Gli autori ringraziano RIKEN per il finanziamento. Ringraziamo anche Cagri Yalgin, Caroline Delandre, e Jay Parrish per le discussioni sui protocolli di genetica e di immunoistochimica.

1.Preparazione di reagenti <ol><li> Preparare Ca + +-libero HL3.1 saline 28. <ol><li> In mM: 70 NaCl, 5 KCl, 20 MgCl 2, 10 NaHCO 3, 5 HEPES, 115 saccarosio, trealosio e 5; pH 7,2. Filtro sterilizzare e conservare a 4 ° C. Nota: Ca + +-senza soluzione impedisce la contrazione muscolare durante la dissezione. </li></ol></li><li> Fai la poli-L-lisina (PLL) coprioggetto. <ol><li> Sciogliere 100mg PLL in acqua 4.2ml e fare aliquote 300μl in tubi Eppendorf e congelare a -20 ° C. </li><li> Prima coprioggetto rivestimento, disgelo prima una aliquota, portarla fino a 10ml in DDH 2 O e aggiungere 20μl di Kodak Photo-Flo, la concentrazione finale PLL è 0,7 mg / ml. </li><li> Coprioggetto Immergere per 30 minuti in una soluzione PLL, rimuovere e asciutto, quindi risciacquare brevemente con acqua e asciugare. Ripetere due volte. Coprioggetto trattati durano circa un mese. </li></ol></li></ol> 2. Incroci genetici <ol><li> To generare cloni MARCM. Ecco un esempio croce usando un pan-DA conducente 11,27: FRT2A x hsFLP; GAL4 109 (2) 80, SUP-mCD8:: GFP; vasca-Gal80 FRT2A/SM5-TM6B </li><li> Per generare Flp-out cloni. Ecco un esempio utilizzando una croce IV classe specifica del driver 8: PPK-GAL4 x yw, hsFLP; UAS-FRT-CD2, y +-stop-FRT-mCD8:: GFP </li></ol> 3. Collezione di embrioni <ol><li> Tenere attraversa Drosophila in una bottiglia di raccolta a 25 ° C e raccogliere embrioni su una piastra di agar succo di mela 29 distribuirlo con un sottile strato di pasta di lievito 30,31. </li></ol> 4. Calore trattamento d&#39;urto degli embrioni <ol><li> Inserire un piatto vuoto in opposizione alla piastra di agar succo di mela su cui gli embrioni sono state stabilite. Tenuta attorno a queste due piastre con Parafilm (Fig. 2). </li><li> Durante shock termico, immergere °piatto e nel bagno d&#39;acqua e tenere premuto con un peso di metallo. </li><li> Per gli embrioni MARCM <ol><li> Raccogliere embrioni per 2h e incubare in un piatto di 10 centimetri di Petri circondato da inumidito tessuti a 25 ° C per 2 ore aggiuntive. Nota: Regolazione del protocollo di shock da calore altera la frequenza con cui vengono generate cloni. </li><li> Per un minor numero di cloni, con l&#39;obiettivo di singoli neuroni isolati, immergere e shock termico nel bagnomaria per 1 ora a 38 ° C. </li><li> Per un numero maggiore di cloni, shock termico per 45 minuti a 38 ° C, recuperare a RT 30 minuti, poi shock termico di nuovo per altri 30 minuti. </li></ol></li><li> Raccogliere Flp-out embrioni per 24 ore, e lo shock termico per 1 ora a 38 ° C. </li></ol> 5. Screening per cloni <ol><li> Dopo la scossa di calore, rimuovere il coperchio della tenuta stagna disposizione e la piastra di agar succo di mela in una capsula di Petri 10 centimetri circondato da tessuti inumidito. Cultura gli embrioni e larve successive a 25 ° C fino a vagarezione 3 ° stadio. Nota: le condizioni di cultura, soprattutto la nutrizione, hanno dimostrato di alterare DA morfologia dendritica pergolato 32,33. Assicurarsi che le larve di crescita hanno accesso a pasta di lievito in ogni momento; monitorare e riempire la pasta di lievito, come richiesto. </li><li> Da questo punto in poi il protocollo, manipolare delicatamente con pinze larve di insetti. </li><li> Brevemente e risciacquare delicatamente le larve in acqua di rubinetto, e poi posto su una piastra di agar. Nota: questo passaggio riduce sfondo auto-fluorescenza da cibo o sporcizia sulla superficie larvale. </li><li> Esaminare le larve