Prima di iniziare <ol><li> Tutte le culture devono essere coltivate a 25 ° C. </li><li> HL3.1 tampone dissezione può essere preparata in anticipo. Prendete un aliquota 50 ml di HL3.1 e tenerlo in ghiaccio. </li><li> Preparare 15 ml di fresca formaldeide 3,5% nel HL3.1. Tenerlo in ghiaccio. </li></ol> Dissezione larvale <ol><li> Mettere una goccia di freddo HL3.1 sul piatto dissezione. Ciò manterrà l&#39;animale da essiccazione e stordire l&#39;animale che lo rende più facile lavorare con. </li><li> Utilizzando le pinze lunghe, selezionare un errante larva terzo instar e inserirlo nel menu di HL3.1. </li><li> In primo luogo, utilizzare pinze breve per cogliere un perno minutien. Posizionare il perno tra gli spiracoli posteriori. L&#39;animale in genere tenta di strisciare via dal perno che si stira fuori e rendendo più facile per voi di inserire un pin dritto nella testa della larva nei pressi della foce ganci. Tratto l&#39;animale in senso longitudinale. Questo vi aiuterà a massimizzare la quantità di parete del corpo esposte si può raggiungere durante il taglio. Nota: Se si sceglie di pin la prima testa, l&#39;animale può avvolgere il corpo in tutto il pin o frusta il suo corpo avanti e indietro, rendendo difficile al pin. Di solito è possibile contrastare questa rimuovendo il HL3.1 e mettere una goccia fresca fredda stupefacente l&#39;animale di nuovo. Lo scienziato ambidestro può solo prendere l&#39;animale e tenerlo in posizione. </li><li> Forbici primavera utilizzando fare una incisione orizzontale appena anteriormente al pin posteriore sul lato dorsale della larva. Luogo una lama delle forbici in incisione e fare un taglio verticale lungo la linea mediana dorsale verso la fine rostrale della larva. Alla tribuna degli animali fanno incisioni orizzontali a sinistra ea destra del perno. Nota: il risultato finale dovrebbe apparire come un io sul lato dorsale della larva lungo l&#39;asse rostrocaudal. </li><li> Rimuovere gli organi. Iniziare mettendo alcuni ulteriori cadute di HL3.1 sulla larva. In questo modo gli organi di galleggiare fuori dal corpo consentendo di rimuoverli molto più facilmente. In primo luogo, rimuovere il sistema tracheale. In secondo luogo, utilizzare la pinza per afferrare il resto degli organi e rimuoverli. </li><li> Durante la fase 3 che hai fatto un flap flap sinistra e destra nella parete del corpo. Pin i lembi in senso orario assicurandosi per allungare la parete del corpo sia orizzontalmente che verticalmente. </li></ol> Fissazione Fissare ogni animale presente nel 3,5% di formaldeide in HL3.1 per 25 minuti. Lavare l&#39;animale due volte in HL3.1 per 5 minuti. Immagazzinamento Rimuovere i perni e trasferire le larve in una provetta contenente 1,5 ml di PBS 1X. Se si prevede di utilizzare l&#39;immagine del NMJ fuso tag fluorescenti, si dovrebbe immagine degli animali entro due giorni. È possibile memorizzare le larve sezionato per un massimo di una settimana a 4 ° C. Rappresentante risultati Ci sono parecchi componenti chiave di una larva adeguatamente sezionato Drosophila. In primo luogo, si deve fare molta attenzione a non danneggiare i muscoli, soprattutto i muscoli 4, 6, e 7. Questi sono i muscoli più popolari di studiare e se sono danneggiati scartare la preparazione filetto e sezionare un altro animale. In secondo luogo, si deve assicurarsi che l&#39;animale è allungato al massimo in modo che ogni muscolo e NMJ possono essere distinti.

<ol>
	<li>Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+the+larval+neuromuscular+junction+in+Drosophila+Melanogaster.">Properties of the larval neuromuscular junction in Drosophila Melanogaster.</a> J. Physiol. 262, 189-214 (1976).</li><li>Johansen, J., Halpern, M. E., Johansen, K. M., Keshishian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotypic+morphology+of+glutamatergic+synapses+on+identified+muscle+cells+of+Drosophila+larvae.">Stereotypic morphology of glutamatergic synapses on identified muscle cells of Drosophila larvae.</a> J Neurosci. 9, 710-725 (1989).</li><li>Kesheshian, H., Broadie, K., Chiba, A., Bate, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+neuromuscular+junction:+a+model+system+for+studying+synaptic+development+and+function.">The Drosophila neuromuscular junction: a model system for studying synaptic development and function.</a> Annu Rev Neurosci. 19, 545-575 (1996).</li><li>Vactor, D. V., Sink, H., Fambrough, D., Tsoo, R., 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=Genes+that+control+neuromuscular+specificity+in+Drosophila.">Genes that control neuromuscular specificity in Drosophila.</a> Cell. , 73-1137 (1993).</li><li>Feng, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+minimal+hemolymph-like+solution,+HL3.1,+for+physiological+recordings+at+the+neuromuscular+junctions+of+normal+and+mutant+Drosophila+larvae.">A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae.</a> J Neurogenet. 18 (2), 377-402 (2004).</li></ol>

La tecnica di dissezione dimostrato in questo video può essere utilizzato per preparare le larve di Drosophila per una varietà di tecniche sperimentali. Se i tag proteina fluorescente sono presenti, le larve possono essere montati e ripreso immediatamente. Altrimenti, immunostaining può essere effettuata in modo da segnare specifici comparti sinaptica. Inoltre, elettrofisiologia può essere facilmente effettuata sulla larve sezionato per valutare il funzionamento della neurotrasmissione. <p class="jove_c...

