Name: analyzing synaptic modulation of drosophila melanogaster photoreceptors after exposure to prolonged light
Uploaded: 2017-02-10T14:00:00
Description: Watch this Scientific Journal Video about Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light at JoVE.com

Vi er taknemmelige for T. Stürner for nyttige rettelser, diskussioner og kommentarer til manuskriptet; SL Zipursky for at give flyve bestande; M Schölling til udførelse billedbehandling. En del af billedet analyse gennemføres ved A. Kakita laboratorium. Dette arbejde blev støttet af Alexander von Humboldt Foundation og JSP&#39;er stipendier til forskning i udlandet (AS), JSP&#39;er Fellows (SH-S.), Grant-In- Støtte til opstart (24.800.024), om innovative områder (25.110.713), Mochida, Takeda Inamori, Daiichi-Sankyo, Toray Foundations (TS), DZNE grundfinansiering (GT) og DZNE Light Microscopy Facility (CM).

De eksperimentelle procedurer beskrevet i denne protokol indebærer udelukkende arbejder med Drosophila og ikke udsættes for dyrevelfærd love i Tyskland og Japan. 1. Lette eksponeringsforhold <ol><li> Forbered fluer, der bærer sensless-flippase (sens-FLP) og bruchpilot-FRT-STOP-FRT-GFP (BRP-FSF-GFP) 15 til visualisering BRP-GFP i fotoreceptor R8 neuroner (R8s). Den genomiske locus kodning BRP inden en Bakterie...

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I denne undersøgelse viste vi, hvordan du forbereder lysforholdene at udsætte fluer i lige lysintensitet. Vi kvantificeres ikke kun antallet af en synaptisk markør puncta men kunne også rumligt løse tætheden af ​​synapser langs axoner og måle flytning niveau af markørproteinet i cytoplasmatiske områder. Disse tre vurderinger giver os mulighed for at vurdere detaljerne i synaptiske dynamik på enkelt neuron niveau i forskellige miljøforhold. Vores protokol kan tilpasses forskellige synaptiske proteiner, men...

<html> Modulering af synaptisk funktion bidrager til den bemærkelsesværdige evne af nervesystemet til præcist reagere eller tilpasse sig skiftende miljømæssige stimuli. Justering af præsynaptiske vesikel release sandsynlighed er en måde at styre synaptisk styrke en. Synaptisk vesikel frigivelse finder sted på den aktive zone (AZ), en specialiseret region i den præsynaptiske membran 2. AZ er kendetegnet ved en kassette af specifikke proteiner 3, 4. De fleste proteiner, der bidrager til AZ samling er stærkt konserverede i nematoder, insekter og pattedyr 5. Nylige undersøgelser tyder på, at niveauet af neuronal aktivitet regulerer den molekylære sammensætning af AZ, hvilket igen bidrager til reguleringen af den funktionelle output både in vitro og in vivo 6, 7,&gt; 8. Vi har tidligere fundet, at fotoreceptor AZS gennemgår molekylær ombygning i Drosophila efter længere tids udsættelse for naturligt omgivende lys 9. I denne tilstand, observerede vi, at antallet af Bruchpilot (BRP) -positiv AZS blev reduceret i fotoreceptor axoner. BRP / CAST / ELKS familiens proteiner er fundamentale byggesten i Azs i hvirveldyr og hvirvelløse synapser 10. I Drosophila balanceansvarlige aktørs mutanter, fremkaldte vesikel frigivelse er undertrykt 11, 12. De 17 C-terminale aminosyrerester af BRP er afgørende for synaptisk vesikel klynger ved Drosophila neuromuskulære forbindelse (NMJ) 13, 14. Disse undersøgelser viste centrale rolle dette molekyle i AZ organisation og funktion. Med en nylig udviklet genetisk værktøj, Synaptic Tagging med Rekombination (stjerne), BRPkan observeres in vivo i specifikke celletyper, på endogene ekspressionsniveauer og ved en enkelt synapse beslutning 15. Dette værktøj gør det muligt at evaluere de endogene dynamik synapser kvantitativt i komplekset centralnervesystemet. Der har været flere undersøgelser, herunder synapse kvantificeringer baseret på data indsamlet fra konfokal mikroskopi. Synaptiske ændringer er blevet evalueret ved at måle længden, området, volumen, densitet og tælle antallet baseret på avancerede programmer. For eksempel, freeware ImageJ giver en kvantificering metode til total synaptisk område og synaptiske foranstaltninger densitet på Drosophila NMJ 16. Antallet af co-lokaliseringstoppe steder af pre og postsynaptiske markører er blevet kvantificeret ved hjælp af plugin &quot;puncta Analyzer&quot; tilgængelige på ImageJ software platform 17. Alternativt, en multi-paradigm numerisk computermiljø baseret program, Synapse Detector (synd), kan automatisk spore dendritter af neuroner mærket med en fluorescerende markør, og derefter kvantificeres de synaptiske proteinniveauer som funktion af afstand fra cellelegemet 18. Den software Synaptic puncta Analysis (SynPAnal), er designet til hurtig analyse af 2D-billeder af neuroner erhvervet fra konfokal eller fluorescerende mikroskopi. Den primære funktion af denne software er den automatisk og hurtig kvantificering af tæthed og intensitet af protein puncta 19. For nylig har en automatisk læring-baserede synapse afsløring algoritme blevet genereret til kvantificering af synaptisk nummer i 3D 20, at drage fordel af 3D Visualisering-Assisted Analysis (Vaa3D) software 21. Kommerciel billedanalyse software er også stærke værktøjer til synaptiske kvantificeringer. For eksempel den fluorescerendemærkede neurotransmitterreceptorer eller en præsynaptisk AZ komponent er blevet kvantificeret i tre dimensioner med en enkelt synapse beslutning i C. elegans 22 eller Drosophila lugtesystemet 23, 24, hvilket tillader hundreder af synapser, der skal hurtigt kendetegnet inden for en enkelt prøve. Her præsenteres en fremgangsmåde ved en tilpasset billedanalyse software plug-in implementeret i en multi-paradigme numerisk computermiljø, der gør det muligt at analysere halvautomatisk flere aspekter af AZS, herunder deres nummer, fordeling og niveauet af berigelse af molekylære bestanddele til AZ. Således er denne komplekse analyse tilladt os at vurdere dynamikken i synaptiske bestanddele i axonterminaler under forskellige miljøforhold. Vi undersøgte effekten af ​​lys eksponering på output synapser af voksne flyve fotoreceptorer. Proceduren udføres i tre trin: 1)forberedelse til lyseksponering, 2) dissektion, immunhistokemi og konfokal billeddannelse, og 3) billedanalyse.

