Gli autori desiderano ringraziare la Banca Ibridoma Developmental Studies presso la University of Iowa per averci inviato anticorpi. Questo lavoro è stato sostenuto da una borsa di studio (n. 29/08) da The Israel Science Foundation. COME è anche supportato da un assegno di ricerca dal DFG (Deutsche Forschungsgemeinschaft).

Prima di iniziare <ol><li> Far crescere le culture mosca desiderati. Tenere i flaconcini poco affollate (circa 30 mosche per flaconcino da 50 ml). Lasciate che le mosche depongono le uova per un solo giorno in ogni fiala. Ciò garantirà l&#39;approvvigionamento alimentare sufficiente larve e permettere loro di raggiungere una dimensione massima prima di strisciare fuori dal cibo. Tenere i flaconcini di temperatura adeguata fino al terzo stadio le larve iniziano a vagare sulla parete flaconcino. </li><li> Preparare un nuovo ceppo di tampone fosfato (PBS) e PBT (0,1% Tween in PBS). Mantenere 10 ml di PBS su ghiaccio. </li><li> Preparare 1-5 ml di fissativo fresco (4% della formaldeide in PBT) e tenerlo in ghiaccio. Usiamo bottiglia di formaldeide 37-38% come soluzione stock. </li></ol> 1. Dissezione e fissazione del Terzo Larve Instar <ol><li> Scegli una larva vagante terzo stadio dalla parete del flaconcino e collocarlo in una diminuzione del 50 microlitri di PBS ghiacciato su un piatto dissezione Sylgard (in Sylgard 184 kit di silicone elastomero, Dow Corning Corporation in untessuto piatto di cultura). </li><li> Tenere il lato dorsale larva alto, vicino ai ganci della bocca, con pinza sottile (Dumont # 5 pinza), bastone e un perno insetto (minutien, 0,1 mm, in acciaio inox) attraverso il cervello della larva. </li><li> Afferra l&#39;estremità posteriore della larva con la pinza, tirare delicatamente e allungare delicatamente la larva della lunghezza. Attaccare un altro pin insetto tra i due spiracoli posteriori. </li><li> Molla con le forbici tagliare due incisioni orizzontali nella parete del corpo (perpendicolare alla asse anteriore-posteriore), vicino ai piedini anteriori e posteriori. </li><li> Utilizzando le forbici molla tagliare la parete larvale corpo lungo la linea mediana dorsale dal anteriore alla posteriore incisione. </li><li> Togliere gli organi interni (intestino, ghiandole salivari, Malpighi tubuli ecc) e la trachea utilizzando una pinza sottile. Lavare delicatamente una o due volte con PBS. </li><li> Prendete i due lembi parete del corpo con le pinze, allungare fuori e li pin alla piastra con due pin di insetti per ogni lato. </li><li> Rimuovere il PBS e aggiungere 50 ul di soluzione di fissaggio (4% formaldeide in PBT). Incubare 20 minuti a temperatura ambiente. </li><li> Rimuovere il fissativo. Lavare due volte con PBS. Estrarre i perni di insetti. Molla con le forbici tagliare la testa larvale e la coda lasciando un filetto rettangolare. Trasferire il tessuto fissato ad un tubo Eppendorf refrigerati con PBS. </li><li> Una volta che un numero sufficiente di larve sono fissati, si può continuare direttamente a colorazione anticorpale. Se la colorazione immediata non è desiderato, le larve fisso deve essere lavata 3 volte, 5 minuti ciascuno, con 95% etanolo e conservato in etanolo al 95% a -20 ° C. </li></ol> 2. Dissezione e fissazione di pupe Parte I <ol><li> Partecipare alle fiale, come per la fissazione delle larve, le larve fino al terzo instar cominciano a pupariate. Esaminare i flaconi ogni ora larve e Mark che hanno pupariated. Lasciare che il pupe marcata a sviluppare 30-40 ore a 24 ° C o 24-27 ore a 29 ° C. </li><li> Utilizzando una pinza sottile raccogliere 20-30 pupe di The l&#39;età appropriata e metterli in un buio di porcellana multi-piatto e dissezione. Fare attenzione a non danneggiare i