Supportato dalla concessione NIH R01 MH 099557

1. fabbricazione di elettrodi di registrazione <ol><li>tirando gli elettrodi di vetro <ol><li>uso il seguente protocollo per l'estrattore di microelettrodi (Vedi Tabella materiali): <ol><li>linea 1: calore 510 tirare - velocità 30 tempo 250; Linea 2: Calore 490 Pull - velocità 30 tempo 250. 
			Nota: Unità di tempo corrisponde a 0,5 ms per unità; le altre unità sono relative. Il valore del calore deve essere regolato per ogni filamento dopo viene eseguito il test rampa.</li>
		</ol></li> <li>Utilizzare un microscopio (ingrandimento x 35) per garantire che il diametro interno dell'elettrodo tirato è nella gamma di 7-10 µm ( Figura 1A). Conservare i capillari in contenitori ben chiusi per prevenire l'accumulo di polvere.</li>
	</ol></li> <li>Fuoco lucidatura <ol><li>capillari di fuoco polacco (80-90% del valore massimo del calore per 1-2 s) utilizzando una micro-forgia (Vedi Tabella materiali). Garantire il diametro interno finale dell'elettrodo lucido è 5 µm ( Figura 1A) 27.</li>
	</ol></li> <li>Piegatura Nota: due curve sono realizzate al fine di posizionare l'elettrodo sulla parte superiore del muscolo sotto un obiettivo ad alto ingrandimento.
	<ol><li>Uso l'apparecchio mostrato in Figura 1B. Difficoltà l'elettrodo nel manipolatore e posizionare la punta sul filamento, non toccando it.</li>
		<li>Set valore al 60-70% del massimo calore e premere il pedale della micro-forgia per 1-2 s (Vedi Tabella materiali) per riscaldare il filamento. Utilizzare un ago a forma di L per tirare delicatamente verso il basso la punta dell'elettrodo ( Figura 1B allargata). Fai la curva a circa 90 o ( Figura 1).</li>
		<li>Tenere l'elettrodo sopra la fiamma della torcia a mano usando il forcipe e fare alla seconda curva ad una distanza di 7-10 mm dal primo tornante e ad un angolo di circa 120 o ( Figura 1).</li>
	</ol></li></ol> <img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56493/56493fig1.jpg" /> Figura 1. Fasi finali di fabbricazione micropipetta. (A) elettrodo suggerimenti dopo tirando e lucidatura di fuoco. (B. 1) l'installazione per la piegatura di punta. (B. 2), l'area circoscritta è mostrato allargata a destra. L'elettrodo e il filamento sono fissati in modo che la punta dell'elettrodo è posizionato leggermente di sopra del filo e non toccare. (C. 1) l'elettrodo di registrazione. (C. 2), l'area circoscritta è mostrato allargata a destra. La prima curva ha un angolo di circa 90°, e la distanza tra la prima curva e la punta dell'elettrodo è di circa 1 mm. scala bar = 3 mm. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig1large.jpg" target="_blank"> Clicca qui per visualizzare una versione più grande di questa figura.</a>
 2. ulteriori passaggi preparatori <ol><li>preparare heamolymph-come (HL3) soluzione (in mM): 70 NaCl, 5 KCl, 20 MgCl 2, 10 NaHCO 3, 5 trealosio, saccarosio 115, 5 HEPES e 1 mM CaCl 2; regolare il pH a 7,3 - 7.4. conservare la soluzione in frigorifero e fanno fresco ogni settimana.</li>
	<li>Rendere pipette di stimolazione lo stesso modo come registrazione vetro pipette, salvo non vi è alcuna necessità di piegarle. 
	Nota: La preparazione di elettrodi di stimolazione è descritto in dettaglio in 28 e 27. Il diametro finale dopo lucidatura a fuoco dovrebbe essere nella gamma di 5-7 µm.</li>
	<li>Inserto una pipetta di stimolazione in un supporto di microelettrodo collegata ad una siringa.</li>
	<li>Piastre di dissezione di fabbricazione da piccole capsule di Petri (35 x 10 mm) rivestiti con resina epossidica di gomma del silicone (Vedi Tabella materiali), come descritto in 28. 
	Nota: Gomma di Silicone dovrebbe essere completamente indurita prima dell'uso.</li>
	<li>Scegli un errante larve di terza-instar e sezionarla come descritto in 27 , 28 , 29 , 30. 
	Nota: Per visualizzare boutons, utilizzare il ceppo volo CD8-GFP (Vedi Tabella materiali) <ol><li>forcipe di utilizzo (Vedi Tabella materiali), pick 3 rd instar larve da un flaconcino.</li>
		<li>Pin le larve, ponendo il primo perno posteriore e un altro pin anteriore (vicino a bocca ganci).</li>
		<li>Soluzione aggiungere HL3.</li>
		<li>Fare un taglio con forbici di primavera (Vedi Tabella materiali) fin dal perno superiore a quella inferiore sul lato dorsale delle larve.</li>
		<li>Pin il filetto di larve, immissione 2 pin aggiuntivi a sinistra e a destra delle larve.</li>
		<li>Rimuovere le budella e trachee utilizzando forcipe.</li>
		<li>Tagliare i nervi appena fuori il cavo ventrale del nervo usando forbici di primavera.</li>
	</ol></li></ol> 3. Le registrazioni elettriche della EJC <img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56493/56493fig2.jpg" /> Figura 2. La configurazione di registrazione. Il campione NMJ appuntato al silicio rivestito di Petri (freccia) è posizionato sopra la fase mobile del microscopio composto verticale dotato di funzionalità di epifluorescenza, un obiettivo di alto ingrandimento e due micromanipolatori. Il microscopio è disposto su un tavolo antivibrazione. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig2large.jpg" target="_blank"> Clicca qui per visualizzare una versione più grande di questa figura.</a>