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	<tbody>
		<tr>
			<td>Vial</td>
			<td>Hightech, Japan</td>
			<td>MKC-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Plug</td>
			<td>Thermo Fisher Sciehtific, USA</td>
			<td>AS-275</td>
			<td></td>
		</tr>
		<tr>
			<td>Customized transparent rack made of acrylic resin&#160;</td>
			<td>Shin-Shin Corporation, Japan</td>
			<td></td>
			<td>a height of 41 cm, a base of 21 cm, a thickness of 4 cm and a height of 13 cm for each step</td>
		</tr>
		<tr>
			<td>Cool incubator</td>
			<td>MITSUBISHI ELECTRIC, Japan</td>
			<td>CN-40A</td>
			<td></td>
		</tr>
		<tr>
			<td>LED panel</td>
			<td>MISUMI, Japan</td>
			<td>LEDXC170-W</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital light meter</td>
			<td>CEM</td>
			<td>DT-1301</td>
			<td></td>
		</tr>
		<tr>
			<td>Fly pad</td>
			<td>Tokken, Japan</td>
			<td>TK-HA03-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (35 x 10 mm)</td>
			<td>Greiner Bio-One International, Germany</td>
			<td>627102</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS tablet</td>
			<td>Takara, Japan</td>
			<td>T900</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Wako, Japan</td>
			<td>160-24751</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml tube</td>
			<td>Sarstedt, Germany</td>
			<td>A. 152X</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde 16%</td>
			<td>NEM, Japan</td>
			<td>3152</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman P-200</td>
			<td>Gilson</td>
			<td>F123601&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman P-20</td>
			<td>Gilson</td>
			<td>F123600</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman P-2</td>
			<td>Gilson</td>
			<td>F144801</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-chaoptin antibody</td>
			<td>DSHB</td>
			<td>24B10</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa568-conjugated anti-mouse antibody</td>
			<td>Life Technologies</td>
			<td>A-11031</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASHIELD Mounting Medium</td>
			<td>Vector Laboratories, Inc.</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide (76 x 26 mm)</td>
			<td>Thermo Fisher Scientific Gerhard Menzel B.V. &#38; Co. KG, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip (18 x 18 mm, 0.17 mm)</td>
			<td>Zeiss, Germany</td>
			<td>474030-9000-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Industrial Microscopes</td>
			<td>Olympus, Japan</td>
			<td>SZ61-C-SET</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope Lighting</td>
			<td>Olympus, Japan</td>
			<td>KL 1600 LED</td>
			<td></td>
		</tr>
		<tr>
			<td>confocal microscopy</td>
			<td>Zeiss, Germany</td>
			<td>LSM780</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris</td>
			<td>Bitplane, Switzerland</td>
			<td></td>
			<td>Version 7.6.4 or above</td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>The MathWorks, Inc., USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Excel for Mac&#160;</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
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</table>

analyzing synaptic modulation of drosophila melanogaster photoreceptors after exposure to prolonged light

