Ringraziamo RIKEN per il finanziamento. P10-GAL4 è stato un gentile dono di Alain Vincent (Université Paul Sabatier di Tolosa, Francia).

1. Preparazione dei reagenti Note prima di iniziare: Dissection e colorazione immunoistochimica si svolgono in una camera magnetica e la larva è immobilizzato mediante perni di insetti forma speciale. Istruzioni dettagliate per la costruzione di una camera magnetica, e la preparazione di questi pin possono essere trovati nei riferimenti relativi 14,15. In breve, un foro quadrato 1x1cm viene tagliato in un foglio magnetico e un copri apposta sul retro del foglio per fare una piccola camera. I lati della camera sono sigillati con la colla epossidica, dopo questa colla ha fissato la camera è lavato più volte con etanolo al 70% prima dell&#39;uso. Perni insetti dissezione vengono preparate piegando secondo la forma desiderata e poi incollate su una scheda di metallo 14,15. In alternativa ad una linguetta di metallo, abbiamo usato una rovesciata a testa piatta puntina da disegno in acciaio con manico fatto da un cut-off punta gialla. Avvalersi di questa facoltà camera magnetica permette uno stretto controllo Over posizionamento pin e stiramento dei tessuti durante la dissezione. Per guidare l&#39;espressione genica giornalista in vari sottoinsiemi di neuroni DA investigatori possono utilizzare diverse GAL4 differenti linee (riassunto da Shimono e colleghi 16). Molte di queste linee sono disponibili presso i centri di magazzino pubblico. In questo protocollo rappresentante, effettuiamo immunocolorazione di una linea in cui due classi contrastanti DA neuroni sono co-marcato: il più semplice di classe I e più complessi di classe IV (P10-GAL4 17,18, UAS-mCD8:: Kusabira-Orange (KO)). <ol><li> Preparare Ca + +-libero HL3.1 salina <ol><li> In mM: 70 NaCl, 5 KCl, 20 MgCl 2, 10 NaHCO 3, 5 HEPES, 115 saccarosio, trealosio e 5; pH 7,2 19. Filtro-sterilizzare e conservare a 4 ° C. Nota: Ca + +-senza soluzione impedisce la contrazione muscolare durante la dissezione. </li></ol></li><li> Preparare 2x tampone PHEM <ol><li> In mM: 130 TUBI, 60 HEPES, 20 EGTA, 4 MgSO4; pH 7,0. Filtro-sterilizzare e conservare a 4 ° C. Nota: Questi materiali non si dissolvono fino a quando il pH si avvicina 7.0. </li></ol></li><li> Preparare il fissativo fresco immediatamente prima della fissazione. <ol><li> Al fine di preparare una soluzione da 25 ml, prima paraformaldeide 2g mix, 100μl di NaOH 1M e 10 ml di acqua in un tubo da 50 ml Falcon. </li><li> Agitare fissativo in un bagno d&#39;acqua ° 55 C con agitatore fino a quando la soluzione è limpida. (Volume totale sarà di circa 11,5 ml.) </li><li> Fissativo raffreddare su ghiaccio. </li><li> Aggiungi 12.5ml tampone 2x PHEM. </li><li> Regolare il pH a 7,0 con HCl 1M. </li><li> Riempire la soluzione con acqua fino a quando il volume è 25ml. </li><li> Filtrare la soluzione con carta Whatman. </li></ol></li></ol> 2. Dissezione larvale Nota prima di iniziare: le reti di microtubuli, in particolare quella dendriti sensoriali, sarà guasto rapidamente dopo l&#39;inizio della dissezione. Achieving dissezione veloce in meno di cinque minuti, seguita da fissazione immediata sono fattori chiave per il successo di questo protocollo. <ol><li> Lavare la larva in acqua e rapidamente muoversi in questa goccia utilizzando un ciclo di capelli. </li><li> Orientare il lato alto larva dorsale ed il lato ventrale sul vetro. Nota: Questo orientamento è quello di esaminare i neuroni gruppo ventrale. Per esaminare i neuroni gruppo dorsale, invertire l&#39;orientamento. </li><li> Utilizzare il centro insetti pin to pin all&#39;estremità anteriore vicino alla bocca ganci. Posizionare il perno vicino alla fine per i migliori risultati. </li><li> Mettere una goccia di soluzione salina HL3.1 nella camera di dissezione. </li><li> Tagliare la punta posteriore della larva fuori con microscissors. Nota: questo passaggio si apre un&#39;apertura alla fine posteriore della larva che permetterà di accesso per il microscissors (punto 2.7). </li><li> Prendete con le pinze la regione del budello che ora spunta fuori l&#39;apertura alla fine posteriore della larva. Estrarre delicatamente l&#39;intestino intero. </li><li> Posizionare ilpunta di una lama di microscissors attraverso l&#39;apertura e tagliare lungo la linea mediana ventrale verso l&#39;anteriore. </li><li> Utilizzando i perni d&#39;angolo, pin agli angoli ora libero, posteriore, poi anteriore. Allo stesso tempo delicatamente tratto aperto il filetto larvale. </li></ol> 3. Fissazione, blocco, colorazione e montaggio di filetti larvale Note prima di iniziare: tutte le fasi di fissazione e colorazione sono svolte nella camera di dissezione. Durante questo processo, fare attenzione a non urtare i perni tenendo la larva dell&#39;insetto in quanto ciò potrebbe provocare danni tissutali. Per evitare che l&#39;esperimento si secchi, fare tutti i passi colorazione in un piccolo contenitore Tupperware circondato da tessuti inumidito. <ol><li> Aspirare il Ca + +-libero HL3.1 buffer utilizzando una punta di giallo. Aggiungere immediatamente il fissativo utilizzando un altro pipetteman. </li><li> Pipettare delicatamente su e giù per miscelare il fissativo con le restanti tracce di Ca + +-libero HL3.1 tamponenella camera. Immediatamente aspirato e quindi aggiungere fissativo fresco nella camera. </li><li> Incubare a temperatura ambiente per 20 