Name: construction of cell-based neurotransmitter fluorescent engineered reporters (cnifers) for optical detection of neurotransmitters in vivo
Uploaded: 2016-05-12T13:00:00
Description: Watch this Scientific Journal Video about Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters CNiFERs for Optical Detection of Neurotransmitters In Vivo at JoVE.com

Vi takker B. Conklin (University of California, San Francisco) for å gi G qi5 og G qs5 cDNA A. Schweitzer for å få hjelp med elektronikken, N. Taylor for å få hjelp med screening av kloner, Ian Glaaser og Robert Rifkin for korrektur og Olivier Griesbeck for TN-XXL. Dette arbeidet ble støttet av forskningsmidler gjennom det amerikanske National Institute on Drug Abuse (NIDA) (DA029706; DA037170), National Institute of Biomedical Imaging og bioteknologi (NIBIB) (EB003832), Hoffman-La Roche (88610A) og &quot;Neuroscience relatert til narkotika av misbruk &quot;trening stipend gjennom NIDA (DA007315).

Alle dyr prosedyrer utført i denne studien er i samsvar med Institutional Animal Care og bruk Committee (IACUC) retningslinjer, og har blitt godkjent av IACUCs på Icahn School of Medicine ved Mount Sinai og University of California, San Diego. 1. Generer GPCR-uttrykke Lentivirus for Transforming HEK293 celler <ol><li> Skaff cDNA for en bestemt GPCR fra en kommersiell kilde, for eksempel, cdna.org. Alternativt kan forsterke den GPCR-genet fra et cDNA-bibliotek ved anvendelse av PCR. Få tak i en lent...

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Opprettelsen av CNiFERs gir en innovativ og unik strategi for optisk måling frigivelse av nevrotransmittere i hjernen in vivo. CNiFERs er ideell for måling extrasynaptic utgivelse, dvs. volum ledning, for nevrotransmittere. Viktigere, hver CNiFER besitter egenskapene til det native GPCR, noe som gir et fysiologisk optisk måling av endringer i nivåene av nevrotransmittere i hjernen. Hittil har CNiFERs blitt opprettet for å oppdage acetylkolin (M1-CNiFER) 6, dopamin (D2-CNiFER) 7</su...

For å forstå hvordan nerveceller kommuniserer i hjernen, er det nødvendig å ha en metode for å måle frigivelsen av nevrotransmittere in vivo. Det er flere veletablerte teknikker for måling av nevrotransmittere in vivo. En vanlig brukt teknikk er mikrodialyse, hvor en kanyle er satt inn i hjernen og et lite volum av spinalvæske blir samlet opp og analysert ved hjelp av væskekromatografi og elektrokjemisk deteksjon en. Mikrodialyse har en romlig oppløsning i størrelsesorden noen få diametere på sonden, f.eks, ~ 0,5 mm for en 200 um diameter mikrosonde. Den tidsmessige oppløsning av denne teknikken, er imidlertid langsom på grunn av samplingsintervaller som vanligvis varer i ~ 5 min eller lengre en. Videre er analyser ikke gjøres i sanntid. En annen teknikk er rask scanning syklisk voltametri (FSCV), som bruker en karbon-fiber sonde som settes inn i hjernen. FSCV har utmerket temporal oppløsning (subsecond), høy følsomhet (nanomolar), og romlig oppløsning med sonde diameter på 5 til 30 mikrometer. Imidlertid er FSCV begrenset til sendere som frembringer en karakteristisk oksydasjon og reduksjon profil med spenning på et karbon potensiometrisk sonde 2. En tredje teknikk for å måle signalstoffer er direkte gjennom genetisk kodet nevrotransmitter (NT) biosensorer 3. Med denne metoden blir et fusjonsprotein laget som inneholder en ligand-bindende domene til en transmitter koblet til en fluorescens-resonans energioverføring (FRET) -basert par av fluoroforer 4 eller en permutert GFP 5. I motsetning til de to foregående fremgangsmåter, er disse biosensorer genetisk kodet og uttrykkes på overflaten av en vertscelle, slik som en nevron, gjennom produksjon av transgene dyr eller akutt med bruk av virale midler for å infisere celler. Hittil har genetisk kodet biosensorer er kun utviklet for detekteringg glutamat og GABA 3-5. Begrensninger med disse teknikkene har vært den lave følsomhet, i nM område, og den manglende evne til å utvide deteksjons til det store antall sendere, f.eks klassiske nevrotransmittere, nevropeptider og neuromodulatorer, som signalerer via G protein-koblede reseptorer (GPCR). Faktisk er det nesten 800 GPCRs i det menneskelige genom. For å møte disse manglene, har vi utviklet et innovativt verktøy til optisk måling utgivelsen av noen nevrotransmitter som signaliserer gjennom en GPCR. CNiFERs (cellebasert nevrotransmitter fluorescerende konstruert journalister) er klonale HEK293 celler konstruert for å uttrykke en bestemt GPCR at når stimulert, utløser en økning i intracellulært [Ca 2+] som er oppdaget av en genetisk kodet FRET baserte Ca 2+ sensor, TN-XXL. Således CNiFERs omforme nevrotransmitter-reseptorbinding til en endring i fluorescens, noe som gir en direkte og real-time optisk rEAD-out av lokale neurotransmitter aktivitet. Ved å benytte den native reseptoren for en gitt nevrotransmitter, CNiFERs beholde den kjemiske spesifisitet, affinitet og tidsmessige dynamikken i endogent uttrykte reseptorer. Til dags dato, har vi laget tre typer CNiFERs, en for detektering av acetylkolin ved hjelp av M1-reseptoren, en for å detektere dopamin ved hjelp av D2-reseptoren, og en for å detektere noradrenalin ved hjelp av α1a reseptoren 6,7. Den CNiFER teknologien er lett utvidbar og skalerbar, slik at det er mottakelig for alle typer GPCR. I denne Jove artikkelen beskriver vi og illustrere metodikken for å designe, realisere, og test in vivo CNiFERs for alle bruksområder.

