberi: un esercizio di laboratorio per studenti

The purpose of the report is to describe dissection techniques for preparing the crayfish hindgut and to demonstrate how to make physiological recordings ...

Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells

The vivid world of odors is recognized by the sense of olfaction. Olfaction in mice is mediated by a repertoire of about 1200 G Protein Coupled Receptors (GPCRs) 1 that are postulated to bind volatile odorant molecules and converting the extracellular signal into an intracellular signal by coupling with G protein G&alpha;olf. Binding of the odorants to the receptors is thought to follow a combinatorial rule, that is, one odorant may bind several receptors and one receptor may bind several odorants to varying degrees 2. Biochemical, signaling and ligand binding studies have been conveniently carried out for most GPCRs using heterologous cells. However use of heterologous cells for study of odorant receptors, was precluded for a long time since on transfection they failed to export to the surface. Saito et al have demonstrated single membrane pass Receptor Transporting Protein (RTP) family chaperones show enhanced expression in the olfactory sensory neurons and act as chaperones to traffic odorant receptors to the surface in heterologous cells, when co transfected together 3. To carry out biochemical assays for receptors using heterologous cells, one must first determine if the receptor shows robust surface expression in the cell line. This can be assayed by overexpressing the receptors with the chaperone RTP1S followed by live cell staining to fluorescently label the extracellular domain or a tag in the extracellular domain exclusively. Here we demonstrate a protocol to carry out live cell staining that can be used to detect odorant receptors on the surface of HEK293T cells conveniently. In addition, it may also be used to assay for surface expression of other chemosensory receptors or GPCRs.

Here we demonstrate a protocol to carry out live cell staining that can be used to detect odorant receptors on the surface of HEK293T cells conveniently. In addition, it may also be used to assay for surface expression of other chemosensory receptors or GPCRs.

Saggio espressione di superficie dei recettori chemosensoriali in cellule eterologhe

The vivid world of odors is recognized by the sense of olfaction. Olfaction in mice is mediated by a repertoire of about 1200 G Protein Coupled Receptors ...

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

Imaging ottico di neuroni nel ganglio Granchio Stomatogastric con tensione sensibili Coloranti

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

Uso di pHluorin per valutare la dinamica dei recettori di guida axon nella coltura cellulare e nell'embrione del pulcino

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Indurre Plasticità di astrociti recettori per manipolazione del neuronali di cottura Prezzi

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to perform receptor autoradiographic mapping.
Tissue specimens are harvested and stored at -80 &#176;C. A cryostat is used to coronally section the tissue (brain) and thaw-mount the sections onto charged slides. The slide-mounted tissue sections are incubated in 125I-SI-Ang II to radiolabel Ang II receptors. Adjacent slides are separated into two sets: &#39;non-specific binding&#39; (NSP) in the presence of a receptor saturating concentration of non-radiolabeled Ang II, or an AT1 Ang II receptor subtype (AT1R) selective Ang II receptor antagonist, and &#39;total binding&#39; with no AT1R antagonist. A saturating concentration of AT2 Ang II receptor subtype (AT2R) antagonist (PD123319, 10 &#181;M) is also present in the incubation buffer to limit 125I-SI-Ang II binding to the AT1R subtype. During a 30 min pre-incubation at ~22 &#176;C, NSP slides are exposed to 10 &#181;M PD123319 and losartan, while &#39;total binding&#39; slides are exposed to 10 &#181;M PD123319. Slides are then incubated with 125I-SI-Ang II in the presence of PD123319 for &#39;total binding&#39;, and PD123319 and losartan for NSP in assay buffer, followed by several &#39;washes&#39; in buffer, and water to remove salt and non-specifically bound radioligand. The slides are dried using blow-dryers, then exposed to autoradiography film using a specialized film and cassette. The film is developed and the images are scanned into a computer for visual and quantitative densitometry using a proprietary imaging system and a spreadsheet. An additional set of slides are thionin-stained for histological comparisons.
The advantage of using receptor autoradiography is the ability to visualize Ang II receptors in situ, within a section of a tissue specimen, and anatomically identify the region of the tissue by comparing it to an adjacent histological reference section.

Here we present a protocol to describe the localization of angiotensin II Type 1 receptors in the rat brain by quantitative, densitometric, in vitro receptor autoradiography using an iodine-125 labeled analog of angiotensin II.

Autoradiografia del recettore Protocollo per la visualizzazione localizzata di recettori dell&#39;angiotensina II

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to ...