sotto un potente microscopio a fluorescenza dissezione. Identificare le larve con GFP-positive neuroni / cellule nella parete del corpo. </li></ol> 6. Imaging in vivo dei dendriti <ol><li> Posizionare la larva in un bicchiere scivolo depressione con una piccola goccia di glicerolo 80%. Mettere un coprioggetti su slitta per immobilizzare la larva. Assicurarsi che non rimanga aria fra la larva e la coverslip. </li><li> Spingere delicatamente il coprioggetto per rotolare la larva per permettere la visualizzazione del neurone di interesse. L&#39;immagine della mCD8:: GFP-etichettata pergolato dendritiche tramite microscopia confocale. </li></ol> 7. Dissezione larvale Nota prima di iniziare: DA dendriti dei neuroni degradare rapidamente dopo l&#39;inizio della dissezione. Sezionare ogni singola larva in meno di 5min per garantire la buona morfologia dendrite. <ol><li> Sezionare vagare 3 ° larve instar su un piatto Sylgard come descritto in precedenza 34 con le seguenti modifiche: <ol><li> Per l&#39;imaging di DA assoni dei neuroni termini, assicurarsi che il sistema nervoso centrale è intatto ei nervi segmentali non sono rotti. Dopo aver aperto le larve, prima accuratamente tagliate le connessioni tracheale alla parete del corpo e poi rimuovere delicatamente l&#39;intestino intero. </li><li> Se immagini il dendriti da solo, rimuovere il CNS per consentire una più piatta di montaggio. </li></ol></li></ol> 8. Fixationi e blocco dei filetti larvale <ol><li> Con la larva ancora inchiodato alla piastra Sylgard, fissarlo in PFA 4% in PBS per 20 minuti a temperatura ambiente su un agitatore rotante con delicatezza. </li><li> Rimuovere le tracce dei tessuti larvali (grasso corporeo, trachea, ecc) dopo la fissazione. </li><li> Lavare in PBST (PBS con 0,1% Triton X-100) 10 minuti 3 volte sul piatto Sylgard e shaker. Se esaminando termini assone, tenere il filetto sul piatto Sylgard. (Se dendriti colorazione è possibile trasferire i filetti larvale di un tubo da 0,5 ml in questa fase 34) </li><li> Bloccare le larve per 20 minuti a temperatura ambiente nel 5% di siero normale asino (NDS) in PBST su un agitatore. </li></ol> 9. Colorazione dei filetti larvale <ol><li> Rimuovere la soluzione di saturazione e incubare in anticorpo primario (nel 5% NDS / PBST) notte a 4 ° C in un piccolo contenitore Tupperware circondata da tessuto inumidito. </li><li> Rimuovere i tubi da 4 ° C e incubare un&#39;altra ora a temperatura ambiente. </li><li> Lavare 10 minuti 6x in PBST. Aggiungere il antib secondariaMelody (nel 5% NDS / PBST), e coperchio per evitare fotometabolismo fluoroforo 34. </li><li> Incubare sia a temperatura ambiente per due ore o una notte a 4 ° C seguita da un&#39;ora a temperatura ambiente. </li><li> Lavare i 10 minuti larve 6x in PBST e procedere al montaggio. </li></ol> 10. Montaggio di filetti larvale per l&#39;esame del pergolato dendrite <ol><li> Montare ogni filetto larvale più piatto possibile con le forbici dissezione o un bisturi per tagliare la testa (compresi i ganci bocca), e la parte posteriore (compresi gli spiracoli) 34. </li><li> Mettere il filetto di larve sul vetrino cuticola rivolto verso il basso, montare in 80% glicerolo, e sigillare i lati del coprioggetto con smalto per un &#39;veloce&#39; montare 34. </li><li> Per un montaggio permanente e un&#39;immagine più chiara <ol><li> Mettere il filetto sezionato larvale muscolare rivolto verso il basso su una goccia di PBS su un vetrino PLL, sarà presto aderire al coprioggetto. </li><li> Rimuovere quanto più liquido possibile dopo il montaggio, however non lasciarlo asciugare completamente. </li><li> Prendere il coprioggetto (con annesso filetto