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			<td height="60" style="height:60px;width:387px;">isoflurane chamber/mask</td>
			<td style="width:191px;">Highland Medical Equipment</td>
			<td style="width:429px;">564-0427, HME 109 Table Top Anesthetic Machine with Isoflurane Vaporizer, O2 Flowmeter, Gang Valve; 564-0852, Induction Chamber 16X7X7.5cm</td>
			<td>surgery and stereotaxic injection</td>
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			<td height="20" style="height:20px;width:387px;">3D scope with arm</td>
			<td style="width:191px;">Zeiss</td>
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			<td>surgery and stereotaxic injection</td>
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			<td height="20" style="height:20px;width:387px;">fiber optic light</td>
			<td style="width:191px;"></td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
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			<td height="20" style="height:20px;">Betadine&#160;</td>
			<td style="width:191px;"></td>
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			<td>surgery and stereotaxic injection</td>
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			<td height="20" style="height:20px;">70 % (v/v) isopropyl alcohol</td>
			<td style="width:191px;"></td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
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			<td height="20" style="height:20px;">Povidone-Iodine Prep Pads</td>
			<td style="width:191px;">dynarex</td>
			<td style="width:429px;">1108</td>
			<td>surgery and stereotaxic injection</td>
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			<td height="20" style="height:20px;">NaCl 0.9% (INJECTION, USP, 918610)</td>
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			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
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			<td height="20" style="height:20px;">CYCLOSPORINE (INJECTION, USP)</td>
			<td style="width:191px;"></td>
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			<td>surgery and stereotaxic injection</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Buprenex (INJECTION) buprenorphine (0.03 &#956;g per g rodent)</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Ophthalmic ointment&#160;</td>
			<td style="width:191px;">Akorn</td>
			<td style="width:429px;">NDC 17478-235-35</td>
			<td>surgery and stereotaxic injection</td>
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			<td height="20" style="height:20px;">Surgifoam</td>
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			<td>surgery and stereotaxic injection</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Grip dental cement</td>
			<td style="width:191px;">Dentsply</td>
			<td style="width:429px;">#675571, 675572</td>
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			<td height="20" style="height:20px;">Instant SuperGlue&#160;</td>
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			<td height="20" style="height:20px;">LOCTITE 4041</td>
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			<td height="20" style="height:20px;">METABOND</td>
			<td style="width:191px;">C&#38;B</td>
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			<td height="20" style="height:20px;">no. 0 cover glass</td>
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			<td height="20" style="height:20px;">stereotaxic frame&#160;</td>
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			<td height="60" style="height:60px;">Rectal probe and heating pad</td>
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			<td style="width:429px;">40-90-8D, DC Temperature Controller,40-90-2-06, 6.5X9.5cm Heating Pad40-90-5D-02, Rectal Thermistor Probe</td>
			<td>surgery and stereotaxic injection</td>
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			<td>surgery and stereotaxic injection</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Mineral oil</td>
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			<td>surgery and stereotaxic injection</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Kwik-Cast (Silicone elastomer)</td>
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			<td height="20" style="height:20px;">Suture&#160;&#160;</td>
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			<td style="width:429px;">18&#8217;&#8217;, 1667, 4-0</td>
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			<td height="20" style="height:20px;">Scissors</td>
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			<td style="width:429px;">91500-09, 15018-10</td>
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			<td style="width:429px;">11252-30; #55, 11295-51; Grafe, 11050-10</td>
			<td>surgery and stereotaxic injection</td>
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			<td style="width:429px;">91308-12</td>
			<td>surgery and stereotaxic injection</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Small Vessel Cauterizer Kit</td>
			<td style="width:191px;">Fine Scientific Tools</td>
			<td style="width:429px;">18000-00</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hot Bead Sterilizers</td>
			<td style="width:191px;">Fine Scientific Tools</td>
			<td style="width:429px;">18000-45</td>
			<td>surgery and stereotaxic injection</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Instrument Case with Silicone Mat</td>
			<td style="width:191px;">Fine Scientific Tools</td>
			<td style="width:429px;">20311-21</td>
			<td>surgery and stereotaxic injection</td>
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			<td height="20" style="height:20px;">Plastic Sterilization Containers with Silicone Mat</td>
			<td style="width:191px;">Fine Scientific Tools</td>
			<td style="width:429px;">20810-01</td>
			<td>surgery and stereotaxic injection</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:387px;"></td>
			<td style="width:191px;"></td>
			<td style="width:429px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;"></td>
			<td style="width:191px;"></td>
			<td style="width:429px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:387px;">2P fixed-stage fluorescence scope for in vivo imaging</td>
			<td style="width:191px;">Olympus</td>
			<td style="width:429px;">FV1200 MPE</td>
			<td>in vivo imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">Multiphoton laser</td>
			<td style="width:191px;">SpectraPhysics</td>
			<td style="width:429px;">Mai Tai DeepSee</td>
			<td>in vivo imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">Green Laser</td>
			<td style="width:191px;">Olympus</td>
			<td style="width:429px;">473 nm Laser</td>
			<td>in vivo imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">xy translation base</td>
			<td style="width:191px;">Scientifica</td>
			<td style="width:429px;">MMBP</td>
			<td>in vivo imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">FRET filter cube for YFP and CFP</td>
			<td style="width:191px;">Olympus</td>
			<td style="width:429px;"></td>
			<td>in vivo imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">25-X water immersion objective</td>
			<td style="width:191px;">Olympus</td>
			<td style="width:429px;"></td>
			<td>in vivo imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">air table</td>
			<td style="width:191px;">Newport</td>
			<td style="width:429px;"></td>
			<td>in vivo imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">custom built light-tight cage</td>
			<td style="width:191px;">Thorlab</td>
			<td style="width:429px;"></td>
			<td>in vivo imaging</td>
		</tr>
	</tbody>
</table>

