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>

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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>

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

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

Accoppiato a pinza registrazioni patch da Motor-neurone e muscolo scheletrico bersaglio in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

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 ...

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP&#8217;s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP&#8217;s and minature excitatory junction potentials (mEJP&#8217;s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP&#8217;s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

Metodi elettrofisiologici per la registrazione Potenziali Synaptic dal NMJ Larve di Drosophila

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

Biologia

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available to evoke excitatory junctional potentials (EJPs) in this preparation involves the use of suction electrodes. In both research and teaching labs, students often have difficulty maneuvering and manipulating this type of stimulating electrode. In the present work, we show how to remotely stimulate synaptic potentials at the larval NMJ without the use of suction electrodes. By expressing channelrhodopsin2 (ChR2) 4,5,6 in Drosophila motor neurons using the GAL4-UAS system 7, and making minor changes to a basic electrophysiology rig, we were able to reliably evoke EJPs with pulses of blue light. This technique could be of particular use in neurophysiology teaching labs where student rig practice time and resources are limited.

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

This procedure uses a blue light-activated algal channel and cell-specific genetic expression tools to evoke synaptic potentials with light pulses at the neuromuscular junction (NMJ) in Drosophila larvae. This technique is an inexpensive and easy-to-use alternative to suction electrode stimulation for synaptic physiology studies in research and teaching laboratories.

Channelrhodopsin2 stimolazione mediata dei potenziali Synaptic a Drosophila Giunzioni neuromuscolari

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available ...

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

Dissezione e registrazione dal bivio C. Elegans neuromuscolare

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

Preparation of Drosophila Central Neurons for in situ Patch Clamping

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience research. Ion channels are the basis of all behavior since they mediate neuronal excitability. The first voltage gated ion channel cloned was the Drosophila voltage gated potassium channel Shaker1,2. Toward understanding the role of ion channels and membrane excitability for nervous system function it is useful to combine powerful genetic tools available in Drosophila with in situ patch clamp recordings. For many years such recordings have been hampered by the small size of the Drosophila CNS. Furthermore, a robust sheath made of glia and collagen constituted obstacles for patch pipette access to central neurons. Removal of this sheath is a necessary precondition for patch clamp recordings from any neuron in the adult Drosophila CNS. In recent years scientists have been able to conduct in situ patch clamp recordings from neurons in the adult brain3,4 and ventral nerve cord of embryonic5,6, larval7,8,9,10, and adult Drosophila11,12,13,14. A stable giga-seal is the main precondition for a good patch and depends on clean contact of the patch pipette with the cell membrane to avoid leak currents. Therefore, for whole cell in situ patch clamp recordings from adult Drosophila neurons must be cleaned thoroughly. In the first step, the ganglionic sheath has to be treated enzymatically and mechanically removed to make the target cells accessible. In the second step, the cell membrane has to be polished so that no layer of glia, collagen or other material may disturb giga-seal formation. This article describes how to prepare an identified central neuron in the Drosophila ventral nerve cord, the flight motoneuron 5 (MN515), for somatic whole cell patch clamp recordings. Identification and visibility of the neuron is achieved by targeted expression of GFP in MN5. We do not aim to explain the patch clamp technique itself.

Preparation of Drosophila Central Neurons for in situ Patch Clamping

In situ patch clamp recordings are used for electrophysiological characterization of neurons in intact circuitry. In the Drosophila genetic model patch clamping is difficult because the CNS is small and surrounded by a robust sheath. This article describes the procedure to remove the sheath and clean neurons for subsequent patch clamp recordings.

Preparazione di<em&gt; Drosophila</em&gt; Neuroni centrali per<em&gt; In situ</em&gt; Patch di serraggio

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

Registrazione elettrofisiologiche in Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

Bloccare Registrazioni patch da cellule embrionali Zebrafish Mauthner

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at the Drosophila NMJ enables researchers to acquire synaptic information, encompassing both quantitative data on synapse abundance and detailed insights into their morphology. However, the diffuse distribution and limited visual range of the TEM present challenges for the ultrastructural analysis. This study introduces an innovative and efficient sample preparation method that surpasses the conventional approach. The procedure begins by placing a metal mesh at the base of a flat-bottomed bottle or test tube, followed by positioning fixed larvae samples onto the mesh. An additional mesh is placed over the samples, ensuring that they are positioned between the two meshes. The fixed samples are thoroughly dehydrated and infiltrated before proceeding with the embedding procedure. Then embedding of the samples in epoxy resin is performed in a flat sheet manner, which allows for the preparation of muscles for positioning and sectioning. Benefiting from these steps, all the muscles of Drosophila larvae can be visualized under light microscopy, therey facilitating subsequent positioning and sectioning. Excess resin is removed after locating the 6th and 7th muscles of body segments A2 and A3. Serial ultra-thin sectioning of the 6th or 7th muscle is performed.

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

This report provides a new sample preparation procedure for visualizing neuromuscular junctions in Drosophila Larvae. This method is more effective in preventing the curling of the samples compared to the traditional method and is particularly useful for Drosophila neuromuscular junction ultrastructural analysis.

Ottimizzazione del processo di preparazione dei campioni per la microscopia elettronica a trasmissione delle giunzioni neuromuscolari nelle larve di Drosophila

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at ...

Drosophila Neuromuscular Junction (NMJ) Quantification: A Method to Assess Synaptic Morphology and Function

Source: Castells-Nobau, A., et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/55395/" target="_blank">Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology</a>. J. Vis. Exp. (2017).
The Drosophila neuromuscular junction (NMJ) is used as a model to study the morphology and function of synapses. This video describes important features that researchers quantify to characterize the NMJ. The example protocol demonstrates a software able to perform the quantification automatically.

drosofila Quantificazione della giunzione neuromuscolare (NMJ): un metodo per valutare la morfologia e la funzione sinaptica

Source: Castells-Nobau, A., et al. Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology. ...