Nervesystemet har den bemærkelsesværdige evne til at tilpasse og svare på forskellige stimuli. Denne neurale justering er i vid udstrækning gennem plasticitet på den synaptiske niveau. Den aktive zone (AZ) er den region på den præsynaptiske membran, der medierer neurotransmitterfrigivelse og er sammensat af en tæt samling af scaffold-proteiner. AZS af Drosophila melanogaster (Drosophila) fotoreceptorer gennemgår molekylær remodellering efter længere tids udsættelse for naturligt omgivende lys. Dermed omfanget af neuronal aktivitet kan omarrangere den molekylære sammensætning af AZ og bidrage til reguleringen af ​​den funktionelle udgang. Med udgangspunkt i den lyseksponering opsætning forberedelse til immunhistokemi, denne protokol detaljer, hvordan at kvantificere nummer, den rumlige fordeling og flytning niveau af synaptiske molekyler på Azs i Drosophila fotoreceptorer. Brug billedanalyse software, klynger af GFP-smeltet AZ komponent Bruchpilot blev identificeret for hver R8 fotoreceptorer (R8) Axon terminalen. Registrerede Bruchpilot spots blev automatisk tildelt individuelle R8 axoner. For at beregne fordelingen af ​​spot frekvens langs Axon, vi implementeret en tilpasset software plugin. Hver axon start-point og endepunkt blev manuelt defineret og positionen af ​​hver Bruchpilot spot blev projiceret op på forbindelseslinje mellem start og slutpunkt. Ud over antallet af Bruchpilot klynger, vi kvantificeret også flytning niveau af Bruchpilot-GFP inden for grupperne. Disse målinger afspejler i detaljer rumligt løst synaptiske dynamik i en enkelt neuron under forskellige miljøforhold på stimuli.

Øjet af Drosophila forbindelsen omfatter ~ 780 ommatidia, som hver indeholder otte typer af fotoreceptorer (R1-8). R7 og R8 projekt deres axoner til anden optik ganglion, medulla, hvor de danner synapser i lag M6 og M3 26. For at undersøge effekten af langvarig udsættelse for lys på den molekylære sammensætning af fotoreceptor R8 aktive zoner, tog vi fordel af Star Metode 15. Endogene ekspressionsniveauer af BRP blev fluoresc...

Watch this Scientific Journal Video about Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light at JoVE.com

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Her viser vi, hvordan at kvantificere antallet og rumlige fordeling af synaptiske aktive zoner i Drosophila melanogaster fotoreceptorer, fremhævet med en genetisk kodet molekylær markør, og deres modulation efter længere tids udsættelse for lys.

Analyse Synaptic Graduering af<em&gt; Drosophila melanogaster</em&gt; Fotoreceptorer efter Udsættelse for Langvarig Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

Research

JoVE Journal

Neuroscience

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

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 ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Optogenetic Stimulering af flugt Opførsel i<em&gt; Drosophila melanogaster</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Bestemmelse af Spontan motorisk aktivitet i<em&gt; Drosophila melanogaster</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

Conditions Affecting Social Space in Drosophila melanogaster

Conditions Affecting Social Space in Drosophila melanogaster

Forhold, der påvirker sociale rum i<em&gt; Drosophila melanogaster</em

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

High Throughput Assay til at undersøge æglæggende præferencer af Individuel<em&gt; Drosophila melanogaster</em

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Elektrofysiologiske Metode til Recording Intracellulær Voltage Responses af<em&gt; Drosophila</em&gt; Fotoreceptorer og interneuroner til Light Stimuli<em&gt; In vivo</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Elektrofysiologisk metode til hele celle spænding clamp optagelser fra<em&gt; Drosophila</em&gt; Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Desensitization and Recovery of Crayfish Photoreceptors Upon Delivery of a Light Stimulus

Desensibilisering og genvinding af krebs-Photoreceptorer ved levering af en let stimulus

A method to study desensitization and recovery of crayfish photoreceptors is presented. We performed intracellular electrical recordings of photoreceptor ...

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Overvågning celle til celle Transmission af Prion-lignende Protein aggregater i Drosophila Melanogaster

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

Metaboliske analyse af Drosophila melanogaster Larval og voksen hjerne

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...