Name: using an adapted microfluidic olfactory chip for the imaging of neuronal activity in response to pheromones in male c. elegans head neurons
Uploaded: 2017-09-07T14:00:00
Description: Watch this Scientific Journal Video about Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons at JoVE.com

Vi vil gjerne takke Manuel Zimmer for å gi oss filen opprinnelige utforming som ble tilpasset for bruk med menn; Frank Schroeder for syntese og forsyning av ascr #3; Ross Lagoy for innsikt og hjelp med bildebehandling og analyse; og Laura Aurilio for master fabrikasjon og som, sammen med Christopher Chute, bidro til gjennomgang av dette manuskriptet. Midler til dette arbeidet ble gitt under den National Institutes of Health grant 1R01DC016058-01 (js), National Science Foundation grant CBET 1605679 (D.R.A.) og Burroughs Wellcome karriere prisen på vitenskapelige Interface (D.R.A.).

1. enheten fabrikasjon Merk: se referanse 1.
 Merk: Silicon master formene ble laget ved hjelp av photolithographic for mønstre SU-8 photoresist på en silicon master 1 , 7. Photomasks for kjeks mønstre ble trykt på 25.000 dpi. De mannlige-tilpasset enhetsfunksjoner en Chronis Olfactory Chip design 19 med en endring i ormen lasting port, tilpasse en desi...

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Hann-tilpasset olfactory chip opptar en sving i en smalere lasting port, som gir mer kontroll over retningen og effektiv overlapping av mannlige C. elegans. Dette gir effekten av både venstre og høyre neuronal bilaterale par, uten behov for z-stabling. Denne kurven fører til en retning fra loddrett 100% av tiden i worms hvor bare ett bilaterale par er mål fluorescerende markøren, som ASH (figur 2D-E)29,30</su...