larvale) attraverso una serie di etanolo: 35% di etanolo, seguito dal 50%, 70%, 95% e, infine, 2x etanolo al 100% per ogni 10 minuti (il 2 ° soluzione 100% EtOH dovrebbe essere cambiato di frequente ), infine, lavare 2-3x 10 minuti in xilene. </li><li> Mettete una goccia di DPX montaggio (Xilene Plasticizer Distyrene) su un vetrino pulito e adagiare il filetto coprioggetto-side-down sulla parte superiore. Conservare al buio, e aspettare un giorno per il DPX per impostare prima di imaging. </li></ol></li></ol> 11. Montaggio di filetti larvale per l&#39;esame dei capolinea degli assoni <ol><li> Mantenere i nervi segmentali in esecuzione tra il PNS e il sistema nervoso centrale intatto durante la dissezione, la fissazione e colorazione per consentire il tracciamento degli assoni periferici dal corpo cellulare dei neuroni sensoriali al VNC. </li><li> Montare ciascun filetto cuticola larvale rivolto verso il basso in 80% glicerolo in una diapositiva depressione. Traccia gli assoni dal corpo cellulare alla DAVNC sotto il microscopio confocale. Nota: i neuroni PNS in ogni progetto segmento del corpo muro al segmento affine of the VNC. </li></ol> 12. Rappresentante dei risultati: Risultati rappresentativi sono riportati nelle figure 3-5. Fig. 3 mostra il pergolato di una intera classe IV da neurone, catturato in vivo al microscopio confocale. Fig. 4 mostra un primo piano di parte del pergolato dendrite di un immunoistochimiche etichettati classe III neurone che è stata correttamente fissata per preservare la morfologia. L&#39;inserto associati mostra la degradazione che possono verificarsi dopo una dissezione senza successo e la fissazione (entrambi colorati con anticorpi anti-GFP). Fig. 5 mostra un unico capolinea classe IV assonale nel sistema nervoso centrale (anti-GFP, verde); tutti i termini IV classe sono co-colorati con anti-CD2 (magenta). <img alt="Figura 1" src="/files/ftp_upload/3111/3111fig1.jpg" /> Figura 1 panoramica protocollo. a) raccogliere e poi heat-shock embrioni. b)<html> Seleziona una larva con GFP-positive DA neurone clone, e poi l&#39;immagine della GFP-etichettata dendrite pergolato nella larva vive. c) Dissect la larva, e poi immunoistochimiche macchia il filetto. d) Montare il filetto colorato, e poi l&#39;immagine del pergolato dendritiche e proiezioni assonali dei cloni dei neuroni DA. <img alt="Figura 2" src="/files/ftp_upload/3111/3111fig2.jpg" /> Figura 2 Preparazione della piastra di agar succo di mela per shock termico. Prendere la piastra (freccia bianca, ab), aggiungere un secondo piatto di Petri in alto (freccia rossa b) e sigillo in giro con Parafilm (freccia blu, b). <img alt="Figura 3" src="/files/ftp_upload/3111/3111fig3.jpg" /> Figura 3 in immagini in vivo del pergolato dendrite di un mCD8:: GFP-etichettata clone MARCM (genotipo come in 2.1) che rappresenta una IV classe (v&#39;ada) neurone di una larva di 3 ° stadio. Barra di scala è 50 micron. d/3111/3111fig4.jpg &quot;/&gt; Figura 4 mCD8:: GFP-etichettata clone MARCM di una categoria III neurone (ddaA) colorati con anticorpi anti-GFP, mostrando buona morfologia dopo la dissezione di successo e di fissazione (verde). L&#39;inserto mostra dendrite degradazione (frecce bianche) che si verificano dopo la dissezione senza successo e la fissazione (magenta). Barra di scala è a 25 micron. <img alt="Figura 5" src="/files/ftp_upload/3111/3111fig5.jpg" /> Figura 5 Il 3 ° instar larvale VNC. Un Flp-out di un singolo clone di classe VI (VDAB) capolinea assone (genotipo come in 2.2) viene rilevata con anticorpi anti-GFP (verde). Anti-CD2 etichette di tutte le classi IV termini (magenta). Barra di scala è a 25 micron.

