Name: electrophysiological recording from drosophila labellar taste sensilla
Uploaded: 2014-02-26T23:00:00
Description: Watch this Scientific Journal Video about Electrophysiological Recording From Drosophila Labellar Taste Sensilla at JoVE.com

Dette arbeidet ble støttet av en NRSA predoctoral stipend 1F31DC012985 (til RD) og ved NIH tilskudd til JC Vi ønsker å takke dr. Linnea Weiss for nyttige kommentarer til manuskriptet, Dr. Ryan Joseph for hjelp kompilere tall, og Dr. Frederic Marion-Poll for nyttig teknisk rådgivning. Vi ønsker også å erkjenne de nyttige kommentarer av fire anmeldelser.

Følgende protokoll i samsvar med alle retningslinjene i Yale University dyr omsorg. En. Reagenser og utstyr Forberedelse <ol><li> Opptaksutstyr oppsett (figur 1A). <ol><li fo:keep-together.within-page="always"> Velg et rom for riggoppsettet som er fritt for store variasjoner i temperatur eller luftfuktighet, og også isolert fra kilder av elektrisk og mekanisk støy, som for eksempel kjøleskap og sentrifuger. <img alt="Figur 1" fo:content-width="5in" fo:src="/files...

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Labellar sensilla varierer i den enkle opptak på grunn av forskjeller i morfologi og anatomisk organisasjon. Noen ganger kan en sensillum ikke svare på eventuelle Tastants, selv en som er kjent for å lokke fram en positiv respons. Hvor ofte dette skjer varierer avhengig sensillum type. L sensilla er mest konsistent følsomme og er relativt lett å åpne på grunn av deres lengde. Generelt, S sensilla er konsekvent responsive, men deres korte lengde og posisjon på labellum får god kontakt utfordrende. I sensilla kan...

Smakssansen gjør at et insekt å oppdage et stort spekter av løselige kjemikalier og spiller en viktig rolle i aksept av et næringsrikt stoff, eller avvisning av en skadelig eller giftig ett. Smak er også antatt å spille en rolle i mate valg, gjennom påvisning av feromoner 1-5. Disse viktige og ulike funksjoner har gjort insekt smak systemet en overbevisende målet for etterforskningen i hvordan sansesystemer sette miljømessige signaler i relevante atferds utganger. Den primære enhet av Drosophila melanogaster smak system er smaken hår, eller sensillum. Molekyler angi sensillum via en pore på spissen sin 2,6. Sensilla er funnet på labellum, bena, vingen margin, og svelget seks. På labellum, er antallet og plasseringen av sensilla stereotyp. Det er tre klasser av morfologiske sensilla basert på lengde: den lange (L), middels (I) og korte (S<html> ) Sensilla 7,8. Hver sensillum inneholder enten to (I-type) eller fire (L-og S-type) gustatory reseptor nevroner (GRNs) 9. Ulike GRNs svare på ulike kategorier av smaks stimuli: bitter, sukker, salt og osmolaritet 7,10 og uttrykke ulike undergrupper av gustatory reseptorer 8,11-13. Bare jeg og S-type sensilla inneholde bitre-responsive GRNs 8,10. Den GRNs prosjektet til subesophageal ganglion (SOG) og deres aktivering ved smaks molekyler blir videresendt til høyere sentralnervesystemet for dekoding, noe som resulterer i en atferdsmessig respons 6.. Den relativt lite antall nerveceller og amenability til molekylær og atferdsanalyse gjør Drosophila smak systemet en utmerket modell for etterforskningen av gustatory systemer generelt. Den relative enkle som systemet kan manipuleres via genetisk mutasjon eller Gal4-UAS uttrykk systemet fungerer også som et verdifullt verktøy 14,15. ontent &quot;&gt; Fordi disse sensilla stikker ut fra overflaten av labellum, de er utmerkede mål for elektrofysiologi. avfyring av GRNs kan overvåkes ved bruk av ekstracellulære opptak. Historisk sett har side-veggen opptaksmetode, som bruker en glasselektrode innsatt i sensillum å ta opp neuronal aktivitet, 26 har vært i bruk. Imidlertid er denne fremgangsmåte er teknisk vanskelig å utføre, og det er vanskelig å ta for lang tid fra hvert preparat. Spissen-opptaksmetoden, som måler responsen av nervecellene med en elektrode som samtidig leverer et smaksmiddel, siden blitt metoden for valg 9,16. det har blitt anvendt for å undersøke smak system av Drosophila melanogaster 8,10,17,18, samt en rekke andre insektarter 19-23. har blitt betydelig lettere ved utviklingen av tastePROBE forsterkeren, som overvant én av de store ulempene ved tip-opptaksmetoden ved å kompensere forden store potensialforskjell mellom referanseelektroden og den insekt sensillum, slik at grn aksjonspotensialer som skal tas opp uten overdreven forsterkning eller filtrering 24. En annen viktig utvikling var bruken av tricholine citrate ettersom opptakselektrolytt 25. TCC undertrykker svar fra osmolariteten følsomme GRN og stimulerer ikke salt-følsomme GRN, gjør responser generert av bitter og sukker Tastants mye lettere å analysere 25. Her beskriver vi hvordan tips innspillingen av Drosophila labellar sensilla er for tiden utføres i Carlson laboratoriet. Denne protokollen vil forklare hvordan å etablere en egnet elektrofysiologi rigg, hvordan å forberede fly, og hvordan du utfører smaks innspillinger. Vi presenterer også noen representative data innhentet ved opptak fra undergrupper av Drosophila sensilla, samt noen vanlige problemer og mulige løsninger som kan oppstå ved bruk av denneteknikk.