HL3 Buffer Dissection: (in mm) 70 NaCl, 5 KCl, 0,2 CaCl2, 20 MgCl2, 10 NaHCO3, 5 trealosio, 115 saccarosio, e 5 HEPES, pH 7.3.<br /<br /&gt; Sylgard Piastra dissezione: Mescolare delicatamente (per evitare le bolle) 10:1 in un bicchiere. Versare il composto nella piastra di Petri (qualsiasi dimensione). Lasciare SylGard per curare una notte in incubatrice 37C.

drosophila larval nmj dissection

Il<em&gt; Drosophila</em&gt; Giunzione neuromuscolare (NMJ) è un sistema modello stabilito usato per lo studio dello sviluppo e della plasticità sinaptica. L&#39;uso diffuso del<em&gt; Drosophila</em&gt; Motore del sistema è dovuta alla sua alta accessibilità. Può essere analizzati con cella singola risoluzione. Ci sono 30 muscoli per hemisegment la cui disposizione all&#39;interno delle mura organo periferico sono noti. Un totale di 35 neuroni motori attribuiscono a questi muscoli in un modello che ha alta fedeltà. Utilizzando biologia molecolare e della genetica, si possono creare animali transgenici o mutanti. Poi, si può studiare le conseguenze dello sviluppo sulla morfologia e la funzione del NMJ. Per poter accedere al NMJ per lo studio, è necessario analizzare con attenzione ogni larva. In questo articolo mostriamo come sezionare correttamente<em&gt; Drosophila</emLarve&gt; per lo studio del NMJ rimuovendo tutti gli organi interni, lasciando intatta la parete del corpo. Questa tecnica è adatta per preparare le larve per l&#39;imaging, immunoistochimica, o elettrofisiologia.

Watch this Scientific Journal Video about Drosophila Larval NMJ Dissection at JoVE.com

Drosophila Larval NMJ Dissection

Questo protocollo dimostra come sezionare<em&gt; Drosophila</emLarve&gt; in preparazione per immunoistochimica e / o immagini della giunzione neuromuscolare.

Drosophila larvali NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Research

JoVE Journal

Biology

34.6K Views.  Columbia University College of Physicians and Surgeons. Questo protocollo dimostra come sezionare Drosophila in preparazione per immunoistochimica e / o immagini della giunzione neuromuscolare.

Video: Drosophila Larval NMJ Dissection

Dissection of Drosophila Ovaries

Dissezione di ovaie Drosophila

Ricerca

Biologia

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

Dissezione del sistema nervoso centrale larvali in Drosophila melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

Dissezione di dischi immaginale da larve 3 Instar Drosophila

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

Drosophila larvali NMJ Immunoistochimica

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

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

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

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

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

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

Il monitoraggio delle funzioni cardiaca in larvali Drosophila melanogaster Per gli Studi fisiologici

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

Dissezione di Oenocytes da adulti Drosophila melanogaster</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

Dissezione e colorazione di Drosophila Larvali ovaie

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

Dissezione di cuore in larvale, Zebrafish giovanile e degli adulti, Danio rerio</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific shapes. Drosophila tracheal terminal cells have become a powerful genetic model to identify and elucidate the roles of genes required for generating cellular morphologies. Terminal cells are a component of a branched tubular network, the tracheal system that functions to supply oxygen to internal tissues. Terminal cells are an excellent model for investigating questions of cell shape as they possess two distinct cellular architectures. First, terminal cells have an elaborate branched morphology, similar to complex neurons; second, terminal cell branches are formed as thin tubes and contain a membrane-bound intracellular lumen. Quantitative analysis of terminal cell branch number, branch organization and individual branch shape, can be used to provide information about the role of specific genetic mechanisms in the making of a branched cell. Analysis of tube formation in these cells can reveal conserved mechanisms of tubulogenesis common to other tubular networks, such as the vertebrate vasculature. Here we describe techniques that can be used to rapidly fix, image, and analyze both branching patterns and tube formation in terminal cells within Drosophila larvae. These techniques can be used to analyze terminal cells in wild-type and mutant animals, or genetic mosaics. Because of the high efficiency of this protocol, it is also well suited for genetic, RNAi-based, or drug screens in the Drosophila tracheal system.

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Here, we present a method for light microscopy analysis of tracheal terminal cells in Drosophila larvae. This method allows for quick examination of branch and lumen morphology in whole animals and would be useful for analysis of individual mutants or screens for mutations affecting terminal cell development.

Esame della<em&gt; Drosophila</em&gt; Larvali tracheali Terminal cellule al microscopio ottico

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific ...