Name: examining monosynaptic connections in drosophila using tetrodotoxin resistant sodium channels
Uploaded: 2018-02-14T14:00:00
Description: Watch this Scientific Journal Video about Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels at JoVE.com

Vi vil gerne takke Joshua Singer samt Jonathan Schenk og tankevækkende korrekturlæsere for kommentarer på manuskriptet. Vi vil også gerne takke Ben White og Harold Zakon diskussioner om teknikken. Jonathan Schenk fastsatte figur 3Adata. Dette arbejde blev støttet af en Whitehall Foundation Grant og en NIH R21 til QG.

1. Forbered fluer og identificere GAL4 promotor linjer
<ol><li>Vælg en GAL4 promoter linje og krydse den med fluer bærer både UAS-NaChBac transgenet og en optogenetic transgen til aktivering. Bemærk: UAS-CsChrimson11 er valgt på grund af sin høje ledningsevne og den lethed, hvormed det aktiverer NaChBac-medieret handling potentialer. UAS-NaChBac og UAS-CsChrimson er tilgængelige fra Bloomington bestandene lagringsstedet.</li>
	<li>Forberede all-trans retinal som en stamopløsning i eth...

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+Cell+Recordings+from+Brain+of+Adult+Drosophila.">Whole Cell Recordings from Brain of Adult Drosophila.</a> J Vis Exp. , (2007).</li><li>Wilson, R. I., Turner, G. C., Laurent, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+olfactory+representations+in+the+Drosophila+antennal+lobe.">Transformation of olfactory representations in the Drosophila antennal lobe.</a> Science. 303, 366-370 (2004).</li><li>Kazama, H., Wilson, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeostatic+Matching+and+Nonlinear+Amplification+at+Identified+Central+Synapses.">Homeostatic Matching and Nonlinear Amplification at Identified Central Synapses.</a> Neuron. 58, 401-413 (2008).</li><li>Becnel, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DREADDs+in+Drosophila:+a+pharmacogenetic+approach+for+controlling+behavior,+neuronal+signaling,+and+physiology+in+the+fly.">DREADDs in Drosophila: a pharmacogenetic approach for controlling behavior, neuronal signaling, and physiology in the fly.</a> Cell Reports. 4, 1049-1059 (2013).</li><li>Armbruster, B. N., Li, X., Pausch, M. H., Herlitze, S., Roth, B. 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TERPS analyse komplimenter aktuelle teknikker bruges til kredsløb revner ved at aktivere påvisning af synapser mellem identificerede neuroner. Specifikt, afslører tilgangen monosynaptic forbindelser af bredt silencing handling potentialer med TTX mens gendannelse ophidselse i en udvalgt population af neuroner med TTX-ufølsom natrium kanalen NaChBac. Synaptic frigivelse er fremkaldt af optogenetic stimulation mens postsynaptiske begivenheder overvåges med hele-celle optagelser. TERPS adskiller sig fra andre metoder s...

Et vigtigt mål for neurovidenskab er at knytte forbindelser mellem neuroner til at forstå, hvordan information flyder gennem kredsløb. Adskillige strategier og ressourcer er blevet tilgængelig til test af funktionelle forbindelsen mellem neuroner i en række model systemer1,2. For at generere de mest præcise ledningsdiagrammer ved hjælp af Elektrofysiologi, er det vigtigt at løse om observerede forbindelser mellem to celler er direkte og monosynaptic versus indirekte og polysynaptic. En guld standard for at gøre denne skelnen i pattedyr neuroner er at måle latenstiden mellem enkelt action potentials i de præsynaptiske celler og udbrud af excitatoriske postsynaptiske strømme (EPSCs) i den anden neuron. Monosynaptic forbindelser skal have kort ventetid for et par millisekunder og lav variabilitet3. Denne tilgang kan være kompliceret i hvirvelløse neuroner, fordi synapser kan forekomme i deres dendritter langt fra webstedet somatiske optagelse, forårsager forsinkelser i registrering på grund af lange electrotonic afstande mellem postsynaptiske conductances og optagelsen elektrode. Sådanne forsinkelser kan indføre tvetydighed med hensyn til poly-versus monosynaptic bidrag. Derudover små synaptic begivenheder kan henfalde før de når webstedet somatiske optagelser, og køre stærkere præsynaptiske aktivitet, er tilbøjelige til at ansætte polysynaptic begivenheder.
Forskellige teknikker er blevet udviklet for at teste for monosynaptic forbindelser i hvirvelløse dyr. Én fremgangsmåde bruger høje divalent kation løsninger (Hi-Di) indeholdende overskydende Mg++ og Ca++. Denne løsning blokerer polysynaptic forbindelser ved at reducere sandsynligheden for udgivelse og stigende aktionspotentialet tærsklen til at favorisere påvisning af monosynaptic input4,5. Bestemmelse af det præcise forhold mellem Mg++ til Ca++ kræves, men er ikke trivielt og polysynaptic bidrag kan vare ved selv beskedne stimulation6. En alternativ metode kaldet normal god landbrugspraksis rekonstitution på tværs af Synaptic partnere (greb) udnytter nærhed mellem præ- og postsynaptiske membraner fundet på synapser at udlede en monosynaptic forbindelse 7. Her, en del af grøn fluorescerende proteiner (NGL) udtrykkes i en neuron, og den supplerende fragment af molekylet er udtrykt i en formodede postsynaptiske partner. Tilstedeværelsen af fluorescens viser, at de to neuroner er tæt nok på til at tillade rekonstituering af normal god landbrugspraksis molekyle og antyder eksistensen af en synapse. GREB kan rapportere synapser fejlagtigt, dog, hvis to celler har tæt apposed membraner, som i en nerve eller fascicle. Varianter af greb fjerne sådanne falske positiver af tethering den præsynaptiske fragment af normal god landbrugspraksis direkte til synaptobrevin, således at rekonstitution kun på aktive synapser8. Mens greb og dens varianter har været medvirkende til bestemmelse af funktionelle connectivity i Drosophila CNS, kan nogle forbindelser ikke gøres synlig ved greb hvis afstande mellem præ- og postsynaptiske partnere er relativt stor. Dette er særligt relevant i vurderingen af volumen transmission tilknyttet Neuromodulationsbehandling9 eller GABAergic hæmning10.
Her, er en supplerende og roman teknik påvist for at teste direkte monosynaptic forbindelser i Drosophila CNS. Denne metode, kaldet Tetrodotoxin manipuleret modstand for sondering synapser (TERPS), anlæg af co udtryk af tetrodotoxin (TTX)-resistente natrium kanal, NaChBac og en optogenetic activator i en præsynaptiske celle mens optagelse fra formodede postsynaptiske partnere9. I nærværelse af TTX, er alle aktionspotentialet-medieret aktivitet undertrykt i cellerne end de giver udtryk for NaChBac. NaChBac kanalen gendanner selektivt ophidselse i den målrettede præsynaptiske celler, tillader lys-fremkaldte synaptisk transmission. Denne metode tillader stærk aktivering af de præsynaptiske neuroner, således at forbindelser kan løses postsynaptically af somatiske optagelse samtidig reducere sandsynligheden for rekruttering polysynaptic kredsløb. Vigtigst, denne teknik tillader undersøgelse af volumen transmission og afslører spredning af senderen frigivet fra en enkelt neuron i et kredsløb. Derudover kan TERPS afsløre den kemiske natur for forbindelsen gennem konventionelle farmakologi. TERPS er egnet til brug i enhver modelsystem, der tillader den transgene udtryk for TTX-ufølsom natrium kanaler.

