Name: single-cell resolution fluorescence live imaging of drosophila circadian clocks in larval brain culture
Uploaded: 2018-01-19T15:00:00
Description: Watch this Scientific Journal Video about Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture at JoVE.com

Wij danken Michael Rosbash voor zijn mentorschap en ondersteuning tijdens de eerste fase van de ontwikkeling van deze methode. Dit werk werd gefinancierd door het JST PRESTO programma, de Swiss National Science Foundation (31003A_149893 en 31003A_169548), de European Research Council (ERC-StG-311194), Novartis Foundation for Medical-biomedisch onderzoek (13A39) en de Universiteit van Genève .

1. voorbereiding van de stamoplossingen onder de motorkap van een cultuur
<ol><li>Bereiden van 400 mL 1 x Schneider actieve Medium (SAM) geoptimaliseerd voor ex vivo cultuur van larvale hersenen (gewijzigd van referentie24,25) (tabel 1). Aliquot tot 5 mL en flash bevriezen in vloeibare stikstof (LN2), op te slaan bij - 80 ° C.</li>
	<li>1 x ontrafeling zoutoplossing (DSS) bereid door verdunning 10 x stockoplossing (gewijzig...

<ol>
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T., Currie, J., Menaker, M., Wijnen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence/luminescence+circadian+imaging+of+complex+tissues+at+single-cell+resolution.">Fluorescence/luminescence circadian imaging of complex tissues at single-cell resolution.</a> J Biol Rhythms. 25 (3), 228-232 (2010).</li><li>Liang, X., Holy, T. E., Taghert, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronous+Drosophila+circadian+pacemakers+display+nonsynchronous+Ca(2)(+)+rhythms+in+vivo.">Synchronous Drosophila circadian pacemakers display nonsynchronous Ca(2)(+) rhythms in vivo.</a> Science. 351 (6276), 976-981 (2016).</li><li>Liang, X., Holy, T. E., Taghert, P. 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A., Campusano, J. M., Su, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+mushroom+body+Kenyon+cells+generate+spontaneous+calcium+transients+mediated+by+PLTX-sensitive+calcium+channels.">Drosophila mushroom body Kenyon cells generate spontaneous calcium transients mediated by PLTX-sensitive calcium channels.</a> J Neurophysiol. 94 (1), 491-500 (2005).</li><li>Hafer, N., Schedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+larval+CNS+in+Drosophila+melanogaster.">Dissection of larval CNS in Drosophila melanogaster.</a> J Vis Exp. 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Hier beschreven we de methode voor de lange termijn fluorescentie time-lapse microscopie van gekweekte larvale hersenen. Het succes van dit soort experimenten hangt van meerdere factoren, zoals de gezondheid van de cultuur, methode voor immobilisatie van de hersenen explant, fluorescentie intensiteit en signaal-/ ruisverhouding van de verslaggever, temporele en ruimtelijke resolutie, en toegankelijkheid van de explant. Deze factoren kunnen uitsluiten. Bijvoorbeeld, verhogen van time-lapse frequentie beïnvloedt de gezond...