Microfluidic enheter gir økt tilgang til nettopp kontrollerte miljøer, der dyr, som Rundormer C. elegans, kan være eksperimentelt manipulert1. Disse studiene inkluderer atferdsmessige analyser, kalsium tenkelig studier eller selv visninger for bestemte fenotyper, noe som resulterer i mer nøyaktige målinger av eksperimentelle resultater1,2,3,4, 5,6. MicroFluidics gir småskala flytende forhold som detaljert eksperimenter kan kjøres mens benytte minimale mengder reagenser. Det er en konstant produksjon av nye microfluidic enheten design, og bruk av hver varierer fra arenaer som tillater naturlig sinusformet bevegelse C. elegans i atferdsdata analyser og nevrale tenkelig studier, å fange enheter i nevrale bildebehandling og olfactory studier, til enheter som tillater for høy gjennomstrømming fenotypiske analyse i genetisk skjermer4,5,6,7. Etter fabrikasjon av en master mold, microfluidic enheter er billig å konstruere-gitt gjenbruk av master- og enkel å bruke, slik at for rask data generasjon via høy gjennomstrømming studier. Fabrikasjon av enheter ved hjelp av polymerer som polydimethylsiloxane (PDMS) tillater opprettelsen av nye enheter innen timer.
Kalsium tenkelig studier bruker genetisk kodet kalsium indikatorer (GECIs) i målcellene for å måle nevrale dynamikken i disse cellene i sanntid8,9,10,11. Gjennomsiktig natur C. elegans tillater innspillingen av fluorescerende nivåene av disse proteinene i levende dyr. Tradisjonelt GECIs stole på grønne fluorescerende protein (GFP)-basert sensoren GFP-Calmodulin-M13 peptid (GCaMP), selv om nyere studier har tilpasset disse sensorer for bedre signal-til-støy-forhold og rød-skiftet eksitasjon profiler. Etter utviklingen av GCaMP3, proteiner med disse spesifikasjonene har variert, inkludert sensorer som GCaMP6s og GCaMP6f (langsom og rask fluorescens av-priser, henholdsvis), samt RFP-Calmodulin-M13 peptid (RCaMP), som har en rød-skiftet aktivisering profil. Kombinasjonen av disse GECIs med C. elegans celle-spesifikke genet promoter sekvenser kan målrette celler av interesse, spesielt sensoriske neurons12,13,14,15 , 16.
Mens brukervennligheten C. elegans i microfluidic studier er tydelig, har nesten alle studier fokusert på hermafroditter. Til tross for menn bare regnskap for 0,01-0,02% av befolkningen vill type, uvurderlig funn kan oppstå fra deres karakteristikk. Mens den fysiske connectome i Hermafroditt nervesystemet er fullt tilordnet for tiår17, fortsatt den mannlige connectome ufullstendig, spesielt i regionen hodet i dyr18. Bruk av kalsium imaging hos menn vil hjelpe generere en forståelse av mannlige nervesystemet og forskjeller som oppstår mellom to kjønnene. Mindre størrelsen på C. elegans voksne hanner forhindrer effektiv og pålitelig overlapping i lasting portene på tradisjonelle olfactory enheter designet for større hermafroditter. For å løse dette, en modifisert versjon av Chronis Olfactory Chip19 ble utviklet med en smalere lasting port, en lavere kanal høyde, og viser i ormen lasting port (som roterer dyret), slik at effekten av bilaterale venstre/høyre neuronal parene. Dette tillater: (1) effektiv overlapping av unge voksne hanner, (2) en sikrere orientering av dyret for visualisering av både medlemmer av bilaterale sammenkoblede neurons, og (3) nøyaktig bildebehandling nevrale aktivitet i mannlige neurons.
Stadig, viser studier at C. elegans menn reagere annerledes enn hermafroditter til en rekke ascarosides (ascr) eller Rundormer feromoner20,21,22,23 ,24. Derfor blitt utvikle en forståelse av nevrale dynamikk og representasjoner i den mannlige connectome enda mer relevant. Mannlige C. elegans inneholder 87 sex-spesifikke neurons finnes ikke i Hermafroditt25,26, endre connectome i som-ennå ubestemt måter. Å kunne bilde disse unike nevrale dynamikken vil tillate oss å bedre forstå sex-spesifikke tiltak og nevrale representasjoner.
Denne protokollen beskriver bruken av en mann-tilpasset olfactory chip for neural avbilding av mannlige C. elegans chemosensation. Nociceptive Nevron aske svarer pålitelig 1 M glyserol inne hanndyr, konsekvent med forrige hermafroditter studier27. Eksponering for ascarosides kan framprovosere reaksjoner som er variabel fra dyr til dyr, krever et større antall dyr skal testes. Responsen av mann-spesifikke CEM neurons har tidligere vist, gjennom både electrophysiology og kalsium imaging studier, svare ulik ascaroside #323.