<table border="0" cellpadding="0" cellspacing="0" width="946">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">UAS-csChrimson</td>
			<td style="width:219px;">Bloomington Drosophila Stock Center</td>
			<td style="width:344px;">55135</td>
			<td style="width:167px;">Used as a neural activator</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">UAS-NaChBac</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>9466</td>
			<td style="width:167px;">Resotores excitibility in cells in TTX</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tetrodotoxin</td>
			<td>Tochris</td>
			<td>1078</td>
			<td style="width:167px;">Special permission may be needed to purchase TTX as it is a controlled substance</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">all trans-Retinal</td>
			<td>Sigma-Aldrichall trans-Retinal</td>
			<td>R2500</td>
			<td style="width:167px;">Require co-factor for channelrhodopsin</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Weldable 321 Stainless Steel&#160;Sheet,&#160;0.002&#34;&#160;Thick,&#160;10&#34;&#160;Wide</td>
			<td>McMaster Carr</td>
			<td>3254K7</td>
			<td style="width:167px;">Used to make custom fly holder. Custom foil can be laser cut at pololu.com from the provided PDF file</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dissection Microscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000-C</td>
			<td style="width:167px;">Used for dissection of preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Waxer</td>
			<td>Almore</td>
			<td>Eectra Waxer 66000</td>
			<td style="width:167px;">Used during dissection to secure fly in foil</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Paraffin Wax</td>
			<td>Joann</td>
			<td>4917217</td>
			<td style="width:167px;">Used with waxer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Number 5 forceps</td>
			<td>Fine Science Tools</td>
			<td>#5CO</td>
			<td style="width:167px;">Used for dissection and desheathing</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dissection Scissors</td>
			<td>Fine Science Tools</td>
			<td>15001-08</td>
			<td style="width:167px;">Used to remove parts of the cuticle during dissection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tungsten wire</td>
			<td>A-M Systems</td>
			<td>797500</td>
			<td style="width:167px;">Use with electrolysis to make sharpened needles for dissection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Reciculating Peristaltic Pump</td>
			<td>Simply Pumps (Amazon)</td>
			<td>PM200S</td>
			<td style="width:167px;">for recirculating TTX</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Speed Controller for peristaltic pump</td>
			<td>Zitrades (Amazon)</td>
			<td style="width:344px;">N/A</td>
			<td style="width:167px;">PWM Dimming Controller For LED Lights or Ribbon, 12 Volt 8 Amp,Adjustable Brightness Light Switch Dimmer Controller DC12V 8A 96W for Led Strip Light B</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Versa-Mount&#160;Precision Compressed Air Regulator</td>
			<td>McMaster Carr</td>
			<td style="width:344px;">1804T1</td>
			<td style="width:167px;">For applying positive pressure during patching</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass capilaries</td>
			<td>World Precision Instruments</td>
			<td style="width:344px;">TW150F-3</td>
			<td style="width:167px;">For patch pipettes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Multipurpose Gauge</td>
			<td>McMaster Carr</td>
			<td>3846K431</td>
			<td style="width:167px;">Gauge for pressure regulator</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electrophysiology Camera</td>
			<td>Dage MTI&#160;</td>
			<td>IR-1000</td>
			<td style="width:167px;">Any camera that works in the IR range (850 nm) will work. You do not want to use red illumination as this can activate csChrimson</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">IR LED</td>
			<td>Thorlabs&#160;</td>
			<td>M850F2</td>
			<td style="width:167px;">For oblique illumination</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">fiber optic for IR LED</td>
			<td>Thorlabs</td>
			<td>M89L01</td>
			<td style="width:167px;">Couples to IR LED</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Objective lens&#160;</td>
			<td>Olympus 40X</td>
			<td>LUMPlanFLN</td>
			<td style="width:167px;">This can be used on most microscipes and works well for visualizing fly neurons.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Amber LED</td>
			<td>Thorlabs</td>
			<td>M590L3d</td>
			<td style="width:167px;">For visualizing RFP and mCherry</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Blue LED</td>
			<td>Thorlabs</td>
			<td>M470L3d</td>
			<td style="width:167px;">For visualizing GFP</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">GFP filter set</td>
			<td>Chroma</td>
			<td>49011</td>
			<td style="width:167px;">For visualizing GFP or stimulating channel2rhodopsin</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Custom mCherry Filter Set</td>
			<td>Chroma</td>
			<td style="width:344px;">&#160;et580/25x and t600lpxr (from the 49306 set) but with an et610lp barrier/emission optic</td>
			<td style="width:167px;">Use only if you wish to patch identified neurons with channel2rhodopsin</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dichroic to combine Amber and blue LED</td>
			<td>Thorlabs</td>
			<td style="width:344px;">DMLP550R</td>
			<td style="width:167px;">Use only patch under mCherry and excite channel2rhodopsin with blue light.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Red LED</td>
			<td>LEDSupply</td>
			<td>Cree XPE 620 - 630 nm</td>
			<td style="width:167px;">Used to drive csChrimson</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">LED Driver</td>
			<td>LEDSupply</td>
			<td>3021-D-E-1000</td>
			<td style="width:167px;">Used to drive LEDs for optogenetic stimulation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Manipulator</td>
			<td>Sutter Instruments</td>
			<td>MP-225</td>
			<td style="width:167px;">Used to position pipette during recordings</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Patchclamp Amplifier</td>
			<td>A-M Systems</td>
			<td>Model 2400</td>
			<td style="width:167px;">An equivalent amplifier is suitable</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bessel Filter</td>
			<td>Warner Instruments&#160;</td>
			<td>LPF 202A</td>
			<td style="width:167px;">Auxillary filter used to filter current trace to oscilloscope during patching.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Data Acquisition System&#160;</td>
			<td>National Instruments</td>
			<td>NI PCIe-6321&#160;&#160;&#160;&#160;&#160;&#160; 781044-01</td>
			<td style="width:167px;">Used to record data from amplifier to computer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Connector Block - BNC Terminal BNC-2090A</td>
			<td>National Instruments</td>
			<td>779556-01</td>
			<td style="width:167px;">Used to connect amplifier to DAQ card.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Steel Foil&#160;</td>
			<td>McMaster Carr</td>
			<td>3254K7</td>
			<td style="width:167px;">Steel foil for custom recording chamber</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Magnets</td>
			<td>K&#38;J Magnetics</td>
			<td>D42</td>
			<td style="width:167px;">To secure recording chamber to ring stand</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;">1/16&#34; Cell Cast Acrylic Clear</td>
			<td>Pololu</td>
			<td></td>
			<td style="width:167px;">Used to make custom recording chamber. Acrylic can be laser cut at pololu.com from the provided PDF file</td>
		</tr>
	</tbody>
</table>

examining monosynaptic connections in drosophila using tetrodotoxin resistant sodium channels