<ol>
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J., Gunawardena, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+the+Embryonic+Nervous+System+in+Whole-mount+Drosophila+Embryos.">Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos.</a> J. Vis. Exp. (46), e2150-e2150 (2010).</li><li>Featherstone, D. E., Chen, K., Broadie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harvesting+and+preparing+Drosophila+embryos+for+electrophysiological+recording+and+other+procedures.">Harvesting and preparing Drosophila embryos for electrophysiological recording and other procedures.</a> J Vis Exp. , (2009).</li><li>Medina, P. M., Swick, L. L., Andersen, R., Blalock, Z., Brenman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+forward+genetic+screen+for+identifying+mutations+affecting+larval+neuronal+dendrite+development+in+Drosophila+melanogaster.">A novel forward genetic screen for identifying mutations affecting larval neuronal dendrite development in Drosophila melanogaster.</a> Genetics. 172, 2325-2335 (2006).</li><li>Mirouse, V., Swick, L. L., Kazgan, N., St Johnston, D., Brenman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LKB1+and+AMPK+maintain+epithelial+cell+polarity+under+energetic+stress.">LKB1 and AMPK maintain epithelial cell polarity under energetic stress.</a> J Cell Biol. 177, 387-392 (2007).</li><li>Brent, J., Werner, K., 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+Immunohistochemistry.">Drosophila Larval NMJ Immunohistochemistry.</a> J. Vis. Exp. 25, e1108-e1108 (2009).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Sugimura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+developmental+modes+and+lesion-induced+reactions+of+dendrites+of+two+classes+of+Drosophila+sensory+neurons.">Distinct developmental modes and lesion-induced reactions of dendrites of two classes of Drosophila sensory neurons.</a> J Neurosci. 23, 3752-3760 (2003).</li><li>Zito, K., Parnas, D., Fetter, R. D., Isacoff, E. Y., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Watching+a+synapse+grow:+noninvasive+confocal+imaging+of+synaptic+growth+in+Drosophila.">Watching a synapse grow: noninvasive confocal imaging of synaptic growth in Drosophila.</a> Neuron. 22, 719-729 (1999).</li><li>Landgraf, M., Sanchez-Soriano, N., Technau, G. M., Urban, J., Prokop, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charting+the+Drosophila+neuropile:+a+strategy+for+the+standardised+characterisation+of+genetically+amenable+neurites.">Charting the Drosophila neuropile: a strategy for the standardised characterisation of genetically amenable neurites.</a> Dev Biol. 260, 207-225 (2003).</li></ol>

Nessun conflitto di interessi dichiarati.

La Drosophila larvale DA modello neurone fornisce un ottimo sistema per investigare i meccanismi genetici che controllano la morfologia dei neuroni e la formazione di circuito. MARCM è generalmente utilizzato per l&#39;etichettatura e per la generazione di mutanti DA cloni neurone. Per MARCM abbiamo utilizzare un pan-neurale (es. GAL4 C155) o DA neurone specifico driver. Utilizzando un pan-neurali del driver è possibile usare direttamente diversi stock ampiamente disponibili presso...

<table border="1"><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </th></tr><tr><td> SZX16 dissezione microscopio a fluorescenza (con GFPHQ filtro) </td><td> Olimpo </td><td> SZX16 </td><td></td></tr><tr><td> Vivere Pinza insetti </td><td> FST </td><td> 26030-10 </td><td></td></tr><tr><td> 26 millimetri x 76mm depressione vetrini </td><td> Toshinriko Co. </td><td> T8-R004 </td><td></td></tr><tr><td> Sylgard 184 (o Silpot 184) </td><td> Dow Corning </td><td> 3097358-1004 </td><td></td></tr><tr><td> Poli-L-lisina </td><td> Sigma </td><td> P-1524 </td><td> Questo prodotto si è dimostrato più efficace </td></tr><tr><td> DPX montaggio medio </td><td> Sigma </td><td> 44581 </td><td></td></tr><tr><td> Coniglio anti-GFP </td><td> Invitrogen </td><td> A-11122 </td><td> Diluizione 1:500 </td></tr><tr><td> Rat anti-CD8 </td><td> Caltag </td><td> 5H10 </td><td> Diluizione 1:200 </td></tr><tr><td> Topo anti-CD2 </td><td> AbD Serotec </td><td> MCA443R </td><td> Diluizione 1:700 </td></tr><tr><td> Topo anti-Fasciclin2 </td><td> DSHB </td><td> 1d4 </td><td> Diluizione 1:10 </td></tr></table>

morphological analysis of drosophila larval peripheral sensory neuron dendrites and axons using genetic mosaics

Sviluppo del sistema nervoso richiede la corretta specificazione della posizione dei neuroni e l&#39;identità, seguita da accurate neurone classe specifico per lo sviluppo dendritiche e cablaggio assonale. Recentemente l&#39;arborizzazione dendritica (DA) neuroni sensoriali del sistema nervoso periferico Drosophila larvale (PNS) sono diventati potenti modelli genetici in cui per chiarire sia i meccanismi generali e specifiche della classe di differenziazione dei neuroni. Ci sono quattro principali classi di neuroni DA (I-IV) 1. Essi sono chiamati in ordine crescente di complessità dendrite pergolato, e sono specifiche della classe differenze nel controllo genetico della loro differenziazione 2-10. Il sistema DA sensoriale è un modello pratico per studiare i meccanismi molecolari alla base il controllo della morfologia dendritica 11-13 perché: 1) si può usufruire di potenti strumenti genetici disponibili nel moscerino della frutta, 2) il procuratore neurone dendrite pergolato si estende in soli 2 dimensioni sotto un ottica CLEar cuticola larvale che la rende facile la visualizzazione ad alta risoluzione in vivo, 3) la classe-specifica diversità nella morfologia dendritica facilita un&#39;analisi comparativa per trovare elementi chiave Controllando la formazione di alberi dendritici semplici vs altamente ramificata, e 4) dendritiche pergolato stereotipata forme differenti di neuroni DA facilitare morfometriche analisi statistiche. DA l&#39;attività dei neuroni modifica l&#39;uscita di un generatore di pattern locomozione larvale centrale 14-16. Le diverse classi di neuroni DA sono distinte modalità sensoriali, e la loro attivazione provoca diverse risposte comportamentali 14,16-20. Inoltre, diverse classi di inviare proiezioni stereotipo assonale nel sistema nervoso centrale Drosophila larvale nel cordone nervoso ventrale (VNC) 21. Queste proiezioni terminare con le rappresentazioni topografiche di entrambe le modalità DA neuroni sensoriali e la posizione nella parete del corpo del campo dendritiche 7,22, 23. Quindi l&#39;esame di proiezioni DA assonale può essere usata per chiarire i meccanismi alla base mappatura topografica 7,22,23, così come il cablaggio di un semplice locomozione larvale circuito modulante 14-17. Vi presentiamo qui una guida pratica per generare e analizzare i mosaici genetici 24 neuroni DA marcatura tramite MARCM (Analisi Mosaico con un marcatore cellulare reprimibile) 1,10,25 e Flp-out 22,26,27 tecniche (riassunti nella fig. 1).