Circadiane klokken helpen organismen aan te passen aan de periodieke veranderingen in het milieu die zijn gegenereerd door de 24u draaiing van de aarde. De interlocked transcriptionele-translationeel feedbacklussen vaak ten grondslag liggen de moleculaire machine van circadiane klokken over soorten1. Het circuit van de circadiane pacemaker bestaat uit de klok-bevattende neuronen integreert tijd van de dag informatie overgebracht door middel van de ecologische signalen, zoals de licht/donker (LD) en temperatuur cycli, om te orkestreren de ritmes van een overvloed aan dagelijks fysiologische en gedragsmatige processen2,3. De coördinatie van de moleculaire ritmes met neuronale inputs en outputs is uitermate belangrijk voor de werking van de circadiane circuit maar blijft slechts ten dele begrepen.
In Drosophila, de kern van de moleculaire klok, de klok/cyclus (CLK/CYC) heterodimer activeert de transcriptie van periode (per) en tijdloze (tim). PER TIM vormen een complex en voeren de kern, waar ze transcriptionele activiteit van CLK/CYC en daarmee hun eigen transcriptie remmen. Post-transcriptional en posttranslationele verordeningen veroorzaken vertragingen tussen CLK/CYC-gemedieerde transcriptie en repressie door PER / TIM, zorgen voor de generatie van circa 24 uur moleculaire oscillaties1,3,4 . Ongeveer 150 neuronen die deze moleculaire klokken-formulier bevat vliegt een circuit tot controle circadiane gedrag van volwassene5. Een veel eenvoudiger maar volledig functionele circadiane circuit dat bestaat uit 3 groepen van neuronen van de klok - 5 ventrale laterale neuronen (LNvs; 4 PDF-positieve LNvs en één PDF-negatieve LNv, zie hieronder), 2 dorsale Neuron 1s (DN1s) en 2 dorsale Neuron 2s (DN2s) - is aanwezig in het larvale hersenen6,7.
Het eenvoudige larvale circadiane circuit biedt een uitstekend model voor het bestuderen van de interacties tussen Inter neuronale communicatie en de moleculaire uurwerk. Met behulp van onze nieuw ontwikkelde fluorescerende verslaggever PER-TDT, die de hoeveelheid PER eiwit en haar subcellular locatie bootst, wilde wij karakteriseren de dynamiek van de moleculaire uurwerk in verschillende klok neuron subgroepen in het larvale circadiane circuit 8. Bovendien, te weten de sleutelrol van de neuropeptide pigment-dispersing factor (PDF) geproduceerd door 4 LNvs bij het reguleren van de circadiane ritmen bij de neuronale niveau9,10,11, we wilden onderzoeken van de directe effect van de PDF op de moleculaire klokken. Te dien einde ontwikkelden we een methode voor het controleren van de circadiane genexpressie ritmes in larvale hersenen over meerdere dagen door time-lapse confocale microscopie explant. Het protocol is ook aangepast voor farmacologische testen om te testen van het effect van PDF of andere verbindingen op het niveau van de PER-TDT. Dus, de aanpassing bestaat uit de hersenen explant cultuur toegankelijk maken voor toepassing van de drug, verhoging van de temporele resolutie en imaging voor een kortere duur.
Ex vivo cultuur van Drosophila hersenen van verschillende ontwikkelingsstadia geweest tevoren vastgelegde12,13,14,15,16,17 ,18. Overwegende dat deze protocollen zijn gebruikt voor imaging-verschillende biologische fenomenen, sommige van hen zijn niet compatibel met beeldvorming bij eencellige resolutie of bieden geen ondersteuning voor de cultuur voor meer dan enkele uren. Alternatieve methoden voor het uitvoeren van langdurige live beeldvorming van circadiane neuronen in Drosophila omvatten bioluminescentie beeldvorming van moleculaire ritmes19,20,21 en fluorescentie beeldvorming van calcium-indicator met licht blad microscopie22,23. Hoewel bioluminescentie imaging hogere temporele resolutie bereiken kan en lichte blad microscopie aangepast voor in vivo imaging worden kan, ze zijn beperkt op de ruimtelijke resolutie en vereisen gespecialiseerde Microscoop systemen.
De hier beschreven methode is toegesneden op het visualiseren van fluorescerende signalen in de hele hersenen cultuur bij eencellige resolutie over meerdere dagen. Deze facile en veelzijdige methode kan worden aangepast naar afbeelding gekweekte volwassen vliegen hersenen en farmacologische experimenten veel verschillende problemen in Drosophila neurobiologie te bestuderen.

<table>
	<tbody>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td>I am not sure they are exactly the same ones we have in the lab. I chose &#34;suitable for insect cell culture&#34; whenever available</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C7902</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4.7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>230391</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+) Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Sigma-Aldrich</td>
			<td>Y1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I0516-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>BIS-TRIS</td>
			<td>Sigma-Aldrich</td>
			<td>B4429</td>
			<td></td>
		</tr>
		<tr>
			<td>L-(&#8722;)-Malic acid</td>
			<td>Sigma-Aldrich</td>
			<td>M7397</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Trehalose dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>T0167</td>
			<td></td>
		</tr>
		<tr>
			<td>Succinic acid</td>
			<td>Sigma-Aldrich</td>
			<td>S9512</td>
			<td></td>
		</tr>
		<tr>
			<td>Fumaric acid</td>
			<td>Sigma-Aldrich</td>
			<td>F8509</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-Ketoglutaric acid</td>
			<td>Sigma-Aldrich</td>
			<td>K1128</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-heat-inactivated, Foetal Calf Serum (FCS) Mycoplasma and Virus screened</td>
			<td>BioConcept Ltd. Amimed</td>
			<td>2-01F30-I</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES-KOH, pH 7.4</td>
			<td>E&#38;K Scientific Products</td>
			<td>EK-654011</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>S5011</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S7903</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen from bovine plasma</td>
			<td>Calbiochem (Merck)</td>
			<td>341573-1GM</td>
			<td>CAUTION: Harmful by inhalation, in contact with skin and if swallowed. Manipulate under laminar flow</td>
		</tr>
		<tr>
			<td>Thrombin from bovine plasma</td>
			<td>Sigma-Aldrich</td>
			<td>T9549</td>
			<td>CAUTION: Health Hazard, use gloves</td>
		</tr>
		<tr>
			<td>PDF, NH2-NSELINSLLSLPKNMNDA-OH</td>
			<td>Chi Scientific</td>
			<td>custom made</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaccum grease</td>
			<td>Sigma-Aldrich</td>
			<td>18405</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Dish, No. 1.5 Coverslip, 20 mm Glass Diameter, Uncoated</td>
			<td>MatTek</td>
			<td>P35G-1.5-20-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Falcon Easy-Grip Tissue Culture Dishes</td>
			<td>fisherscientific</td>
			<td>08-772A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 500 mL Steritop-GP 33 mm threaded bottle top filter, 0.22 &#956;m</td>
			<td>Millipore</td>
			<td>SCGPS05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Polytetrafluoroethylene (PTFE) film</td>
			<td>Dupont</td>
			<td>200A Teflon FEP Film</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-HV Syringe Filter Unit, 0.45&#160;&#181;m, PVDF, 33&#160;mm, gamma sterilized</td>
			<td>Millipore</td>
			<td>SLHV033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-GV Syringe Filter Unit, 0.22&#160;&#181;m, PVDF, 33&#160;mm, gamma sterilized</td>
			<td>Millipore</td>
			<td>SLGV033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Three-well glass dissection dish</td>
			<td>Any company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps, size 5, Dumont</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Tandem scanner inverted TCS SP5 confocal microscope, with resonant scanner and HyD photo-multiplier detectors</td>
			<td>Leica microsystem CMS GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature control chamber</td>
			<td>Life Imaging Services</td>
			<td>The CUBE &#38; BOX temperature control system, custom designed</td>
			<td></td>
		</tr>
		<tr>
			<td>Stage-top humidity controller</td>
			<td>Life Imaging Services</td>
			<td>custom made</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Immersion Micro Dispenser: dispenser, extended micro-pump MP6 series and Autoimmersion Objective Controller software</td>
			<td>Leica microsystem CMS GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SUM-stack creation and 3D correction drift plugin</td>
			<td>ImageJ software</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10x iterative deconvolution</td>
			<td>AutoQuant and Imaris software</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell resolution fluorescence live imaging of drosophila circadian clocks in larval brain culture