<ol><li>Registrazione <ol><li>posto di Petri con la preparazione sul tavolino del microscopio ( Figura 2). Inserire l'elettrodo di riferimento nel bagno.</li>
		<li>Riempire l'elettrodo di registrazione con HL3 soluzione. Nell'ambito dell'obiettivo 10x (Vedi Tabella materiali), immergere l'elettrodo nel bagno e posto sopra i muscoli 6 e 7 dei segmenti addominali 2, 3 o 4 utilizzando il micromanipolatore (Vedi Tabella materiali) ( Figura 3 A. 1 e a. 2).</li>
		<li>Passare l'obiettivo a 60 x (Vedi Tabella materiali). Concentrarsi sulla zona di interesse utilizzando epi-fluorescenza o ottica DIC. Posizionare la punta dell'elettrodo in cima bouton sinaptico ( Figura 3 b. 1-3).</li>
		<li>Premere l'elettrodo molto delicatamente sul muscolo. Una pressione eccessiva può danneggiare la giunzione neuromuscolare o indurre un aumento nell'attività sinaptica spontanea. Assicurarsi che la punta dell'elettrodo non sia intasato - in questo caso, sostituire it.</li>
		<li>Accendere l'amplificatore, scheda A/D e il computer.</li>
		<li>Scegliere la modalità di morsetto di tensione dell'amplificatore.</li>
		<li>Avviare il software di acquisizione e scegliere il &#39; privo di gap &#39; modalità.</li>
		<li>Osservare la comparsa di mEJCs sullo schermo del computer.</li>
		<li>Assicurarsi che l'ampiezza del mEJCs è nella gamma di 0,2 - 0,7 na. 
		Nota: EJC più piccoli indicano che l'elettrodo di registrazione ha difetti o che non è posizionato correttamente.</li>
	</ol></li></ol> <img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56493/56493fig3.jpg" /> Figura 3. Visualizzazione dei boutons sinaptici. (A), A immagine di campo chiaro di un hemi-segmento sotto i 10 x ingrandimento (a. 1) e l'area circoscritta allargata che mostra i muscoli 6 e 7 (a. 2, la freccia contrassegna l'elettrodo di registrazione). Barra della scala = 50 µm. (B) boutons sinaptici sono visualizzati in una riga di Drosophila con un marcatore neuronale geneticamente codificato (CD8-GFP) utilizzando l'imaging epi-fluorescenza (b. 1) o DIC ottica (b. 2). Immagini sono prese con il 60 x obiettivo e il cubo di filtro per l'imaging di GFP (Vedi Tabella materiali). Synaptiboutons c sono contrassegnati con le frecce, e una sovrapposizione di fluorescente e DIC immagini è mostrata in b. 3. Barra della scala = 10 µm. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig3large.jpg" target="_blank"> Clicca qui per visualizzare una versione più grande di questa figura.</a>
<ol start="2"> <li>stimolazione <ol><li>utilizzando un micromanipolatore, sotto un controllo visivo, posizionare l'elettrodo di stimolazione vicino l'assone che innervano segmenti addominali 2-4.</li>
		<li>Applica pressione negativa tirando il pistone della siringa collegato alla pinza porta elettrodo, in modo che l'axon è tirato dentro l'elettrodo ( Figura 2).</li>
		<li>Accendere lo stimolatore. Ruotare la manopola sull'unità di isolamento (Vedi Tabella materiali) impostare una corrente pari a zero, e poi delicatamente aumentare, fino a quando appaiono EJC (o fino a quando non viene raggiunta la soglia).</li>
		<li>Eseguire la stimolazione in un regime di suprathreshold, con la corrente di stimolazione aumentata circa due volte, rispetto alla soglia per l'osservazione della EJC. 
		Nota: Nella nostra esperienza, tale intensità di stimolazione è ottimale per evitare errori di potenziale di azione e di infornamento del potenziale di azione. Ad esempio, se EJC comparire la corrente di stimolazione di 0.2 mA, utilizzare una corrente di 0,4 mA in tutto l'esperimento.</li>
	</ol></li> <li>Guarnizione di <ol><li>misurare la resistenza di tenuta dell'elettrodo macropatch registrazione girando l'interruttore di resistenza dell'elettrodo dell'amplificatore a &#34; prova di tenuta &#34; posizione. Osservare che il valore della resistenza di guarnizione in GΩ verrà visualizzato nel &#34; attuale &#34; finestra.</li>
		<li>Assicurarsi che la resistenza di tenuta è nella gamma di 0.5 - 2 M &#8486;. I valori fuori di questo intervallo indicano che l'elettrodo è lucidato non correttamente o non correttamente posizionato.</li>
		<li>Monitorare la resistenza di tenuta durante la registrazione, facendo attenzione che rimanga costante in tutto l'esperimento.</li>
	</ol></li></ol> 4. Analisi <ol><li>analizza le registrazioni che impiegano software per analisi personalizzato (Vedi Tabella materiali). 
	Nota: Il nostro laboratorio utilizza software in-House Quantan 31, che è personalizzato per il rilevamento della EJC e mEJCs registrato focale ( Figura 4). Questo software include un filtro gaussiano digitale e permette la rilevazione dei picchi quantal sovrapposte eventi multi-quantali ( Figura 4A). Altri approcci sono descritti in 17.</li>
</ol> <img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56493/56493fig4.jpg" /> Figura 4. Analisi quantale. Rilevamento di mEJCs (A) ed EJC (B) da Quantan software. L'area eventi è contrassegnato in verde, e picchi sono contrassegnati da frecce rosse. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig4large.jpg" target="_blank"> Clicca qui per visualizzare una versione più grande di questa figura.</a>