tessuti di interesse. </li><li> Estrarre il pupe dal caso pupa. Iniziate staccando l&#39;opercolo e continuare fino a quando la pupa è libero. Mettere la pupa in un pozzo pieno di PBT. Continua l&#39;estrazione di tutte le pupe. Passi 2,4-2,6 dovrebbe essere fatto in piccoli lotti di cinque pupe alla volta. </li><li> Posizionare cinque pupe su una superficie piana tra i pozzetti del piatto dissezione. Con un coltello micro dissezione tagliare la punta della testa e la punta posteriore dell&#39;addome (in alternativa, è possibile utilizzare due coppie di pinze per forato ad entrambe le estremità della pupe). </li><li> Tenere la pupa in posizione utilizzando una pinza sottile e usare una siringa da 1 ml per lavare gli organi interni della pupa, iniettando PBT attraverso l&#39;apertura anteriore. </li><li> Lavare brevemente sezionato pupa immergendolo in un pozzo riempito con PBS e spostarla nella fissativo freddo in un tubo Eppendorf mantenuto in ghiaccio. </li><li> Incubare (ON) a 4 ° C. </li></ol> Parte II <ol start="8"><li> Eliminare il fissativo e lavare le pupe per tre volte, a 5 minuti ciascuno, con PBT. Tenere il pupe lavato su ghiaccio. </li><li> Riempire due pozzi del piatto con la dissezione PBT. Utilizzo di un polietilene Pasteur pupe trasferire più pipetta ad uno dei pozzetti. </li><li> Spostare una pupa alla volta nel secondo pozzo. Utilizzando due coppie di pinze con punte affilate perfettamente allineati, staccare la cuticola trasparente pupa dell&#39;ala: fissare il pupa al fondo del pozzetto usando una coppia di pinze, e delicatamente strappare la cuticola utilizzando la seconda coppia di pinze. Una volta che la cuticola è strappata, staccatelo l&#39;ala. Fare attenzione a non staccare l&#39;ala dalla pupa. Dopo aver tolto la cuticola dalla lama ala, continuano peeling la cuticola della cerniera ala (dove i c&#39;os ala si trovano). </li><li> Dopo aver tolto la cuticola dell&#39;ala, si può tentare staccando la cuticola gamba in modo simile.Molte le gambe sono suscettibili di essere perso nel processo, ma nonostante la bassa resa, questo passaggio è altamente consigliata dato che migliora notevolmente la colorazione dei Chos gambe. I Chos del HALTERE e dell&#39;addome possono essere visualizzati senza ulteriori staccando la cuticola. </li><li> Posizionare il &#39;sfogliato&#39; pupa in una provetta Eppendorf riempito con metanolo e mantenere a temperatura ambiente. Continua staccando la cuticola di tutti i pupe, e aggiungerli al tubo stesso. Almeno 10 le pupe ben pelati devono essere raccolti per ogni colorazione. </li><li> Togliere il metanolo e lavare tre volte, a 5 minuti ciascuno, con il 95% di etanolo. Pupe fisso può essere conservati per lunghi periodi di tempo in 95% etanolo a -20 ° C. </li></ol> 3. Immunostaining di larve e pupe <ol><li> Lavare il tessuto fissato con PBT tre volte, 30 minuti ciascuna, a temperatura ambiente. Le larve possono essere lavati con agitando delicatamente su un piatto rotante. Pupe vengono lavati senza agitare. </li><li> Sostituire il PBT con il tampone di bloccaggio (PBT + 5% nsiero di capra Ormal) e incubare ON a 4 ° C. </li><li> Rimuovere il tampone bloccante e incubare con anticorpi primari (diluito in tampone di bloccaggio) ON a 4 ° C. </li><li> Lavare quattro volte con PBT come descritto al punto 3.1. </li><li> Rimuovere il PBT e incubare con anticorpi secondari (diluito in tampone di bloccaggio) ON a 4 ° C. </li><li> Lavare due volte con PBT e una volta con PBS come descritto al punto 3.1. </li><li> Sostituire il PBS con il mezzo di montaggio (Dako Medio fluorescente di montaggio (Cytomation Dako, Glostrup, Danimarca) e incubare a 4 ° C ON. </li></ol> 4. Montaggio di larve <ol><li> Le