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		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">Stainless Steel Blunt Needle 23 Gauge, Polyprolylene Luer</td>
			<td>Component Supply Company</td>
			<td>NE-231PL-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">Stopcocks with Luer connections; 3-way; male lock; 5 flow pattern; non-sterile</td>
			<td>Cole-Palmer</td>
			<td>EW-30600-07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">Fisherfinest Premium Cover Glass</td>
			<td>Fisher Scientific</td>
			<td>12-548-5M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">Mercator Control System LF-5 Plasma System</td>
			<td>Mercator</td>
			<td>LF-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">Scotch Tape</td>
			<td>Scotch</td>
			<td>BSN43575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">Series 20 Chamber</td>
			<td>Warner Instruments</td>
			<td>P-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">Vacuum Desicator</td>
			<td>Bel-Art Scienceware</td>
			<td style="width:205px;">420250000</td>
			<td>24 cm inner diameter.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">Weigh Boats</td>
			<td>Cole-Palmer</td>
			<td style="width:205px;">EW-01017-27</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">Classic Plus Balance</td>
			<td>Mettler Toledo</td>
			<td style="width:205px;">PB1501-S/FACT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">Glass Pasteur Pipettes</td>
			<td>Cole-Palmer</td>
			<td style="width:205px;">EW-25554-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">Transfer pipettes</td>
			<td>Genesee Scientific</td>
			<td style="width:205px;">30-202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">Oven</td>
			<td>Sheldon Manufacturing Inc</td>
			<td style="width:205px;">9120993</td>
			<td>Model Number: 1500E.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">60 mm, non-vented, sharp edge Petri dishes</td>
			<td>TriTech Research</td>
			<td>T3308</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">Zeiss Axio Observer.A1</td>
			<td>Zeiss</td>
			<td style="width:205px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">Hammamatsu Orca Flash 4.0 Digital CMOS</td>
			<td>Hammamatsu</td>
			<td style="width:205px;">C11440-22CU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">Blue Fluorescent Light</td>
			<td>Lumencor</td>
			<td style="width:205px;">SOLA SM6-LCR-SA</td>
			<td>24-30V/7.9A DC.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">Illumination Adaptor</td>
			<td>Zeiss</td>
			<td style="width:205px;">423302-0000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">Series 1 and 2 Miniature Inert PTFE Isolation Valve</td>
			<td>Parker</td>
			<td style="width:205px;">001-0017-900</td>
			<td>3-way valve for controlling flow.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">ValveLink8.2&#174;</td>
			<td>AutoMate Scientific</td>
			<td style="width:205px;">01-18</td>
			<td>Flow Switch Controller</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">Micro Manager</td>
			<td>Micro-Manager</td>
			<td style="width:205px;">-</td>
			<td>Free software, can be downloaded at: https://www.micro-manager.org/wiki/Download_Micro-Manager_Latest_Release</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">ImageJ</td>
			<td>ImageJ</td>
			<td style="width:205px;">-</td>
			<td>Free software, can be downloaded at: https://imagej.nih.gov/ij/download.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">Agar, Bacteriological Grade</td>
			<td>Apex</td>
			<td>9012-36-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">Peptone</td>
			<td>Apex</td>
			<td>20-260</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="width:527px;height:25px;">CaCl2</td>
			<td>VWR</td>
			<td>BDH0224-1KG</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="width:527px;height:25px;">MgSO4</td>
			<td>Sigma-Aldrich</td>
			<td>230391-1kg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">Cholesterol</td>
			<td>Alfa Aesar</td>
			<td>A11470</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>270741-4L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">Tetramisole</td>
			<td>Sigma-Aldrich</td>
			<td style="width:205px;">L9756-10(G)</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">Fluorescein</td>
			<td>Sigma-Aldrich</td>
			<td style="width:205px;">FD2000S-250mg</td>
			<td>Light Sensitive. Store in photoprotective vials.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td style="width:205px;">G6279-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">Ascaroside #3</td>
			<td>-</td>
			<td style="width:205px;">-</td>
			<td>Synthesized in the Schroeder Lab (Cornell University).</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">NaCl</td>
			<td>Genesee Scientific</td>
			<td style="width:205px;">18-215</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="width:527px;height:25px;">KH2PO4</td>
			<td>BDH</td>
			<td>BDH9268.25</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="width:527px;height:25px;">K2HPO4</td>
			<td>J.T. Baker</td>
			<td>3252-025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">ASH GCaMP3 line</td>
			<td>-</td>
			<td style="width:205px;">-</td>
			<td>CX10979 (KyEx2865 [psra-6::GCAMP3 @ 100 ng/uL]). Developed in Bargmann lab. Provided from Albrecht Lab library.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">CEM GCaMP6 line</td>
			<td>-</td>
			<td style="width:205px;">-</td>
			<td>JSR49 (FkEx98[ppkd-2::GCaMP::SL2::dsRED + pBX-1]; pha-1(e2123ts); him-5(e1490); lite-1(ce314)). Developed by Robyn Lints. Provided from Srinivasan Lab library.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">E. coli (OP50)</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>OP50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:527px;height:21px;">&#34;Reservoir&#34;</td>
			<td>-</td>
			<td>-</td>
			<td style="width:1260px;">To create a Reservoir: A &#34;30 mL syringe&#34;, is connected to a &#34;Stopcock with Luer connections; 3-way; male lock; 5 flow pattern; non-sterile&#34;, which is connected to a &#34;3 mL syringe&#34; and a &#34;Stainless Steel Blunt Needle 23 Gauge, Polyprolylene Luer&#34;. The &#34;Stainless Steel Blunt Needle 23 Gauge, Polyprolylene Luer&#34; is then inserted into &#34;Soft Tubing&#34; approximately 1/3 of the way down the needle.</td>
		</tr>
	</tbody>
</table>

using an adapted microfluidic olfactory chip for the imaging of neuronal activity in response to pheromones in male c. elegans head neurons

