Il progetto descritto è stato sostenuto dal Codice di Autorizzazione R01HD050725 dal National Institute of Child Health e lo Sviluppo Umano di TAG Il contenuto è di esclusiva responsabilità degli autori e non rappresentano necessariamente la posizione ufficiale del National Institute of Child Health e Sviluppo Umano o il National Institutes of Health. Ringraziamo i membri del laboratorio Godenschwege così come Barbara Schreader per il loro contributo al manoscritto e video.

1. Dissezione del cordone nervoso ventrale Dissezione e tintura di riempimento del GF può essere effettuata a temperatura ambiente. Per tutti dissezione steps freddo (4 ° C) soluzione salina dovrebbe essere usato per rallentare il metabolismo cellulare e mantenere in vita i neuroni più a lungo. <ol><li> Anestetizzare le mosche adulte (4 giorni o più) con CO 2 o freddo li su ghiaccio fino a che non sono immobilizzati. Anestesia con ghiaccio richiederà più tempo (fino a 30 min), ma non mancherà di tenere le mosche immobilizzato a lungo. </li><li> Mettere una mosca in un piatto rivestito Sylgard Petri. Questa dissezione richiede l&#39;uso di un alto ingrandimento (80-100X) stereomicroscopio dissezione. </li><li> Con le forbici Vännäs, rimuovere le gambe, proboscide, e le ali. </li><li> Utilizzare pinze grosse e fini perni insetti (pin minutien) per orientare il lato volare dorsale fino nel piatto Sylgard. Perno l&#39;addome e poi allungare l&#39;animale un po &#39;mettendo due perni su entrambi i lati del collo, dietro la testa, in modo che il collo è leggermente allungato e accessibile (vedi Figura 2). </li><li> Immergere la mosca in acqua dolce, salina fredda Drosophila. Più comunemente usati saline Drosophila sono sufficienti per la dissezione, comunque in questo caso abbiamo utilizzato una soluzione salina descritto in Gu e O&#39;Dowd, 2006 4. Una bolla d&#39;aria che circonda la mosca. Rimuovere l&#39;aria con una siringa. </li><li> Fare una breve incisione laterale nel fine dorsale dell&#39;addome. A partire l&#39;incisione, fare un superficiale, taglio dritto lungo la linea mediana dorsale del torace verso l&#39;addome. Si prosegue poi il superficiale, taglio longitudinale al torace il più precisamente possibile lungo la linea mediana verso il connettivo. È fondamentale per rendere i tagli superficiali per non danneggiare il VNC (Figura 1). </li><li> Utilizzare due perni insetti multa per aprire con attenzione il torace. Diffondere la metà del corpo a parte con le spine e le spine luogo attraverso i muscoli del volo (Figura 2). Il torace non deve essere aperto ulteriormente come illustrato nella figura 2 per evitare lo strappo della cuticola. Per garantire un orientamento dritto of the VNC i pin devono essere collocati nella stessa posizione antero posteriore su ogni lato. </li><li> L&#39;accesso al VNC per l&#39;iniezione tintura e di imaging corretta può essere ostacolato da organi interni. Pertanto, l&#39;uso pinze e forbici per rimuovere lo stomaco, intestino, cuore e organi riproduttivi. Estrarre le ghiandole salivari sdraiata su entrambi i lati del VNC. Sciacquare con cura la dissezione con soluzione salina fresca per rimuovere il tessuto galleggiante e grasso. </li><li> Togliere il grasso corporeo da sotto il connettivo cervicale (CVC). Il corpo grasso è meglio visibile con un ingrandimento elevato. Utilizzare una pinza sottile di cogliere con attenzione il tessuto dal lato ed estrarlo. </li></ol> La dissezione può essere effettuato entro 5 o 10 minuti. Utilizzando fresco, la soluzione fisiologica fredda sezionato, sistema nervoso danneggiato rimarrà in vita per almeno un&#39;ora. 2. Intracellulare GF riempire Si raccomanda di eseguire la tintura riempire il più presto possibile dopo la dissezione. <ol><li> Porre la capsula Sylgard con la mosca sezionato al microscopio verticale con una fase fissa (Figura 3) e un obiettivo 10x aria. Centro il campione con la fine posteriore della mosca posizionati nei confronti del titolare colorante elettrodo di riempimento. Utilizzare l&#39;interferenza differenziale contrasto (DIC) modalità e concentrarsi sul CVC. Gli assoni grande del GF deve essere chiaramente visibile sulla superficie dorsale del CVC. </li><li> Diluire Lucifero Giallo CH sale dilitio in H2O deionizzata a una concentrazione di 1%. Tirare un elettrodo di vetro borosilicato con filamento di una resistenza di ca. 60-80 MW. La punta del elettrodo di vetro deve essere lungo e affilato. Riempire la punta dell&#39;elettrodo con 1% di soluzione Lucifer Yellow ponendo l&#39;elettrodo con l&#39;estremità opposta della punta dell&#39;elettrodo nella soluzione Lucifero Giallo per ca. 30-60 secondi. Poi l&#39;elettrodo con riempimento 3M LiCl usando una siringa Hamilton lasciando una bolla d&#39;aria tra il colorante e LiCl. Collegare l&#39;elettrodo colorante per riempire lo stadio