minuti. </li><li> Lavare 10 minuti 6x in PBST (0,1% Triton X-100 in PBS). </li><li> Blocco con siero di capra al 5% in PBST per 20 minuti a RT. </li><li> Sostituire la soluzione di saturazione con anticorpi primari diluiti in siero di capra al 5% in PBST. Gli anticorpi primari utilizzati sono topo anti-α-tubulina (DM1A) e nel ratto anti-CD8 (5H10) sia diluito 1 / 1000. Nota: L&#39;investigatore può voler sostituire il mouse anti-α-tubulina (DM1A) con il mouse anti-Futsch (22C10) 20,21 diluito 1 / 1000 in alcune circostanze (vedi la discussione). </li><li> Incubare per una notte (per almeno 16h) a 4 ° C. </li><li> Lavare 10 minuti 6x in PBST. </li><li> Aggiungere la soluzione secondaria di anticorpi nel siero di capra diluito al 5% in PBST. Gli anticorpi secondari utilizzati sono Alexa Fluor 488 capra anti-topo IgG e Cy3 asino anti-topo IgG. Conservare il campione coperto per evitare fluoroforofotometabolismo 22. </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 10 minuti 6x in PBST. </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 monte &#39;veloce&#39;. Nota: Per una migliore compensazione dei tessuti, e un campione permanente stabile, montare in DPX come descritto in precedenza 23. </li></ol> 4. Rappresentante dei risultati: Colorazione fluorescente è stato esaminato al microscopio confocale. Nelle figure 1-2, rami diversi all&#39;interno di un pergolato dendrite hanno diverse organizzazione del citoscheletro. La figura 1 mostra una regione del pergolato di una IV classe DA neurone al 1 ° stadio larvale instar. Il pergolato tutto è contrassegnato da mCD8:: KO e individuata tramite un anticorpo anti-CD8 e fluorescenti secondario (Cy3). Tubulina viene rilevato utilizzando anticorpi anti α-tubulina anticorpo e fluoroescent secondaria (Alexa Fluor 488). I rami principali sono positivi per la tubulina, alcuni rami laterali sottili sono tubulina-negativi. Movie 1 è un insieme di sezioni seriali attraverso una colorazione simile di una classe I DA neurone. La Figura 2 mostra una regione del pergolato di una IV classe DA neuroni colorati con anticorpi contro Futsch e CD8 al 1 ° stadio larvale instar. I rami principali sono Futsch-positivi, alcuni rami laterali sottili sono Futsch-negativi. Figura 3. Trachee nella parete del corpo larvale mostrano una complessa organizzazione dei microtubuli. Film 2 e 3 mostrano sezioni seriali di colorazione attraverso muscoli della parete del corpo e della trachea. <img alt="Figura 1" src="/files/ftp_upload/3662/3662fig1.jpg" /> Figura 1. Fig. 1 mostra una regione del pergolato di una IV classe DA neurone al 1 ° stadio larvale instar. Il pergolato tutto è contrassegnato da mCD8:: KO e rilevati utilizzando anticorpi anti-CD8 e fluorescenti secondario (Cy3). Tubulina viene rilevato mediante anti-&amp;-tubulin anticorpi e fluorescenti secondarie (Alexa 488). Pannelli AC sequenziale confocale z-sezioni (0.5μm), C &#39;C &quot;, la colorazione degli anticorpi singolo pannello C. Esempio di rami con (freccia gialla) o senza (viola punta di freccia) microtubuli sono evidenziati. Red microtubuli evidenziano punte di freccia nel sottostante cellule epiteliali. <img alt="Figura 2" src="/files/ftp_upload/3662/3662fig2.jpg" /> Figura 2. Fig. 2 mostra una regione simile del pergolato di una IV classe da neuroni colorati con anticorpi contro Futsch e CD8 al 1 ° stadio larvale instar. Il pergolato tutto è contrassegnato da mCD8:: KO e rilevati utilizzando anticorpi anti-CD8 e fluorescenti secondario (Cy3). Futsch viene rilevato utilizzando anticorpi anti-Futsch e fluorescenti secondarie (Alexa 488). I rami principali sono Futsch-positivi, alcuni rami laterali sottili sono Futsch-negativi. Esempio di rami con (freccia gialla) o senza (viola punta di freccia) Futsch sono evidenziati. Figura 3. Trachea colorati con l&#39;anti-tubulina protocollo sopra descritto. Questa larva è terzo instar e sezionato come descritto in precedenza 23,24. Movie 3 mostra allargata sezioni seriali dal campo contrassegnato da un quadrato. Sezioni Movie 1. Serial tracciamento colorazione tubulina in tutto il pergolato dendritiche di una classe I neurone. Il pergolato tutto è contrassegnato da mCD8:: KO (Magenta) e rilevata con anticorpi anti-CD8 e fluorescenti secondario (Cy3). Tubulina (verde) viene rilevata con un anti-α-tubulina e anticorpi secondari fluorescenti (Alexa Fluor 488). Scala: da un lato dell&#39;immagine video corrisponde alla 46.88μm nella sezione. <a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie1.avi">Clicca qui per visualizzare il filmato.</a> Sezioni Movie 2. Serial tracciamento colorazione tubulina a body muscoli della parete di una larva di terzo stadio. Tubulina viene rilevato mediante anti-α-tubulina e anticorpi secondari fluorescenti (Alexa Fluor 488). Scala: da un lato dell&#39;immagine video corrisponde alla 46.88μm nella sezione. <a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie2.avi">Clicca qui per visualizzare il filmato.</a> Movie 3. Sezioni seriali tracciamento colorazione tubulina in un corpo trachea muro di una larva di terzo instar, contrassegnato con un quadrato in Figure.3. Tubulina viene rilevato mediante anti-α-tubulina e anticorpi secondari fluorescenti (Alexa Fluor 488). Scala: da un lato dell&#39;immagine video corrisponde alla 46.88μm nella sezione. <a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie3.avi">Clicca qui per visualizzare il filmato.</a>

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Per capire come forme complesse cellule che sono raggiunto è importante essere in grado di accuratezza dell&#39;organizzazione test microtubuli. Qui si descrive un metodo robusto etichettatura immunoistologiche all&#39;organizzazione microtubuli dosaggio dei dendriti arborizzazione dendritica neurone sensoriale. Oltre alla colorazione neuroni sensoriali, questo metodo ottiene robusto colorazione immunoistologiche della trachea, dei muscoli e altri tessuti parete del corpo. Noi utilizziamo q...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Catalogo numero </th><th> Commenti (Opzionale) </th></tr><tr><td> Forcipe </td><td> Dumont </td><td> 11251-20 </td><td></td></tr><tr><td> Microscissors </td><td> FST </td><td> 15000-08 </td><td></td></tr><tr><td> Topo anti-α-tubulina (Clone: ​​DM1A) </td><td> Sigma </td><td> T9026 </td><td> Diluizione 1 / 1000 </td></tr><tr><td> Topo anti-Futsch (Clone: ​​22C10), surnatante </td><td> Sviluppo Studi Ibridomi Banca </td><td> 22C10 </td><td> Diluizione 1 / 1000 </td></tr><tr><td> Rat anti-CD8 (Clone: ​​5H10) </td><td> Caltag </td><td> MCD0800 </td><td> Diluizione 1 / 1000 </td></tr><tr><td> Alexa Fluor 488 IgG anti-topo </td><td> Invitrogen </td><td> A-11001 </td><td> Diluizione 1 / 500 </td></tr><tr><td> Cy3 IgG anti-Rat </td><td> Jackson ImmunoResearch </td><td> 712-166-150 </td><td> Diluizione 1 / 200 </td></tr></tbody></table>

immunohistological labeling of microtubules in sensory neuron dendrites, tracheae, and muscles in the drosophila larva body wall