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			<td height="84" style="height:84px;width:168px;">Stereo Zoom Microscope</td>
			<td style="width:187px;">Olympus&#160;</td>
			<td style="width:211px;">SZX12 DFPLFL1.6x PF eyepieces: WHN10x-H/22</td>
			<td style="width:219px;">capable of ~150x magnification with long working distance table mount stand</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Anti-vibration Table</td>
			<td style="width:187px;">Kinetic Systems</td>
			<td>BenchMate2210</td>
			<td></td>
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			<td height="21" style="height:21px;width:168px;">Micromanipulators</td>
			<td style="width:187px;">Narishige</td>
			<td>NMN-21</td>
			<td></td>
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			<td height="21" style="height:21px;width:168px;">Magnetic stands</td>
			<td style="width:187px;">ENCO</td>
			<td>Model #625-0930</td>
			<td></td>
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			<td height="42" style="height:42px;width:168px;">Reference Electrode Holder</td>
			<td style="width:187px;">Harvard Apparatus</td>
			<td style="width:211px;">ESP/W-F10N</td>
			<td>Can be mounted on 5ml serological pipette for extended range</td>
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			<td height="42" style="height:42px;width:168px;">&#160;Silver Wire</td>
			<td style="width:187px;">World Precision Instruments</td>
			<td style="width:211px;">AGW1510</td>
			<td>0.3-0.5mm diameter</td>
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			<td height="21" style="height:21px;width:168px;">Retort Stand</td>
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			<td>generic</td>
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			<td height="21" style="height:21px;width:168px;">Outlet Plastic Tube</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>generic, 1cm diameter</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Flexible Plastic Tubing</td>
			<td style="width:187px;">Nalgene&#160;</td>
			<td>8000-0060</td>
			<td style="width:219px;">VI grade 1/4 in internal diameter&#160;</td>
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			<td height="21" style="height:21px;width:168px;">500 ml Conical Flask</td>
			<td style="width:187px;"></td>
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			<td>generic,&#160; with side arm</td>
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			<td height="42" style="height:42px;width:168px;">Aquarium Pump</td>
			<td style="width:187px;">Aquatic Gardens</td>
			<td style="width:211px;">Airpump 2000</td>
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			<td height="42" style="height:42px;width:168px;">Fiber Optic Light Source</td>
			<td style="width:187px;">Dolan-Jenner Industries</td>
			<td>Fiber-Lite 2100</td>
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			<td height="21" style="height:21px;width:168px;">White Card/Paper</td>
			<td style="width:187px;">Whatman</td>
			<td style="width:211px;">1001-110</td>
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			<td height="42" style="height:42px;width:168px;">Digital Acquisition System</td>
			<td style="width:187px;">Syntech</td>
			<td style="width:211px;">IDAC-4</td>
			<td>Alternative: National Instruments NI-6251 &#160;</td>
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			<td height="21" style="height:21px;width:168px;">Headstage</td>
			<td style="width:187px;">Syntech</td>
			<td style="width:211px;">DTP-1</td>
			<td>Tasteprobe</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Tasteprobe Amplifier</td>
			<td style="width:187px;">Syntech</td>
			<td style="width:211px;">DTP-1</td>
			<td>Tasteprobe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Alligator Clips</td>
			<td style="width:187px;">Grainger</td>
			<td style="width:211px;">1XWN7</td>
			<td>Any brand is fine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Insulated Electrical Wire</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>Generic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Gold Connector Pins</td>
			<td style="width:187px;">World Precision Instruments</td>
			<td align="right" style="width:211px;">5482</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Personal Computer</td>
			<td style="width:187px;">Dell&#160;</td>
			<td style="width:211px;">Vostro</td>
			<td>Check for compatibility with digital acquisition system and software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Acquisition Software</td>
			<td style="width:187px;">Syntech</td>
			<td style="width:211px;">Autospike</td>
			<td>Autospike works with IDAC-4; alternatively, use Labview with NI-6251</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Aluminum Foil and/or Faraday Cage</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>Electro-magnetic noise shielding</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;"></td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Borosilicate Glass Capillaries</td>
			<td style="width:187px;">World Precision Instruments</td>
			<td style="width:211px;">1B100F-4</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Pipette Puller</td>
			<td style="width:187px;">Sutter Instrument Company</td>
			<td style="width:211px;">Model P-87 Flaming/Brown Micropipette Puller</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;"></td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Beadle and Ephrussi Ringer Solution</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>See recipe in protocol section</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Tricholine citrate, 65%&#160;</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:211px;">T0252-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;"></td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Stereo Microscope</td>
			<td style="width:187px;">Olympus</td>
			<td style="width:211px;">VMZ 1x-4x</td>
			<td style="width:219px;">Capable of 10x-40x magnification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Ice Bucket</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>Generic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">p200 Pipette Tips</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>Generic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Spinal Needle</td>
			<td style="width:187px;">Terumo</td>
			<td style="width:211px;">SN*2590</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">1ml Syringe</td>
			<td style="width:187px;">Beckton-Dickenson</td>
			<td align="right" style="width:211px;">301025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Fly Aspirator</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>Assembled from P1000 pipette tips, flexible plastic tubing, and mesh</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Modeling Clay</td>
			<td style="width:187px;"></td>
			<td style="width:211px;"></td>
			<td>Generic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:168px;">Forceps</td>
			<td style="width:187px;">Fine Science Tools By Dumont</td>
			<td style="width:211px;">11252-00</td>
			<td>#5SF (super-fine tips)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">10ml Syringe&#160;</td>
			<td style="width:187px;">Beckton-Dickinson</td>
			<td align="right" style="width:211px;">301029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Plastic Tubing</td>
			<td style="width:187px;">Tygon</td>
			<td style="width:211px;">R-3603</td>
			<td></td>
		</tr>
	</tbody>
</table>