<ol>
	<li>Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+the+larval+neuromuscular+junction+in+Drosophila+melanogaster.">Properties of the larval neuromuscular junction in Drosophila melanogaster.</a> J Physiol. 262 (1), 189-214 (1976).</li><li>Katz, B., Miledi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+temperature+on+the+synaptic+delay+at+the+neuromuscular+junction.">The effect of temperature on the synaptic delay at the neuromuscular junction.</a> J Physiol. 181 (3), 656-670 (1965).</li><li>Macleod, G. T., Gan, J., Bennett, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicle-associated+proteins+and+quantal+release+at+single+active+zones+of+amphibian+(Bufo+marinus)+motor-nerve+terminals.">Vesicle-associated proteins and quantal release at single active zones of amphibian (Bufo marinus) motor-nerve terminals.</a> J Neurophysiol. 82 (3), 1133-1146 (1999).</li><li>Macleod, G. T., Farnell, L., Gibson, W. G., Bennett, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantal+secretion+and+nerve-terminal+cable+properties+at+neuromuscular+junctions+in+an+amphibian+(Bufo+marinus).">Quantal secretion and nerve-terminal cable properties at neuromuscular junctions in an amphibian (Bufo marinus).</a> J Neurophysiol. 81 (3), 1135-1146 (1999).</li><li>Zefirov, A., Benish, T., Fatkullin, N., Cheranov, S., Khazipov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+active+zones.">Localization of active zones.</a> Nature. 376 (6539), 393-394 (1995).</li><li>Macleod, G. T., Lavidis, N. A., Bennett, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+dependence+of+quantal+secretion+from+visualized+sympathetic+nerve+varicosities+on+the+mouse+vas+deferens.">Calcium dependence of quantal secretion from visualized sympathetic nerve varicosities on the mouse vas deferens.</a> J Physiol. 480 (Pt 1), 61-70 (1994).</li><li>Samigullin, D., Bill, C. A., Coleman, W. L., Bykhovskaia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+transmitter+release+by+synapsin+II+in+mouse+motor+terminals.">Regulation of transmitter release by synapsin II in mouse motor terminals.</a> J Physiol. 561 (Pt 1), 149-158 (2004).</li><li>Coleman, W. L., Bykhovskaia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab3a-mediated+vesicle+recruitment+regulates+short-term+plasticity+at+the+mouse+diaphragm+synapse.">Rab3a-mediated vesicle recruitment regulates short-term plasticity at the mouse diaphragm synapse.</a> Mol Cell Neurosci. 41 (2), 286-296 (2009).</li><li>Atwood, H. L., Parnas, H., Parnas, I., Wojtowicz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantal+currents+evoked+by+graded+intracellular+depolarization+of+crayfish+motor+axon+terminals.">Quantal currents evoked by graded intracellular depolarization of crayfish motor axon terminals.</a> J Physiol. 383, 587-599 (1987).</li><li>Parnas, H., Dudel, J., Parnas, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotransmitter+release+and+its+facilitation+in+crayfish.+I.+Saturation+kinetics+of+release,+and+of+entry+and+removal+of+calcium.">Neurotransmitter release and its facilitation in crayfish. I. Saturation kinetics of release, and of entry and removal of calcium.</a> Pflugers Arch. 393 (1), 1-14 (1982).</li><li>Wojtowicz, J. M., Marin, L., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-induced+changes+in+synaptic+release+sites+at+the+crayfish+neuromuscular+junction.">Activity-induced changes in synaptic release sites at the crayfish neuromuscular junction.</a> J Neurosci. 14 (6), 3688-3703 (1994).</li><li>Zucker, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crayfish+neuromuscular+facilitation+activated+by+constant+presynaptic+action+potentials+and+depolarizing+pulses.">Crayfish neuromuscular facilitation activated by constant presynaptic action potentials and depolarizing pulses.</a> J Physiol. 241 (1), 69-89 (1974).</li><li>Zucker, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+statistics+of+transmitter+release+during+facilitation.">Changes in the statistics of transmitter release during facilitation.</a> J Physiol. 229 (3), 787-810 (1973).</li><li>Worden, M. K., Bykhovskaia, M., Hackett, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facilitation+at+the+lobster+neuromuscular+junction:+a+stimulus-dependent+mobilization+model.">Facilitation at the lobster neuromuscular junction: a stimulus-dependent mobilization model.</a> J Neurophysiol. 78 (1), 417-428 (1997).</li><li>Bykhovskaia, M., Hackett, J. T., Worden, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asynchrony+of+quantal+events+in+evoked+multiquantal+responses+indicates+presynaptic+quantal+interaction.">Asynchrony of quantal events in evoked multiquantal responses indicates presynaptic quantal interaction.</a> J Neurophysiol. 81 (5), 2234-2242 (1999).</li><li>Bykhovskaia, M., Polagaeva, E., Hackett, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechnisms+underlying+different+facilitation+forms+at+the+lobster+neuromuscular+synapse.">Mechnisms underlying different facilitation forms at the lobster neuromuscular synapse.</a> Brain Res. 1019 (1-2), 10-21 (2004).</li><li>Cooper, R. L., Stewart, B. A., Wojtowicz, J. M., Wang, S., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantal+measurement+and+analysis+methods+compared+for+crayfish+and+Drosophila+neuromuscular+junctions,+and+rat+hippocampus.">Quantal measurement and analysis methods compared for crayfish and Drosophila neuromuscular junctions, and rat hippocampus.