testa. </li><li> Inserire un filo elettrodo rivestito di cloruro d&#39;argento di terra in soluzione fisiologica e posizionare l&#39;elettrodo colorante riempire al di sopra del parallelo volare verso il VNC utilizzando un micromanipolatore 3D idraulico. L&#39;elettrodo tintura di riempimento dovrebbe essere collegato ad un amplificatore che consente il passaggio di corrente al fine di rilasciare negativamente o positivamente coloranti addebitato l&#39;elettrodo passando iper-o depolarizzanti corrente, rispettivamente. </li><li> Passare a una lente 40x immersione in acqua e abbassare la punta dell&#39;elettrodo colorante riempire su una delle GFS. Gli assoni GF si trovava vicino alla superficie dorsale del CVC. Utilizzare un in avanti (verso la testa), movimento per inserire l&#39;elettrodo nella GF. </li><li> Cambia la tua fonte di luce in luce epifluorescente e passare a un filtro giallo Lucifero. Applicare una corrente iperpolarizzante all&#39;elettrodo colorante riempire con l&#39;amplificatore per iniettare il Giallo di Lucifero nel GF. La quantità di colorante iniettata può essere controllato da ªquantità di corrente e passato così come la durata è passata la corrente. Per evitare che il colorante dalla fuoriuscita del sito d&#39;iniezione o danneggiare la GF, si raccomanda di iniettare con una corrente bassa (circa 3-5 nA) oltre 1-5 minuti. </li><li> Applicare corrente fino a quando il terminale GF nel VNC si può vedere chiaramente (Figura 4A). Per trans-sinaptica dei neuroni postsinaptici colorazione, continuano a passare la corrente fino a colorante può essere visto anche nei neuroni postsinaptici. Spegnere la corrente e togliere l&#39;elettrodo colorante riempimento. </li></ol> 3. Rappresentante dei risultati: Un colorante rappresentante riempire per una GF con Lucifero giallo è mostrato nella figura 4A. L&#39;immagine è stata acquisita come una serie di immagini nel software Nikon Elementi utilizzando una telecamera montata sul posto il microscopio. In questo campione in esame è stato passato attraverso l&#39;elettrodo per circa 2 minuti. Il GF è completamente riempito e il terminale GF grande nel torace è ben marcato (4A, ellisse). Nessun neuroni postsinaptici sono visibili in questo caso, sebbene Lucifero giallo è abbastanza piccolo da passare attraverso giunzioni gap. Se il colorante non viene iniettato abbastanza a lungo nel GF poi trans-sinaptica riempie dei neuroni post-sinaptici non possono stata vista perché il segnale è troppo debole per essere rilevata. I neuroni postsinaptici al GF (Figura 4B, punte di frecce) possono essere visualizzati in modo più affidabile anche utilizzando traccianti neuronali più piccoli, come ad esempio Neurobiotin. L&#39;iniezione di un Neurobiotin 7% in 2 soluzione di acetato di M di potassio con una corrente depolarizzante per 2 minuti è sufficiente per etichettare i neuroni postsinaptici fortemente. Neurobiotin possono essere visualizzati per la microscopia confocale utilizzando streptavidina accoppiato ai coloranti di diverse lunghezze d&#39;onda. Perché Neurobiotin non è un colorante fluorescente, co-iniezione di Rhodamin-destrano o Idrazidi Alexa nella stessa soluzione, può essere utilizzato per monitorare una iniezione di successo nel GF (Figura 4C, da Boerner e Godenschwege, 2010 5). <img src="/files/ftp_upload/3080/3080fig1.jpg" alt="Figura 1" /> Figura 1. Disegno schematico di una mosca vista lateralmente. Il torace è visualizzato con una incisione longitudinale di visualizzare le strutture interne. Durante la dissezione, i muscoli dorsali volo longitudinale (DLMS, blu) sono stati tagliati senza danneggiare il midollo ventrale del nervo (VNC, profondo rosso), che si trova ventrale ai muscoli del volo. L&#39;intestino (verde) si trova al di sopra del VNC. <img src="/files/ftp_upload/3080/3080fig2.jpg" alt="Figura 2" /> Figura 2. Vista dorsale di una mosca sezionato. Due perni sono posizionati dietro la testa per posizionare al volo. I perni apposta per i muscoli del volo (DLM) tenere il torace aperto. Organi interni sono stati rimossi per esporre il midollo ventrale del nervo (VNC). <img src="/files/ftp_upload/3080/3080fig3.jpg" alt="Figura 3" /> Figura 3. Configurazione Dye riempimento. <img src="/files/ftp_upload/3080/3080fig4.jpg" alt="Figura 4" /> Figura 4. A) Z-proiezione della parte posteriore del VNC mostrando uno GF pieno con Lucifero Giallo. L&#39;immagine è stata acquisita subito dopo la tintura di riempimento con una macchina fotografica posto. In wild-type animali terminale (ellisse) nel torace è ben visibile. B) Immagine confocale di un colorante GF si riempiono di Neurobiotin in una wild-type volare. Entrambi GF (frecce) e neuroni postsinaptici al GF (teste di freccia) sono etichettati. Neurobiotin è stata visualizzata con streptavidina accoppiato ad Cy3. C) Immagine confocale di wild-type GF pieno di Alexa555.

<ol>
	<li>Power, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thoracico-abdominal+nervous+system+of+an+adult+insect+Drosophila+melanogaster.">The thoracico-abdominal nervous system of an adult insect Drosophila melanogaster.</a> J. Comp. Neurol. 88, 347-409 (1948).</li><li>Allen, M. J., Godenschwege, T. A., Tanouye, T. A., Phelan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+an+escape:+development+and+function+of+the+Drosophila+giant+fibre+system.">Making an escape: development and function of the Drosophila giant fibre system.</a> Semin. Cell Dev. Biol. 17, 31-41 (2006).</li><li>Boerner, J., Duch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Average+shape+standard+atlas+for+the+adult+Drosophila+ventral+nerve+cord.">Average shape standard atlas for the adult Drosophila ventral nerve cord.</a> J. Comp. Neurol. 518, 2437-2455 (2010).</li><li>Gu, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+synaptic+transmission+in+adult+Drosophila+Kenyon+cells+in+sity.">Cholinergic synaptic transmission in adult Drosophila Kenyon cells in sity.</a> J. Neurosci. 26, 265-272 (2006).</li><li>Boerner, J., Godenschwege, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+for+the+Drosophila+ventral+nerve+cord+standard+in+neuronal+circuit+reconstruction+and+in-depth+analysis+of+mutant+morphology.">Application for the Drosophila ventral nerve cord standard in neuronal circuit reconstruction and in-depth analysis of mutant morphology.</a> J. Neurogent. 24, 158-1567 (2010).</li><li>Godenschwege, T. A., Kristiansen, L. V., Uthaman, S. B., Hortsch, M., Murphey, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conderved+role+for+Drosophila+Neuroglian+and+human+L1-CAM+in+central-synapse+formation.">A conderved role for Drosophila Neuroglian and human L1-CAM in central-synapse formation.</a> Curr. Biol. 16, 12-23 (2006).</li><li>Uthaman, S. B., Godenschwege, T. A., Murphey, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanism+distinct+from+highwire+for+the+Drosophila+ubiquitin+conjugase+bendless+in+synaptic+growth+and+maturation.">A mechanism distinct from highwire for the Drosophila ubiquitin conjugase bendless in synaptic growth and maturation.</a> J. Neurosci. 28, 8615-8623 (2008).</li><li>Storkebaum, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dominant+mutations+in+the+tyrosyl-tRNA+synthetase+gene+recapitulate+in+Drosophila+features+of+human+Charcot-Marie-Tooth+neuropathy.">Dominant mutations in the tyrosyl-tRNA synthetase gene recapitulate in Drosophila features of human Charcot-Marie-Tooth neuropathy.</a> Proc. Natl. Acad. Sci. USA. 106, 11782-11787 (2009).</li><li>Allen, M. J., Godenschwege, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+recordings+from+the+Drosophila+giant+fiber+system+(GFS).">Electrophysiological recordings from the Drosophila giant fiber system (GFS).</a> Cold Spring Harb. Protoc. 2010, pdb.prot5453-pdb.prot5453 (2010).</li><li>Augustin, H., Allen, M. J., Partridge, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+Recordings+from+the+Giant+Fiber+Pathway+of+D.+melanogaster.">Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster.</a> J. Vis. Exp. , (2011).</li></ol>