Bruk av kalsium indikatorer har kraftig forbedret vår forståelse av nevrale dynamikk og regulering. Rundormer Caenorhabditis elegans, helt tilordnet nervesystemet og gjennomsiktig anatomi, presenterer en ideell modell for å forstå sanntid nevrale dynamics bruke kalsium indikatorer. I kombinasjon med microfluidic teknologi og eksperimentell design, er kalsium-imaging studier med disse indikatorene utført i både fritt bevegelige og fanget dyrene. Men har de fleste tidligere studier utnytte fangst enheter, for eksempel olfactory chip beskrevet i Chronis et al., enheter designet for bruk i den vanligste Hermafroditt, mindre vanlig hannen er begge morphologically og strukturelt ulike. En tilpasset olfactory chip ble designet og laget for økt effektivitet på mannlige neuronal bildebehandling med bruke ung voksen dyr. En sving ble innlemmet i ormen lasting port rotere dyrene og tillate for separasjon av individuelle neurons med et bilateralt par i 2D bildebehandling. Ormer er utsatt for en kontrollert flyt av odorant i microfluidic enheten, som beskrevet i tidligere Hermafroditt studier. Kalsium transienter analyseres deretter ved hjelp av åpen kildekode programvare ImageJ. Fremgangsmåten som er beskrevet her bør tillate økt mengde hann-baserte C. elegans kalsium imaging studier, utdype vår forståelse av mekanismer for sex-spesifikke neuronal signalering.

Et eksempel på den samlede Enhetsinnstillinger ses i figur 1A-B. Figur 1A viser riktig reservoaret konstruksjon og installasjon. Figur 1B viser forbindelsen med reservoarene til den microfluidic enheten. Figur 1 c viser en microfluidic enhet med individuelle porter merket for klarhet.
Utformin...

Watch this Scientific Journal Video about Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons at JoVE.com

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

Bruke en tilpasset Microfluidic Olfactory Chip for avbilding av Neuronal aktivitet svar feromoner i mannlige C. Elegans hodet nerveceller

Bruk av en tilpasset "olfactory chip" for effektiv kalsium avbilding av C. elegans menn er beskrevet her. Studier av mannlige eksponering til glyserol og en feromon vises også.

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its ...

Research

JoVE Journal

Neuroscience

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

In vivo Laser Axotomy in C. elegans

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

En molekylær Avlesning av Long-term olfactory Tilpasning i<em&gt; C. elegans</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

Multi-enheten opptaket Metoder for å karakterisere nevrale aktiviteten i Locust (<em&gt; Schistocerca Americana</em&gt;) Olfactory kretser

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

In vivo Neuronal Calcium Imaging in C. elegans

In vivo Neuronal Calcium Imaging in C. elegans

In vivo Neuronal Kalsium Imaging i C. elegans

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Transplantasjon av Olfactory Ensheathing Cells å evaluere Funksjonell Recovery etter perifer nerveskade

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

Fast Micro-iontoforese av glutamat og GABA: Et nyttig verktøy for å etterforske Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

In vivo avbildning av Dauer-spesifikke nevronale ombygging i C. elegans

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of ...

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Olfactory Nevroner innhentet gjennom Nasal Biopsi Kombinert med Laser-Capture mikrodisseksjon: En Potential tilnærming til Study Behandling Response i psykiske lidelser

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

Utnytte Kombinert Metoder for å definere rollen til plasmamembranen Levering Under Axon Forgrening og neuronal morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Opptak Temperatur-indusert neuronal aktivitet gjennom Overvåking Kalsium endringer i luktelappen av<em&gt; Xenopus laevis</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...