larve sono montati su un vetrino da microscopio in una goccia di mezzi di montaggio con la loro cuticola rivolto verso il basso e muscoli della parete del corpo rivolta verso l&#39;alto. Metti una piccola goccia di mezzo di montaggio su un vetrino pulito e usarlo per coprire la preparazione. Non applicare alcuna pressione sulle larve montato. </li></ol> 5. Montaggio di Pupae <ol><li> Preparare due vetrini da microscopiocon una goccia di mezzo di fissaggio, uno per la dissezione e la seconda per il montaggio. </li><li> Mettere pupe più su una delle diapositive. </li><li> Molla con le forbici togliere la testa e la parte posteriore dell&#39;addome. </li><li> Separare la parte dorsale del torace dalla metà ventrale tagliando tra le ali e le gambe. </li><li> Collocare le parti del corpo sezionati del pupa sul vetrino secondo una goccia di soluzione di montaggio. Assicurarsi che le ali e le gambe sono tese e cercare di ridurre al minimo la sovrapposizione dei tessuti, per quanto possibile. </li><li> Mettere una goccia di mezzo di montaggio su un vetrino e collocarlo sul campione. Non applicare una pressione sul campione montato. </li></ol> 6. Risultati rappresentativi Un esempio di immuno-macchiati c&#39;os di larva terzo stadio è illustrato nella figura 1. Questo esempio mostra un segmento addominale ben tesa, in cui sette degli otto Chos sono chiaramente visibili. I neuroni sono labELED con MAb22C10 (1:20, ottenuto dalla Banca Developmental Studies Ibridoma presso la University of Iowa), il tappo, legamento, cap-attachment e le cellule di attacco dei legamenti vengono etichettati con anti-αTub85E (1:10, Klein et al,. 2010). Anticorpi secondari per la colorazione fluorescente erano Cy3 o Cy5-coniugato anti-mouse/rabbit (1:200, ImmunoResearch Jackson Laboratories). I campioni sono stati visualizzati mediante microscopia confocale (LSM 510, Zeiss). Un gruppo di Chos ala al vena ventrale radiale di 35 hr-vecchio pupa è mostrato in Figura 2. <img alt="Figura 1" src="/files/ftp_upload/3846/3846fig1.jpg" /> Figura 1 (A) Illustrazione schematica dei sei tipi di cellule che costituiscono un singolo organo: Ch. Attaccamento cap (CA), il tappo (C), scolopale (S), neurone (N), legamento (L) e di attaccamento legamento (LA ). Il LCh5 organo, in cui sono raggruppati cinque c&#39;os, è teso il gruppo di muscoli laterali trasversali (LT1-4). Il muscolo laterale longitudinale (LL1), ventrali muscoli longitudinali (VL1-4) e laterale muscolo obliquo (LO1) sono anche illustrati. Cellule tendinee sono illustrati come sfere marroni (Tratto da Klein et al., 2010). (B) Un segmento addominale di una larva di terzo stadio visti con un ingrandimento 10X. Sette degli otto Chos presenti in ciascun segmento addominale sono evidenti: cinque organi laterali che formano l&#39;organo pentascolopidial (LCh5), un unico organo laterale (LCh1) e uno dei due Chos ventrali (VChB). I neuroni (N) del C&#39;os sono etichettati con il marcatore neuronale MAb 22C10 (rosso). Il legamento (L), il tappo (C) e le cellule di attacco (Los Angeles, CA) vengono etichettati con anti-αTub-85E (blu). Le cellule tappo e legamenti sono inoltre etichettati con un Cho-specifica reporter GFP ospitare un elemento normativo del locus dei (Nachman et al, dati non pubblicati). <img alt="Figura 2" src="/files/ftp_upload/3846/3846fig2.jpg" /> Figura 2. </sTrong&gt; Gli organi ala cordotonali alla vena ventrale radiale sono visti con un ingrandimento 40X. I neuroni (N) sono contrassegnati con le 22C10 marcatori neuronali (Red, B). Il legamento (L) e il tappo (C), le cellule sono co-etichettati con anticorpi anti-αTub85E (Blu, C) e un Cho-specifica GFP giornalista transgene (Nachman et al, dati non pubblicati).