Het circuit van de circadiane pacemaker regisseert ritmische gedrags- en fysiologische uitgangen gecoördineerd met milieu signalen, zoals dag/nacht cyclus. De moleculaire klok binnen elke pacemaker neuron genereert circadiane ritmen in genexpressie, ten grondslag liggen, de ritmische neuronale functies noodzakelijk zijn voor de werking van het circuit. Onderzoek naar de eigenschappen van de individuele moleculaire oscillatoren in verschillende subklassen van pacemaker neuronen en hun interactie met de neuronale signalering levert een beter begrip van het circadiane pacemaker-circuit. Hier presenteren we een time-lapse fluorescent microscopie aanpak ontwikkeld voor het controleren van de moleculaire uurwerk in klok neuronen van gekweekte Drosophila larvale hersenen. Deze methode staat de meerdaagse opname van de ritmes van genetisch gecodeerde fluorescerende circadiane verslaggevers bij eencellige resolutie. Deze setup kan worden gecombineerd met farmacologische manipulaties nauw analyseren real-time respons van de moleculaire klok op verschillende verbindingen. Buiten de circadiane ritmes biedt deze multifunctionele methode in combinatie met krachtige Drosophila genetische technieken de mogelijkheid om te bestuderen van de diverse neuronale of moleculaire processen in levende hersenweefsel.

Hier, laten we de representatieve resultaten van het langdurige opname van een circadiane fluorescerende verslaggever in ex vivo larvale hersenen cultuur, en de levende imaging resultaten van PDF Bad toepassing op verslaggever expressie.
Non-zwerven L3 larven uiting geven aan de moleculaire klok verslaggever PER-TDT en UAS-mCD8::YFP gedreven door een klok neuron driver Clk (856)-gal4 (Figuu...

Watch this Scientific Journal Video about Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture at JoVE.com

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

Het doel van dit protocol is om de ex vivo Drosophila larvale hersenen cultuur, geoptimaliseerd voor het controleren van de circadiane moleculaire ritmes met langdurige fluorescentie time-lapse beeldvorming. De toepassing van deze methode op farmacologische testen wordt ook besproken.

Eencellige resolutie fluorescentie leven beeldvorming van Drosophila circadiane klokken in larvale hersenen cultuur

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. ...

Research

JoVE Journal

Neuroscience

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Visualisatie van larvale Segmentale zenuwen in 3 Rd Instar Drosophila Larvale Voorbereidingen

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

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

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

Live-cell imaging van zintuig voorloper cellen in Intact Drosophila Poppen

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 ...

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

Een snelle aanpak van High-Resolution Imaging fluorescentie in Semi-Dik Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

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

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

Morfologische Analyse van de Drosophila Larvale Perifere sensorische neuron dendrieten en axonen het gebruik van genetische Mozaïeken

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

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

Niet-Laser Capture Microscopie Aanpak voor de Microdissection van Discrete Mouse Brain Regio&#39;s voor Totaal RNA isolatie en Downstream next-generation sequencing en Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

Met een hoge resolutie Live-Imaging van cel gedrag in de ontwikkelingslanden Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Ex vivo Live-Imaging van Single Cell Divisies in Muis neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Live-Imaging van mitose in het ontwikkelen van muis embryonale Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Super-resolution Imaging of Neuronal Dense-core Vesicles

Super-resolution Imaging van Neuronal Dense-core Blaasjes

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and ...

Live Imaging of Drosophila Larval Neuroblasts

Live Imaging of Drosophila Larval Neuroblasts

Live-Imaging van<em&gt; Drosophila</em&gt; Larvale neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...