</a> J Neurosci Methods. 61 (1-2), 67-78 (1995).</li><li>Stewart, B. A., Atwood, H. L., Renger, J. J., Wang, J., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+stability+of+Drosophila+larval+neuromuscular+preparations+in+haemolymph-like+physiological+solutions.">Improved stability of Drosophila larval neuromuscular preparations in haemolymph-like physiological solutions.</a> J Comp Physiol A. 175 (2), 179-191 (1994).</li><li>Pawlu, C., DiAntonio, A., Heckmann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postfusional+control+of+quantal+current+shape.">Postfusional control of quantal current shape.</a> Neuron. 42 (4), 607-618 (2004).</li><li>Akbergenova, Y., Bykhovskaia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapsin+maintains+the+reserve+vesicle+pool+and+spatial+segregation+of+the+recycling+pool+in+Drosophila+presynaptic+boutons.">Synapsin maintains the reserve vesicle pool and spatial segregation of the recycling pool in Drosophila presynaptic boutons.</a> Brain Res. 1178, 52-64 (2007).</li><li>Akbergenova, Y., Bykhovskaia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+the+endosomal+endocytic+pathway+increases+quantal+size.">Enhancement of the endosomal endocytic pathway increases quantal size.</a> Mol Cell Neurosci. 40 (2), 199-206 (2009).</li><li>Vasin, A., Volfson, D., Littleton, J. T., Bykhovskaia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+the+Complexin+Accessory+Helix+with+Synaptobrevin+Regulates+Spontaneous+Fusion.">Interaction of the Complexin Accessory Helix with Synaptobrevin Regulates Spontaneous Fusion.</a> Biophys J. 111 (9), 1954-1964 (2016).</li><li>Wong, K., Karunanithi, S., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantal+unit+populations+at+the+Drosophila+larval+neuromuscular+junction.">Quantal unit populations at the Drosophila larval neuromuscular junction.</a> J Neurophysiol. 82 (3), 1497-1511 (1999).</li><li>Dudel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+reduced+calcium+on+quantal+unit+current+and+release+at+the+crayfish+neuromuscular+junction.">The effect of reduced calcium on quantal unit current and release at the crayfish neuromuscular junction.</a> Pflugers Arch. 391 (1), 35-40 (1981).</li><li>Dudel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+Ca2++inflow+to+quantal,+phasic+transmitter+release+from+nerve+terminals+of+frog+muscle.">Contribution of Ca2+ inflow to quantal, phasic transmitter release from nerve terminals of frog muscle.</a> Pflugers Arch. 422 (2), 129-142 (1992).</li><li>Marrero, H. G., Lemos, J. 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> Loose-Patch-Clamp method. , (2007).</li><li>Wu, W. 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=Physiological+recordings+of+high+and+low+output+NMJs+on+the+crayfish+leg+extensor+muscle.">Physiological recordings of high and low output NMJs on the crayfish leg extensor muscle.</a> J Vis Exp. (45), (2010).</li><li>Verstreken, P., Ohyama, T., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FM+1-43+labeling+of+synaptic+vesicle+pools+at+the+Drosophila+neuromuscular+junction.">FM 1-43 labeling of synaptic vesicle pools at the Drosophila neuromuscular junction.</a> Methods Mol Biol. 440, 349-369 (2008).</li><li>Brent, J. R., Werner, K. M., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+larval+NMJ+dissection.">Drosophila larval NMJ dissection.</a> J Vis Exp. (24), (2009).</li><li>Imlach, W., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+methods+for+recording+synaptic+potentials+from+the+NMJ+of+Drosophila+larvae.">Electrophysiological methods for recording synaptic potentials from the NMJ of Drosophila larvae.</a> J Vis Exp. (24), (2009).</li><li>Bykhovskaia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+quantal+analysis+more+convenient,+fast,+and+accurate:+user-friendly+software+QUANTAN.">Making quantal analysis more convenient, fast, and accurate: user-friendly software QUANTAN.</a> J Neurosci Methods. 168 (2), 500-513 (2008).</li><li>Huntwork, S., Littleton, J. 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+complexin+fusion+clamp+regulates+spontaneous+neurotransmitter+release+and+synaptic+growth.">A complexin fusion clamp regulates spontaneous neurotransmitter release and synaptic growth.</a> Nat Neurosci. 10 (10), 1235-1237 (2007).</li><li>Zhong, Y., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+synaptic+plasticity+in+Drosophila+memory+mutants+with+a+defective+cyclic+AMP+cascade.">Altered synaptic plasticity in Drosophila memory mutants with a defective cyclic AMP cascade.</a> Science. 251 (4990), 198-201 (1991).</li><li>Delgado, R., Maureira, C., Oliva, C., Kidokoro, Y., Labarca, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+of+vesicle+pools,+rates+of+mobilization,+and+recycling+at+neuromuscular+synapses+of+a+Drosophila+mutant,+shibire.">Size of vesicle pools, rates of mobilization, and recycling at neuromuscular synapses of a Drosophila mutant, shibire.</a> Neuron. 28 (3), 941-953 (2000).</li><li>Melom, J. E., Akbergenova, Y., Gavornik, J. P., Littleton, J. 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+evoked+release+are+independently+regulated+at+individual+active+zones.">Spontaneous and evoked release are independently regulated at individual active zones.</a> J Neurosci. 33 (44), 17253-17263 (2013).</li></ol>

Gli autori non hanno nulla a rivelare.

Drosophila rappresenta un organismo modello vantaggioso per studiare la trasmissione sinaptica. Sono state utilizzate diverse configurazioni di registrazione alle NMJ larvale, tra cui registrazioni intracellulari di potenziali sinaptici, registrazioni di correnti sinaptiche con due elettrodi tensione morsetto33,34e focale macropatch registrazioni di correnti sinaptiche descritte qui. La seconda tecnica permette la quantificazione precisa della trasmissio...

Drosophila è un sistema eccellente modello per studiare i meccanismi molecolari che controllano trasmissione sinaptica. Il sistema neuromuscolare in Drosophila è glutamatergici, e pertanto la drosofila giunzione neuromuscolare (NMJ) può essere utilizzata per studiare le caratteristiche conservate di glutamatergic rilascio. Da Jan e Jan Studio1, le larve instar terza è stato largamente utilizzato per studiare la trasmissione sinaptica spontanea ed evocata da monitoraggio potenziali eccitatori giunzione (giuridiche) o correnti (EJC). Giuridiche sono comunemente registrate intracellulare con un micro-elettrodo di vetro taglienti, e riflettono l'attività di NMJ intera, compresi tutti i boutons fare sinapsi al data fibra muscolare.
Al contrario, l'attività di un numero limitato di siti di rilascio possa essere registrata focale posizionando un suggerimento micropipetta vicino terminali neuronali o varicosità sinaptica. Questa tecnica è stata impiegata originariamente da Katz e Miledi2e focale registrazioni extracellulari sono state impiegate con successo in diverse preparazioni di NMJ, tra cui rana3,4,5, mouse6 , 7 , 8, crostaceo9,10,11,12,13,14,15,16e Drosofila17,18,19,20,21,22,23. Questo approccio è stato ulteriormente sviluppato da Dudel, che ottimizzato macropatch ricodifica elettrodi24,25. Nell'implementazione di Dudel, questa tecnica non si discostano l' allentato-patch-clamp metodo26.
NMJ larvale Drosophila ha chiaramente definito boutons sinaptici e linee transgeniche con tag fluorescente neuronale geneticamente codificato (Vedi Tabella materiali) sono prontamente disponibili. Questi vantaggi ci ha permesso di registrare EJC e mEJCs da un bouton sinaptico selezionato20,21,22. Qui, descriviamo questa tecnica in dettaglio.

<table width="1251">
	<tbody>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Sutter P-97</td>
			<td style="width:288px;">Sutter instrument</td>
			<td style="width:180px;">P-97</td>
			<td style="width:495px;">Microelectrode puller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Narishige MF-830</td>
			<td style="width:288px;">Narishige</td>
			<td style="width:180px;">MF-830</td>
			<td>Microforge</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">WPI MF200</td>
			<td style="width:288px;">WPI</td>
			<td>MF200</td>
			<td>Microforge</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Glass capilaries</td>
			<td style="width:288px;">WPI</td>
			<td>B150-86-10</td>
			<td style="width:495px;">Glass capilaries</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microtorch 1WG61</td>
			<td style="width:288px;">Grainer</td>
			<td style="width:180px;">1WG61</td>
			<td>Microtorch</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td style="width:288px;">Dow Corning</td>
			<td style="width:180px;">SYLGARD 184</td>
			<td>Silicone for dissection plates preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Dissection pins</td>
			<td style="width:288px;">Amazon</td>
			<td>B00J5PMPJA</td>
			<td>Pins for larvae positioning</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Tweezers</td>
			<td style="width:288px;">WPIINC</td>
			<td>500342</td>
			<td>Tweezers for placing pins, removing the guts and tracheas.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Scissors</td>
			<td style="width:288px;">WPIINC</td>
			<td>501778</td>
			<td>Scissors for cutting the cuticula of the larvae and nerves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Olympus BX61WI</td>
			<td style="width:288px;">Olympus</td>
			<td style="width:180px;">BX61WI</td>
			<td>Upright microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Olympus Lumplan FL N 60x</td>
			<td style="width:288px;">Olympus</td>
			<td style="width:180px;">UPLFLN 60X</td>
			<td>Microscope objective 60X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus UPlan FL N 10x</td>
			<td style="width:288px;">Olympus</td>
			<td style="width:180px;">Uplanfl N 10X</td>
			<td>Microscope objective 10X</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Narishige Micromanipulator</td>
			<td style="width:288px;">Narishige</td>
			<td style="width:180px;">MHW-3</td>
			<td>Three-axis Water Hydraulic Micromanipulator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">npi Electronic GmbH ELC-03XS</td>
			<td style="width:288px;">npi Electronic GmbH</td>
			<td style="width:180px;">ELC-03XS</td>
			<td>Electrophysiological amplifier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A.M.P.I Master 8</td>
			<td style="width:288px;">A.M.P.I.</td>
			<td style="width:180px;">Master 8</td>
			<td>Electrical stimulator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A.M.P.I Iso-Flex</td>
			<td style="width:288px;">A.M.P.I.</td>
			<td style="width:180px;">Iso-Flex</td>
			<td>Stimulus isolator</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">TMC antivibration table</td>
			<td style="width:288px;">TMC</td>
			<td style="width:180px;">63-9090</td>
			<td>Antivibration table</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TMC Faraday cage</td>
			<td style="width:288px;">TMC</td>
			<td style="width:180px;">81-333-90</td>
			<td>Faraday cage</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Digidata 1322A</td>
			<td style="width:288px;">Axon Instruments</td>
			<td style="width:180px;">Digidata 1322A</td>
			<td>Digidata</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Computer</td>
			<td style="width:288px;">Dell</td>
			<td style="width:180px;">Dell Dimension 5150</td>
			<td>Computer with Win XP OS&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Electrode holder</td>
			<td style="width:288px;">WPI</td>
			<td>MEH3SW&#160;</td>
			<td style="width:495px;">Electrode holder</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Optical filter</td>
			<td style="width:288px;">Omega optical</td>
			<td style="width:180px;">XF 115-2</td>
			<td style="width:495px;">Filter cube for Green Fluorescent Protein (GFP) detection&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">pCLAMP 8</td>
			<td style="width:288px;">Axon Instruments</td>
			<td style="width:180px;">8.0.0.81</td>
			<td>Software for signal recording</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Quantan</td>
			<td style="width:288px;">In-house software</td>
			<td style="width:180px;">-</td>
			<td>Software for signal processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Canton-S (Wildtype)</td>
			<td style="width:288px;">Bloomington Stock Center</td>
			<td>64349</td>
			<td>Control fly line</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">cpx SH1</td>
			<td style="width:288px;">Generous Gift of J.T. Littleton</td>
			<td style="width:180px;">-</td>
			<td>Complexin knock-out fly line with increased spontaneous exocytosis</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">CD8-GFP</td>
			<td style="width:288px;">Bloomington Stock Center</td>
			<td style="width:180px;">5137</td>
			<td>Fly line with neuronal fluorescent (GFP) Tag</td>
		</tr>
	</tbody>
</table>

