Name: morphological analysis of drosophila larval peripheral sensory neuron dendrites and axons using genetic mosaics
Uploaded: 2011-11-07T12:00:00
Description: Watch this Scientific Journal Video about Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics at JoVE.com

Forfatterne takker Riken til finansiering. Vi takker også Cagri Yalgin, Caroline Delandre, og Jay Parrish til diskussioner om genetiske og immunhistokemi protokoller.

1.Preparation af reagenser <ol><li> Forbered Ca + +-fri HL3.1 saltvand 28. <ol><li> I mm: 70 NaCl, 5 KCl, 20 MgCl 2, 10 NaHCO 3, 5 HEPES, 115 saccharose, og 5 trehalose, pH 7,2. Filter steriliseres og opbevares ved 4 ° C. Bemærk: Ca + +-fri løsning forebygger muskel sammentrækning under dissektion. </li></ol></li><li> Gør poly-L-lysin (PLL) dækglas. <ol><li> Opløs 100mg PLL i 4.2ml vand og 300μl aliquoter i Eppendorf rør og fryses ved -20 ° C. </l...

<ol>
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Den Drosophila Larvetilstand DA neuron model giver en fremragende genetiske system til at undersøge mekanismerne, der styrer neuron morfologi og kredsløb dannelse. MARCM er generelt bruges til mærkning og for at skabe mutant DA neuron kloner. Til MARCM bruger vi enten en pan-neurale (f.eks Gal4 C155) eller DA neuron-specifik driver. Ved hjælp af en pan-neurale driver det er muligt direkte at bruge flere lagre almindeligt tilgængelige fra offentlige lager centre. Men ved hjælp a...

<table border="1"><tr><th> Navn på reagenset </th><th> Firma </th><th> Katalog nummer </th><th> Kommentarer (valgfri) </th></tr><tr><td> SZX16 fluorescens dissektion mikroskop (med GFPHQ filter) </td><td> Olympus </td><td> SZX16 </td><td></td></tr><tr><td> Live-Insect Tang </td><td> FST </td><td> 26030-10 </td><td></td></tr><tr><td> 26mm x 76mm depression slide glas </td><td> Toshinriko Co </td><td> T8-R004 </td><td></td></tr><tr><td> Sylgard 184 (eller Silpot 184) </td><td> Dow Corning </td><td> 3097358-1004 </td><td></td></tr><tr><td> Poly-L-lysin </td><td> Sigma </td><td> P-1524 </td><td> Dette produkt har vist sig mest effektive </td></tr><tr><td> DPX Mounting Medium </td><td> Sigma </td><td> 44581 </td><td></td></tr><tr><td> Kanin anti-GFP </td><td> Invitrogen </td><td> A-11122 </td><td> Fortynding 1:500 </td></tr><tr><td> Rat anti-CD8 </td><td> Caltag </td><td> 5H10 </td><td> Fortynding 1:200 </td></tr><tr><td> Muse anti-CD2 </td><td> AbD serotec </td><td> MCA443R </td><td> Fortynding 1:700 </td></tr><tr><td> Muse anti-Fasciclin2 </td><td> DSHB </td><td> 1D4 </td><td> Fortynding 1:10 </td></tr></table>

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

Nervesystemets udvikling kræver den korrekte specifikation af neuron position og identitet, efterfulgt af præcise neuron klasse-specifikke dendritiske udvikling og axonal ledninger. For nylig dendritiske arborization (DA) sensoriske neuroner i det Drosophila larvestadiet perifere nervesystem (PNS) er blevet magtfulde genetiske modeller til at belyse både generelle og klasse-specifikke mekanismer neuron differentiering. Der er fire vigtigste DA neuron klasser (I-IV) 1. De er opkaldt efter stigende dendritceller Arbor kompleksitet, og har klasse-specifikke forskelle i den genetiske kontrol over deres differentiering 2-10. The Da sensoriske system er en praktisk model til at undersøge de molekylære mekanismer bag kontrol af dendritiske morfologi 11-13 fordi: 1) det kan drage fordel af den kraftige genetiske værktøjer til rådighed i bananfluen, 2) DA neuron dendritceller arbor breder sig i kun 2 dimensioner under et optisk Clear larver neglebånd gør det let at visualisere med høj opløsning in vivo, 3) den klasse-specifikke mangfoldighed i dendritiske morfologi letter en sammenlignende analyse for at finde centrale elementer styre dannelsen af simple vs stærkt forgrenede dendritiske træer, og 4) dendritiske arbor stereotype figurer af forskellige DA neuroner lette morfometriske statistiske analyser. DA neuron aktivitet ændrer produktionen af en larvestadiet bevægelse central mønster generator 14-16. De forskellige DA neuron klasser har forskellige sanser, og deres aktivering udløser forskellige adfærdsmæssige reaktioner 14,16-20. Desuden forskellige klasser sende aksonal fremskrivninger stereotypically i Drosophila larvestadiet centrale nervesystem i ventrale nerve ledning (VNC) 21. Disse fremskrivninger opsige med topografiske fremstillinger af både DA neuron sensorisk modalitet og placeringen i kroppen væggen af dendritiske feltet 7,22, 23. Derfor undersøgelse af DA axonal fremskrivninger kan bruges til at belyse mekanismerne bag topografisk kortlægning 7,22,23, samt ledningsføring af et simpelt kredsløb modulerende larver bevægelse 14-17. Vi præsenterer her en praktisk guide til at generere og analysere genetiske mosaikker 24 mærkning DA neuroner via MARCM (Mosaik analyse med en Repressible Cell Marker) 1,10,25 og FLP-out 22,26,27 teknikker (opsummeret i Fig. 1).

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

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

De dendritiske arborization sensoriske neuroner i det<em&gt; Drosophila</em&gt; Larver perifere nervesystem er nyttige modeller til at belyse både generelle og neuron klasse-specifikke mekanismer neuron differentiering. Vi præsenterer en praktisk guide til at generere og analysere dendritiske arborization neuron genetiske mosaikker.

Morfologiske Analyse af Drosophila Larver Perifer sensorisk Neuron dendritter og axoner anvende genetiske Mosaikker

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

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

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

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

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

Immunohistological Mærkning af mikrotubuli i Sensorisk Neuron dendritter, tracheae, og muskler i Drosophila Larver Krop Wall

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

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

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

Visualisering og genetisk manipulering af dendritter og udløberne i mus cerebrale cortex og hippocampus under anvendelse<em&gt; I utero</em&gt; Elektroporation

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

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

Spændingsfølsomme Dye Optagelse fra axoner, dendritter og dendrit udløberne af individuelle neuroner i hjernen Slices

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

Functional Analysis of the Larval Feeding Circuit in Drosophila

Functional Analysis of the Larval Feeding Circuit in Drosophila

Funktionel analyse af larve Fodring Circuit i Drosophila</i

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

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Hele Mount immunolabeling af olfaktoriske receptor neuroner i<em&gt; Drosophila</em&gt; Antenne

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

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

Produktion og Isolering af Axoner fra sensoriske neuroner for Biokemisk Analyse Brug porøse filtre

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

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

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

fjernelse af<em&gt; Drosophila</em&gt; Muskel Væv fra larver Filet for Immunofluorescens analyse af sensoriske neuroner og epidermale celler

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

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Brug Linear Agarose Kanaler til at studere<em&gt; Drosophila</em&gt; Larve Crawling Behavior

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

A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

Et Microbiomechanical System for at studere Varicosity dannelse og opsving i centrale Neuron Axons

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with ...