Per capire come le differenze nelle forme cellulari complessi siano raggiunti, è importante seguire con precisione l&#39;organizzazione dei microtubuli. Il corpo Drosophila larvale parete contiene diversi tipi di cellule che sono modelli per lo studio delle cellule e la morfogenesi dei tessuti. Per esempio trachee sono utilizzati per esaminare la morfogenesi del tubo 1, e la arborizzazione dendritica (DA) neuroni sensoriali della larva di Drosophila sono diventate un sistema primario per la delucidazione dei generali e dei neuroni-classe-specifici meccanismi di 2-5 differenziazione dendritica e degenerazione 6 . La forma di rami dendrite può variare notevolmente tra le classi di neuroni, e anche tra i diversi rami di un singolo neurone 7,8. Studi genetici nei neuroni DA suggeriscono che la differenza di organizzazione del citoscheletro possono essere alla base delle differenze morfologiche di forma ramo dendritiche 4,9-11. Forniamo un robusto metodo di etichettatura immunologico ad unssay nell&#39;organizzazione dei microtubuli vivo nei neuroni sensoriali DA dendrite pergolato (Figure 1, 2, Movie 1). Questo protocollo illustra la dissezione e immunocolorazione della prima larva instar, una fase in cui attivo escrescenza dendriti dei neuroni sensoriali e organizzazione ramificazione sta avvenendo 12,13. Oltre alla colorazione neuroni sensoriali, questo metodo ottiene l&#39;etichettatura robusto di organizzazione dei microtubuli nei muscoli (Film 2, 3), trachea (Figura 3, Movie 3), e di altri tessuti parete del corpo. E &#39;utile per gli investigatori che desiderano analizzare l&#39;organizzazione dei microtubuli in situ nella parete del corpo durante l&#39;analisi dei meccanismi di controllo che il tessuto e la forma delle cellule.

Watch this Scientific Journal Video about Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall at JoVE.com

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

Per capire come forme cellulari complessi, come dendriti neuronali, vengono raggiunti durante lo sviluppo, è importante essere in grado di accuratezza dell&#39;organizzazione test microtubuli. Qui si descrive un metodo robusto etichettatura immunoistologiche di esaminare l&#39;organizzazione dei microtubuli dei dendriti arborizzazione dendritica neurone sensoriale, trachea, dei muscoli, e gli altri<em&gt; Drosophila</em&gt; Larva tessuti parete del corpo.

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 ...

immunohistological-labeling-microtubules-sensory-neuron-dendrites

Research

JoVE Journal

Neuroscience

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

12.3K Views.  RIKEN Brain Science Institute. Per capire come forme cellulari complessi, come dendriti neuronali, vengono raggiunti durante lo sviluppo, è importante essere in grado di accuratezza dell'organizzazione test microtubuli. Qui si descrive un metodo robusto etichettatura immunoistologiche di esaminare l'organizzazione dei microtubuli dei dendriti arborizzazione dendritica neurone sensoriale, trachea, dei muscoli, e gli altri Drosophila Larva tessuti parete del corpo.

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

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Live-cellula Imaging di cellule sensoriali Precursore in Organo intatto Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Ricerca

Neuroscienze

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

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

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

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

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 ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Visualizzazione dei Propriorecettori in<em&gt; Drosophila</em&gt; Larve e pupe

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Retrograda Etichettatura fluorescente Consente mirata extracellulare singola unità di registrazione da parte dei neuroni identificati<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

Etichettatura di singole cellule nel sistema nervoso centrale di<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of 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 ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

Comportamenti olfattive analizzati da Computer di monitoraggio di<em&gt; Drosophila</em&gt; In un Olfattometro quattro quadranti

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

Un semplice passo-dissezione Protocollo per tutto il montaggio Preparazione per adulti<em&gt; Drosophila</em&gt; Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

Etichettatura e imaging di placche amiloidi nel tessuto cerebrale utilizzando la curcumina polifenolo naturale

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

Etichettatura balistica dei neuroni piramidali nelle sezioni cerebrali e nella cultura cellulare primaria

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

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

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Capitoli in questo video

Live-cellula Imaging di cellule sensoriali Precursore in Organo intatto Drosophila Pupae

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

Visualizzazione dei Propriorecettori in

Retrograda Etichettatura fluorescente Consente mirata extracellulare singola unità di registrazione da parte dei neuroni identificati

Etichettatura di singole cellule nel sistema nervoso centrale di

Tutta Monte immunomarcatura di recettore olfattivo neuroni nel

Comportamenti olfattive analizzati da Computer di monitoraggio di

Un semplice passo-dissezione Protocollo per tutto il montaggio Preparazione per adulti

Etichettatura e imaging di placche amiloidi nel tessuto cerebrale utilizzando la curcumina polifenolo naturale

Etichettatura balistica dei neuroni piramidali nelle sezioni cerebrali e nella cultura cellulare primaria