focal macropatch recordings of synaptic currents from the drosophila larval neuromuscular junction

Drosophila della giunzione neuromuscolare (NMJ) è un sistema eccellente modello per studiare la trasmissione sinaptica glutamatergic. Descriviamo la tecnica delle registrazioni macropatch focale delle correnti sinaptiche da boutons visualizzati alle NMJ larvale della drosofila . Questa tecnica richiede la realizzazione personalizzata di registrazione micropipette, come pure un microscopio composto dotato di un alto ingrandimento, obiettivo a immersione in acqua a lunga distanza, ottica (DIC) contrasto differenziale di interferenza e una fluorescente allegato. L'elettrodo di registrazione è posizionato sulla sommità di una bouton sinaptica selezionata visualizzata con ottica DIC, epi-fluorescenza o entrambi. Il vantaggio di questa tecnica è che permette di monitorare l'attività sinaptica di un numero limitato di siti di rilascio. L'elettrodo di registrazione ha un diametro di alcuni micron, e i siti di rilascio posizionati di fuori del cerchio dell'elettrodo non influenzano significativamente le correnti registrate. Le correnti sinaptiche registrate hanno cinetica veloce e possono essere facilmente risolto. Questi vantaggi sono particolarmente importanti per gli studi di linee mutanti volare con maggiore attività sinaptica spontanea o asincrona.

Macropatch focale registrazioni attivare monitoraggio attività sinaptica da selezionati boutons sinaptici (Figura 5). Quando l'elettrodo è posizionato sulla sommità di una bouton sinaptica (Figura 5A, sito 1), il mEJCs registrato (Figura 5, sito 1) hanno un ampiezze significativamente superiore al livello di rumore e affilate lievitazione (in una gamma di millisecondo). Quando l'elettrodo di regis...

Watch this Scientific Journal Video about Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction at JoVE.com

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Macropatch focale registrazioni delle correnti sinaptiche da giunzione neuromuscolare larvale della drosofila

Delle correnti sinaptiche possono essere registrate focale da visualizzati boutons sinaptici alla giunzione neuromuscolare larve instar terza della drosofila . Questa tecnica consente il monitoraggio dell'attività di una singola bouton sinaptica.

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

focal-macropatch-recordings-synaptic-currents-from-drosophila-larval

Research

JoVE Journal

Neuroscience

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

6.0K Views.  Wayne State University. Delle correnti sinaptiche possono essere registrate focale da visualizzati boutons sinaptici alla giunzione neuromuscolare larve instar terza della drosofila . Questa tecnica consente il monitoraggio dell'attività di una singola bouton sinaptica.

Video: Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

Nella visualizzazione in vivo delle vescicole sinaptiche In Drosophila assoni larvali segmentale

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Ricerca

Neuroscienze

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

Dissezione e immagini di zone attive nel Drosophila Giunzione neuromuscolare

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

Sequential Photo-sbiancante per Delineare cellule di Schwann singoli sulla giunzione neuromuscolare

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

Derivando La durata di Liquidazione glutammato con una analisi Deconvoluzione delle Correnti Transporter astrociti

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

Rilevamento di<em&gt; In Situ</em&gt; Complessi proteina-proteina del<em&gt; Drosophila</em&gt; Larvale neuromuscolare Junction Utilizzando prossimità legatura Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

La giunzione neuromuscolare: Synapse Misurazione circonferenza, la frammentazione e Variazioni Synaptic Proteine ​​Densità Uso confocale microscopia a fluorescenza

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

Perché Quantificazione Matters: Caratterizzazione dei fenotipi al<em&gt; Drosophila</em&gt; Larvale Giunzione neuromuscolare

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.