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Il protocollo descritto in questo video fornisce un modo per visualizzare Chos propriocettivi durante le fasi larvali e pupe. Gli studi sulla struttura e lo sviluppo di Chos propriocettivi sono stati finora limitati principalmente agli stadi embrionali e larvali dischi immaginali. Così, molti aspetti delle fasi successive di sviluppo, di Chos sia larvali e adulti, rimangono in gran parte sconosciuto. Il protocollo descritto, insieme con i comuni strumenti genetici in Drosophila, può essere utilizzato per identificare e studiare nuovi geni e le vie che giocano un ruolo nelle fasi successive di sviluppo Chos. Questo protocollo è stato ottimizzato per la visualizzazione di Cho, ma può essere adattato per la colorazione di tessuti, quali alette 6 pupa.

<table border="1"><tbody><tr><td> Nome del reagente / strumento </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> Dumont # 5 (o # 55) pinza, Biologie punta </td><td> FST </td><td> 11252-20 o 11252-10 (Pinza o simili) </td><td></td></tr><tr><td> Austerlitz inox perni in acciaio, insetti minutiens 0.1mm </td><td> Roboz Co strumento chirurgico </td><td> RS-6083-10 </td><td></td></tr><tr><td> Sylgard 184 silicone kit elastomero </td><td> Dow Corning Corporation </td><td> 240.4019862 </td><td></td></tr><tr><td> Vännäs forbici micro (dritto, 7,5 cm, lama 3 mm) </td><td> AS Mdeizintechnik GmbH </td><td> 11-590-00 </td><td> Forbici primavera Vännäs con specifiche identiche possono essere acquistati presso Instrument Co. Roboz chirurgico </td></tr><tr><td> Agitatore orbitale - Rotamax-120 </td><td&gt; Heidolph </td><td> N / A </td><td></td></tr><tr><td> Dako fluorescente Mezzo di montaggio </td><td> Cytomation Dako, Glostrup, Danimarca </td><td> DK-5302392 </td><td></td></tr><tr><td> X10 PBS formulazione </td><td colspan="2"> 2 gr / lit KCl, 2 gr / lit KH 2 PO 4, 80 gr / lit NaCl, 21,7 gr / lit Na 2 HPO4.7H 2 O </td><td> La qualità di PBS è critico per il successo di questo protocollo </td></tr><tr><td> PBT </td><td colspan="2"> X1 PBS + 0,1% Tween 20 </td><td></td></tr></tbody></table>

visualization of proprioceptors in drosophila larvae and pupae

La propriocezione è la capacità di percepire il movimento, o la posizione, di parti del corpo, rispondendo agli stimoli derivanti all&#39;interno del corpo. In moscerini e altri insetti propriocezione è fornita da personale specializzato organi sensoriali chiamati organi cordotonali (c&#39;os) 2. Come molti altri organi in Drosophila, Chos sviluppare due volte durante il ciclo di vita della mosca. In primo luogo, il Chos larve si sviluppano durante l&#39;embriogenesi. Poi, i Chos adulti cominciare a sviluppare nei dischi immaginali larvali e continuano a differenziare durante la metamorfosi. Lo sviluppo di Chos larvali durante l&#39;embriogenesi è stata ampiamente studiata 10,11,13,15,16. Il fulcro di ogni Cho è una unità sensoriale composto da un neurone e una cellula scolopale. L&#39;unità sensoriale è teso tra due tipi di cellule accessorie che si attaccano alla cuticola tramite cellule specializzate di attacco epidermiche 1,9,14. Quando una larva di mosca si muove, lo sfollamento e della relativacellule di fissaggio pidermal porta allo stiramento dell&#39;unità sensoriale e la conseguente apertura specifici transitori recettore vanilloide potenziale (TRPV) canali al segmento esterno del dendrite 8,12. Il segnale suscitato viene poi trasferito al circuito generatore di modello di locomotore centrale nel sistema nervoso centrale. Chos multiple sono state descritte nella mosca adulta 7. Questi sono situati vicino alle articolazioni delle appendici mosca adulta (gambe, ali e cavezze) e nel torace e nell&#39;addome. Inoltre, diverse centinaia di Chos collettivamente formano l&#39;organo di Johnston nel antenna adulto che trasducono acustica in energia meccanica 3,5,17,4. In contrasto con la conoscenza approfondita sullo sviluppo di Chos in stadi embrionali, molto poco si sa circa la morfologia di questi organi durante le fasi larvali. Inoltre, ad eccezione di Chos femorale 18 e organo Johnston, il conoscenge sullo sviluppo e la struttura del Chos nella mosca adulta è molto frammentaria. Qui si descrive un metodo per la colorazione e la visualizzazione chos in terza larve instar e pupe. Questo metodo può essere applicato con strumenti genetici per caratterizzare meglio comprendere la morfologia e lo sviluppo del C&#39;os vari la mosca.