Watch this Scientific Journal Video about Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics at JoVE.com

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

I neuroni sensoriali arborizzazione dendritica del<em&gt; Drosophila</em&gt; Larvale sistema nervoso periferico sono modelli utili per chiarire sia generale e classe di neuroni specifici meccanismi di differenziazione dei neuroni. Vi presentiamo una guida pratica per generare e analizzare arborizzazione dendritica dei neuroni mosaici genetici.

Analisi morfologica del Drosophila Larvale dendriti sensoriali e periferico Neuron assoni utilizzo Mosaici genetici

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

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Research

JoVE Journal

Neuroscience

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

15.2K Views.  RIKEN Brain Science Institute. I neuroni sensoriali arborizzazione dendritica del Drosophila Larvale sistema nervoso periferico sono modelli utili per chiarire sia generale e classe di neuroni specifici meccanismi di differenziazione dei neuroni. Vi presentiamo una guida pratica per generare e analizzare arborizzazione dendritica dei neuroni mosaici genetici.

Video: Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

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

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval body wall contains several cell types that are models to study cell and tissue morphogenesis. For example tracheae are used to examine tube morphogenesis1, and the dendritic arborization (DA) sensory neurons of the Drosophila larva have become a primary system for the elucidation of general and neuron-class-specific mechanisms of dendritic differentiation2-5 and degeneration6.
The shape of dendrite branches can vary significantly between neuron classes, and even among different branches of a single neuron7,8. Genetic studies in DA neurons suggest that differential cytoskeletal organization can underlie morphological differences in dendritic branch shape4,9-11. We provide a robust immunological labeling method to assay in vivo microtubule organization in DA sensory neuron dendrite arbor (Figures 1, 2, Movie 1). This protocol illustrates the dissection and immunostaining of first instar larva, a stage when active sensory neuron dendrite outgrowth and branching organization is occurring 12,13.
In addition to staining sensory neurons, this method achieves robust labeling of microtubule organization in muscles (Movies 2, 3), trachea (Figure 3, Movie 3), and other body wall tissues. It is valuable for investigators wishing to analyze microtubule organization in situ in the body wall when investigating mechanisms that control tissue and cell shape.

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

To understand how complex cell shapes, such as neuronal dendrites, are achieved during development, it is important to be able to accurately assay microtubule organization. Here we describe a robust immunohistological labeling method to examine microtubule organization of dendritic arborization neuron sensory dendrites, trachea, muscle, and other Drosophila larva body wall tissues.

Etichettatura immunoistologiche di microtubuli in Neuron dendriti sensoriali, trachee, e Muscoli in Drosophila Larva parete del corpo

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Visualizzazione e manipolazione genetica di dendriti e spine nella corteccia cerebrale e nell&#39;ippocampo mouse utilizzando<em&gt; In utero</em&gt; Elettroporazione

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

Tensione sensibile registrazione Dye da assoni, dendriti e spine dendritiche dei singoli neuroni in fettine di cervello

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

Analisi funzionale del circuito di alimentazione larvale in Drosophila</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

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 ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

Produzione e isolamento degli assoni dai neuroni sensoriali per l&#39;analisi biochimica Uso dei filtri porosi

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in communication with the epidermal cells they innervate and thus their analysis benefits from in situ visualization of both neuronally and epidermally expressed proteins by immunofluorescence. Traditional methods of preparing larval fillets for immunofluorescence experiments leave intact the muscle tissue that covers most of the body wall, presenting several challenges to imaging neuronal and epidermal proteins. Here we describe a method for removing muscle tissue from Drosophila larval fillets. This protocol enables imaging of proteins that are otherwise obscured by muscle tissue, improves signal to noise ratio, and facilitates the use of super-resolution microscopy to study da neuron development.

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall.

Rimozione di<em&gt; Drosophila</em&gt; Tessuto muscolare da larvali Filetti per l&#39;analisi di immunofluorescenza dei neuroni sensoriali e cellule epidermiche

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Utilizzando canali lineari Agarose per studiare<em&gt; Drosophila</em&gt; Comportamento larvale Crawling

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

Analisi metabolica di Drosophila melanogaster Larval e cervello adulto

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...