electrophysiological recording from drosophila labellar taste sensilla

Det perifere smak respons av insekter kan være kraftfullt undersøkt med elektrofysiologiske teknikker. Metoden som beskrives her tillater forskeren å måle gustatory svar direkte og kvantitativt, som gjenspeiler den sanseinntrykk at insektet nervesystemet mottar fra smaks stimuli i omgivelsene. Denne protokollen skisserer alle viktige skritt i å utføre denne teknikken. De kritiske trinnene i å sette sammen en elektrofysiologi rigg, som for eksempel valg av nødvendig utstyr og et egnet miljø for opptak, er avgrenset. Vi beskriver også hvordan å forberede for opptak ved å gjøre riktige referanse og opptak elektroder, og smaksmiddel løsninger. Vi beskriver i detalj den metode som brukes for fremstilling av insekt ved innsetting av en glassreferanseelektrode i fly for å immobilisere snabel. Vi viser spor av de elektriske impulser avfyrt av smaksnerveceller i respons til et sukker og en bitter forbindelse. Aspekter ved protokollen er technically utfordrende og vi har en omfattende beskrivelse av noen vanlige tekniske utfordringer som kan oppstå, for eksempel manglende signal eller støy i systemet, og mulige løsninger. Teknikken har begrensninger, slik som manglende evne til å levere timelig komplekse stimuli, observerer bakgrunn avfyring umiddelbart før stimulus levering, eller bruke vannuløselige smaksforbindelser beleilig. Til tross for disse begrensningene, er denne teknikken (inkludert mindre variasjoner referert i protokollen) en standard, bredt akseptert prosedyre for opptak Drosophila nevrale responser å smake forbindelser.

Figur 5A viser responsen av en L sensillum til et sukker, sukrose. Det samme sensillum ikke svare på en bitter sammensatte, viser berberin. Figur 5B at en jeg typen sensillum, som inneholder en bitter responsive nevron, viser større amplitude toppene i respons til berberine, og mindre amplitude toppene i respons til sukrose. L sensilla viser en minimal bakgrunns respons til det løsningsmiddel-kontroll, TCC, mens jeg sensilla viser praktisk talt ingen reaksjon TCC (figur 5).</...

Watch this Scientific Journal Video about Electrophysiological Recording From Drosophila Labellar Taste Sensilla at JoVE.com

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

Denne protokollen beskriver ekstracellulære opptak av handlings potensielle responser avfyrt av labellar smaks nevroner i Drosophila.

Elektro Opptak fra<em&gt; Drosophila</em&gt; Labellar Smak sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

Research

JoVE Journal

Neuroscience

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

Electrophysiological Measurements from a Moth Olfactory System

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

Samtidig opptak av kalsium signaler fra Identifiserte Nerveceller og fôring atferd Drosophila melanogaster</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Forebrain Electrophysiological Recording in Larval Zebrafish

Forebrain Elektrofysiologisk opptak i larve Sebrafisk

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Samtidig Pre-og postsynaptiske Elektrofysiologisk Opptak fra<em&gt; Xenopus</em&gt; Nerve-muskel Co-kulturer

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Elektro opptak i hjernen til Intakt Adult Sebrafisk

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

A Method for Systematic Electrochemical and Electrophysiological Evaluation of Neural Recording Electrodes

En metode for systematisk Elektrokjemisk og Elektro Evaluering av Neural Recording Elektroder

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant ...

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

Elektro Metode for opptak Intracellulær spenningsreaksjoner av<em&gt; Drosophila</em&gt; Fotoreseptorer og interneurons til lys stimuli<em&gt; I Vivo</em

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

Taste Preference Assay for Adult Drosophila

Taste Preference Assay for Adult Drosophila

Smak Preference Assay for voksne<em&gt; Drosophila</em

Olfactory and gustatory perception of the environment is vital for animal survival.  The most obvious application of these chemosenses is to be able to ...