Nessun conflitto di interessi dichiarati.

Il sistema nervoso può essere sezionati senza estrarre dal corpo volare. Questo ha due vantaggi, in primo luogo, la dissezione provoca pochi danni al sistema nervoso, e la seconda, più i nervi per rimanere attaccati i muscoli e gli organi sensoriali. Esecuzione della dissezione, come descritto, si prepara il campione per dritto in avanti tintura di etichettatura del GF. Per comodità, i motoneuroni postsinaptica al GF rimanere attaccati ai muscoli. Assoni, quindi, non sono danneggiati, mantenendo in vita i neuroni pi?...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti </th></tr><tr><td> Sylgard 184 Kit silicone elastomero </td><td> Dow Corning </td><td></td><td></td></tr><tr><td> Dissezione Microscopio </td><td> AmScope </td><td> SM-2TZ </td><td></td></tr><tr><td> Vännäs Forbici Superfine </td><td> Strumenti accademico </td><td> VS1023 </td><td></td></tr><tr><td> Dumont Pinza Dumontstar 55 </td><td> Strumenti Scienza multa </td><td> 11295-51 </td><td></td></tr><tr><td> Austerlitz insetti perni </td><td> Strumenti Scienza multa </td><td> 26002-10 </td><td> Ø 0,1 millimetri </td></tr><tr><td> Elettrodi di vetro borosilicato </td><td> Mondo Strumenti di precisione </td><td> 1B100F-4 </td><td></td></tr><tr><td> Verticale Pipettare Puller 700c </td><td> David Kopf Instruments </td><td> Modello 700C </td><td></td></tr><tr><td> Lucifero Giallo CH dilitio sale </td><td> Sigma </td><td> L0259 </td><td> 1% in H 2 O </td></tr><tr><td> Fase Microscopio fisso verticale &amp; Camera </td><td> Nikon </td><td> FN-1 e DS-U2 Telecamera </td><td></td></tr><tr><td> Piano Fluor Obiettivo Air 10X </td><td> Nikon </td><td> CFI Plan Fluor 10X NA 16 millimetri 0,3 WD </td><td></td></tr><tr><td> Acqua Fluor 40X Obiettivo immersione </td><td> Nikon </td><td> CFI Fluor 40X W NA 0,80 WD 2,0 millimetri </td><td></td></tr><tr><td> 3D Micromanipolatore idraulico </td><td> Narishige internazionale </td><td> Modello MW0-3 </td><td></td></tr><tr><td> Amplificatore </td><td> Come Instruments, Inc. </td><td> Modello 5A </td><td></td></tr></tbody></table>

whole mount preparation of the adult drosophila ventral nerve cord for giant fiber dye injection