Here, we describe a method for loading a calcium-sensitive dye through the frog nerve stump into the nerve endings. We also present a protocol for the recording and analysis of fast calcium transients in the peripheral nerve endings.

Caricamento di un colorante di calcio in terminazioni di nervi della rana attraverso il ceppo del nervo: registrazione transitoria del calcio nella giunzione neuromuscolare della rana

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

Funzionalità di misurazione della giunzione neuromuscolare

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The Drosophila larval neuromuscular junction (NMJ), a well-established model for glutamatergic synapses, has been extensively studied for decades. Identification of mutations causing NMJ morphological defects revealed a repertoire of genes that regulate synapse development and function. Many of these were identified in large-scale studies that focused on qualitative approaches to detect morphological abnormalities of the Drosophila NMJ. A drawback of qualitative analyses is that many subtle players contributing to NMJ morphology likely remain unnoticed. Whereas quantitative analyses are required to detect the subtler morphological differences, such analyses are not yet commonly performed because they are laborious. This protocol describes in detail two image analysis algorithms "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics", available as Fiji-compatible macros, for quantitative, accurate and objective morphometric analysis of the Drosophila NMJ. This methodology is developed to analyze NMJ terminals immunolabeled with the commonly used markers Dlg-1 and Brp. Additionally, its wider application to other markers such as Hrp, Csp and Syt is presented in this protocol. The macros are able to assess nine morphological NMJ features: NMJ area, NMJ perimeter, number of boutons, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones in the NMJ terminal.

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Two image analysis algorithms, "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics" were created, to automatically quantify nine morphological features of the Drosophila neuromuscular junction (NMJ).

Due Algoritmi per high-throughput e multi-parametrica Quantificazione degli<em&gt; Drosophila</em&gt; Giunzione neuromuscolare Morfologia

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The ...

Macropatch focale registrazioni delle correnti sinaptiche da giunzione neuromuscolare larvale della drosofila

Riepilogo

Esplora Altri Video

Capitoli in questo video

Nella visualizzazione in vivo delle vescicole sinaptiche In Drosophila assoni larvali segmentale

Dissezione e immagini di zone attive nel Drosophila Giunzione neuromuscolare

Sequential Photo-sbiancante per Delineare cellule di Schwann singoli sulla giunzione neuromuscolare

Derivando La durata di Liquidazione glutammato con una analisi Deconvoluzione delle Correnti Transporter astrociti

Rilevamento di

La giunzione neuromuscolare: Synapse Misurazione circonferenza, la frammentazione e Variazioni Synaptic Proteine Densità Uso confocale microscopia a fluorescenza

Perché Quantificazione Matters: Caratterizzazione dei fenotipi al

Caricamento di un colorante di calcio in terminazioni di nervi della rana attraverso il ceppo del nervo: registrazione transitoria del calcio nella giunzione neuromuscolare della rana

Funzionalità di misurazione della giunzione neuromuscolare

Due Algoritmi per high-throughput e multi-parametrica Quantificazione degli

Macropatch focale registrazioni delle correnti sinaptiche da giunzione neuromuscolare larvale della drosofila

Riepilogo

Esplora Altri Video

Capitoli in questo video

Nella visualizzazione in vivo delle vescicole sinaptiche In Drosophila assoni larvali segmentale

Dissezione e immagini di zone attive nel Drosophila Giunzione neuromuscolare

Sequential Photo-sbiancante per Delineare cellule di Schwann singoli sulla giunzione neuromuscolare

Derivando La durata di Liquidazione glutammato con una analisi Deconvoluzione delle Correnti Transporter astrociti

Rilevamento di

La giunzione neuromuscolare: Synapse Misurazione circonferenza, la frammentazione e Variazioni Synaptic Proteine ​​Densità Uso confocale microscopia a fluorescenza

Perché Quantificazione Matters: Caratterizzazione dei fenotipi al

Caricamento di un colorante di calcio in terminazioni di nervi della rana attraverso il ceppo del nervo: registrazione transitoria del calcio nella giunzione neuromuscolare della rana

Funzionalità di misurazione della giunzione neuromuscolare

Due Algoritmi per high-throughput e multi-parametrica Quantificazione degli

La giunzione neuromuscolare: Synapse Misurazione circonferenza, la frammentazione e Variazioni Synaptic Proteine Densità Uso confocale microscopia a fluorescenza