Watch this Scientific Journal Video about Visualization of Proprioceptors in Drosophila Larvae and Pupae at JoVE.com

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Un metodo per immunostain e visualizzare organi cordotonali in larve e delle pupe<em&gt; Drosophila melanogaster</em&gt; Descritto.

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 ...

visualization-of-proprioceptors-in-drosophila-larvae-and-pupae

Research

JoVE Journal

Neuroscience

Visualization of Proprioceptors in Drosophila Larvae and Pupae

11.9K Views.  Technion-Israel Institute of Technology. Un metodo per immunostain e visualizzare organi cordotonali in larve e delle pupe Drosophila melanogaster Descritto.

Video: Visualization of Proprioceptors in Drosophila Larvae and Pupae

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

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.
The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species1,2. The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology4-6 In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus8. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9 in addition to their recently discovered role as photoreceptors10. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells11,12. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11 where they may connect to second-order neurons that project to the brain.
Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates.

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.

Metodi locale e globale valutazione nocicezione termica in<em&gt; Drosophila</em&gt; Le larve

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

Appetitivo apprendimento associativo olfattiva in<em&gt; Drosophila</em&gt; Le larve

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer basic questions on how light information is processed and shared between rapid and circadian behaviors. Drosophila larvae display a stereotypical avoidance behavior when exposed to light. To investigate light dependent behaviors comparably simple light-dark preference tests can be applied. In vertebrates and arthropods the neural pathways involved in sensing and processing visual inputs partially overlap with those processing photic circadian information. The fascinating question of how the light sensing system and the circadian system interact to keep behavioral outputs coordinated remains largely unexplored. Drosophila is an impacting biological model to approach these questions, due to a small number of neurons in the brain and the availability of genetic tools for neuronal manipulation. The presented light-dark preference assay allows the investigation of a range of visual behaviors including circadian control of phototaxis.

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.

Luce Preferenza Assay per studiare Innata e circadiano Photobehavior Regolamentato<em&gt; Drosophila</em&gt; Larve

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits1-6. Application of optogenetics7,8 in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways9-13. Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system14,15. In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin16,17 or halorhodopsin18-20, respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Here we describe a protocol for optogenetic manipulation of motoneuronal activity while monitoring changes in motor output (muscle contraction) in semi-intact Drosophila larvae using lasers within a conventional confocal microscope. This technique enables researchers to achieve local perturbation of neural activity in a few neuromeres to elucidate the dynamics of motor circuits.

Optogenetic Perturbazione di attività neurale con illuminazione laser in semi-intatte<em&gt; Drosophila</em&gt; Larve in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

Foraggiamento protocollo percorso di lunghezza per<em&gt; Drosophila melanogaster</em&gt; Le larve

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

Nuovo metodo di analisi per Cold nocicezione in<em&gt; Drosophila</em&gt; Le larve

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to&#160;larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

Un'analisi di gradiente di temperatura per determinare le preferenze termiche delle larve di Drosophila

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

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

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

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

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

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

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

Visualizzazione delle reti di microtubuli nelle giunzioni neuromuscolari e nelle cellule muscolari di Drosophila

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of &#945;-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

Autore in primo piano: Comprensione della rete di microtubuli nelle giunzioni neuromuscolari di Drosophila 

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.