Per analizzare la morfologia assonali e dendritiche dei neuroni è essenziale per ottenere un&#39;accurata etichettatura delle strutture neuronali. Preparazione dei campioni ben etichettati con poco o nessun danno ai tessuti ci permette di analizzare la morfologia cellulare e di confrontare singoli campioni l&#39;uno all&#39;altro, permettendo quindi l&#39;identificazione di anomalie mutante. Nel metodo dissezione dimostrato il sistema nervoso rimane per lo più all&#39;interno della mosca adulta. Attraverso una incisione dorsale, l&#39;addome e il torace sono aperti e la maggior parte degli organi interni vengono rimossi. Solo il lato dorsale del cordone nervoso ventrale (VNC) e il connettivo cervicale (CVC) contenente gli assoni grande delle fibre giganti (GF) 1 sono esposte, mentre il cervello che contiene il corpo cellulare GF e dendriti rimane 2 in testa intatta . In questa preparazione più nervi of the VNC dovrebbe rimanere attaccati alla loro muscoli. Dopo la dissezione, il riempimento intracellulare della fibra gigante (GF) con un colorante fluorescente è dimostrata. Nel CVC gli assoni GF si trovano sulla superficie dorsale e quindi possono essere facilmente visualizzati al microscopio con contrasto di interferenza differenziale (DIC) ottica. Questo permette l&#39;iniezione degli assoni GF con colorante in questo sito per etichettare il GF intero compreso gli assoni e la loro terminali nel VNC. Questo metodo si traduce in colorazione affidabile e forte del GF che permette ai neuroni di essere ripreso immediatamente dopo il riempimento con un microscopio epifluorescente. In alternativa, il segnale fluorescente può essere migliorata utilizzando procedure di immunoistochimica standard 3 adatto per la microscopia confocale ad alta risoluzione.

Watch this Scientific Journal Video about Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection at JoVE.com

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

Un<em&gt; In vivo</emDissezione&gt; dell&#39;adulto<em&gt; Drosophila</emCordone nervoso&gt; ventrale (VNC) è dimostrata. Questo metodo di dissezione particolare provoca pochi danni alla VNC che permette l&#39;etichettatura successivo dei neuroni fibra gigante con colorante fluorescente per l&#39;imaging ad alta risoluzione.

Monte Preparazione tutto il Adulti Drosophila Cordone nervoso ventrale per iniezione Giant Dye Fibra

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

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Research

JoVE Journal

Neuroscience

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

18.9K Views.  Florida Atlantic University. Un In vivo dell'adulto Drosophila ventrale (VNC) è dimostrata. Questo metodo di dissezione particolare provoca pochi danni alla VNC che permette l'etichettatura successivo dei neuroni fibra gigante con colorante fluorescente per l'imaging ad alta risoluzione.

Video: Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

Visualizzazione del sistema nervoso embrionale in Whole-mount Drosophila Embrioni

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

Ricerca

Neuroscienze

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

Le registrazioni elettrofisiologiche dal Percorso di Fiber Gigante D. melanogaster</em

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

Nanoinjection associato e test Elettrofisiologia allo schermo per la bioattività dei composti con il Drosophila melanogaster Sistema Fiber Giant

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Riproducibile nervo sciatico Crush mouse e successiva valutazione da parte della Rigenerazione intera analisi Muscle Monte

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

DII-Etichettatura di neuroni DRG per studiare Branching assonale in un monte intero Preparazione di Spinal Cord embrionali di topo

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

Dissezione del muscolo Transversus Abdominis per l'analisi della giunzione neuromuscolare a montaggio intero

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Tutta Monte immunomarcatura di recettore olfattivo neuroni nel<em&gt; Drosophila</em&gt; Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons are involved in proprioceptor signaling and also report on metabolic state and injury related events. We describe an adult mouse in vitro muscle-nerve preparation for studies on stretch-activated muscle afferents.

Un<em&gt; In Vitro</emPreparazione muscolare-nervosa&gt; Adult mouse per studiare le proprietà di cottura delle afferenze muscolari

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

Tutta Monte dissezione e immunofluorescenza del topo adulto Coclea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

La preparazione di Oblique Spinal Cord Fette per ventrale Root Stimolazione

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...