Her, er en ny teknik, der kaldes Tetrotoxin (TTX) udviklet resistens over for sondering synapser (TERPS) anvendt til at teste for monosynaptic forbindelser mellem target neuroner. Metoden bygger på fælles udtryk for en transgene aktivator med tetrodotoxin-resistente natrium kanalen, NaChBac, i en bestemt præsynaptiske neuron. Forbindelser med formodede post synaptic partnere bestemmes af hele-celle optagelser i overværelse af TTX, som blokerer for elektriske aktivitet i neuroner, der ikke udtryk NaChBac. Denne tilgang kan ændres for at arbejde med alle aktivator eller calcium imaging som en reporter af forbindelser. TERPS tilføjer til den voksende sæt af værktøjer til rådighed til bestemmelse af tilslutningsmuligheder inden for netværk. TERPS er imidlertid unik, idet den rapporterer også pålideligt bulk eller mængde transmission og spillover transmission.

TERPS bruges til at skelne mellem mono- og polysynaptic bidrag i synaptiske forbindelser mellem neuroner. Mens svage stimulering af en celle kan bruges til at teste direkte forbindelser, rekrutter kørsel større præsynaptiske aktivitet ofte polysynaptic forbindelser (figur 1A). TERPS virker ved co udtrykker TTX-ufølsom natrium-kanalen NaChBac og en optogenetic aktivator, og teste forbindelser i overværelse af TTX at eliminere polysynaptic forbindelser (<s...

Watch this Scientific Journal Video about Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels at JoVE.com

Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels

Denne artikel indeholder en metode til at teste de monosynaptic forbindelser mellem neuroner ved hjælp af tetrodotoxin og tetrodotoxin-resistente natrium kanalen, NaChBac.

Undersøge Monosynaptic forbindelser i Drosophila ved hjælp af Tetrodotoxin resistente natrium kanaler

Here, a new technique termed Tetrotoxin (TTX) Engineered Resistance for Probing Synapses (TERPS) is applied to test for monosynaptic connections between ...

Research

JoVE Journal

Neuroscience

Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels

Examining Local Network Processing using Multi-contact Laminar Electrode Recording

Cortical layers are ubiquitous structures throughout neocortex1-4 that consist of highly recurrent local networks. In recent years, significant progress ...

Correlating Behavioral Responses to fMRI Signals from Human Prefrontal Cortex: Examining Cognitive Processes Using Task Analysis

Korrelere adfærdsmæssige reaktioner på fMRI Signaler fra Menneskelig præfrontale cortex: Undersøgelse Kognitive processer ved hjælp af Task Analysis

The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Visualisering af proprioceptorer i<em&gt; Drosophila</em&gt; Larver og pupper

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 ...

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Multi-foton Intracellulær Natrium Imaging Kombineret med UV-medieret Focal Uncaging af glutamat i CA1 pyramideneuroner

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high ...

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 Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

En simpel måde at måle Ændringer i Reward-søger Behavior Brug<em&gt; Drosophila melanogaster</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

Kvantitative cellebiologi af Neurodegeneration i Drosophila gennem saglig analyse af Fluorescently mærket proteiner ved hjælp af ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Kvantificering af makronæringsstoffer Indtag i en termogentisk neuronal skærm ved hjælp af Drosophila Larver

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating ...

Unraveling the Role of Discrete Areas of the Rat Brain in the Regulation of Ovulation through Reversible Inactivation by Tetrodotoxin Microinjections

Optrævling af de diskrete områder af rottehjernen i reguleringen af ægløsning gennem reversibel inaktivering af Tetrodotoxin Microinjections

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and ...

A Standardized Pipeline for Examining Human Cerebellar Grey Matter Morphometry using Structural Magnetic Resonance Imaging

En standardiseret pipeline til undersøgelse af menneskelig cerebellær gråstofmorfometri ved hjælp af strukturel magnetisk resonansbilleddannelse

Multiple lines of research provide compelling evidence for a role of the cerebellum in a wide array of cognitive and affective functions, going far beyond ...