construction of cell-based neurotransmitter fluorescent engineered reporters (cnifers) for optical detection of neurotransmitters in vivo

Cell-baserte nevrotransmitter fluorescerende konstruerte journalister (CNiFERs) gi et nytt verktøy for nevrologer til optisk oppdage utgivelsen av nevrotransmittere i hjernen in vivo. En spesifikk CNiFER er opprettet fra et humane embryonale nyrecelle som stabilt uttrykker en bestemt G protein-koblet reseptor, som kobler til G q / 11 g proteiner, og en FRET basert Ca 2+-detektoren, TN-XXL. Aktivering av reseptoren fører til en økning i den FRET signal. CNiFERs har nM følsomhet og en tidsmessig respons sekunder fordi en CNiFER klone benytter native reseptoren for en bestemt nevrotransmitter, f.eks D2R for dopamin. CNiFERs er direkte implantert i hjernen, slik at de kan avføle neurotransmitterfrigjørelse med en romlig oppløsning på mindre enn ett hundre um, noe som gjør dem ideelle for å måle volumet overføring in vivo. CNiFERs kan også brukes til å screene andre rusmidler for potensiell kryssreaktivitet i vivo. Vi har nylig utvidet familien til CNiFERs å inkludere GPCR som par til G i / o G-proteiner. CNiFERs er tilgjengelige for påvisning av acetylkolin (ACh), dopamin (DA) og norepinefrin (NE). Gitt at en GPCR kan brukes til å skape en ny CNiFER, og at det finnes omtrent 800 GPCR i det humane genom, vi beskriver her den generelle fremgangsmåten for å utforme, å realisere, og teste alle typer CNiFER.

En CNiFER er avledet fra en human embryonisk nyre (HEK293) celle som er konstruert til å stabilt uttrykke minst to proteiner: en spesifikk G-proteinkoblet reseptor (GPCR) og en genetisk kodet [Ca2 +] sensor, TN-XXL. TN-XXL undergår fluorescens resonans energioverføring (FRET) mellom cyan og gult fluorescerende proteiner, henholdsvis eCFP og Citrine,, som respons på Ca 2+ 6,15. Aktivering av GPCR som par for å endogen G q G-proteiner føre ...

Watch this Scientific Journal Video about Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters CNiFERs for Optical Detection of Neurotransmitters In Vivo at JoVE.com

Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters CNiFERs for Optical Detection of Neurotransmitters In Vivo

Vi presenterer en protokoll for å skape celle-basert signalstoffet fluorescerende konstruerte journalister (CNiFERs) for optisk deteksjon av volumetrisk neurotransmitter utgivelsen.

Bygging av Cell-baserte Nevrotransmitter Fluorescent Engineered Reporters (CNiFERs) for optisk deteksjon av Nevrotransmittere<em&gt; I Vivo</em

Cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) provide a new tool for neuroscientists to optically detect the release of ...

Research

JoVE Journal

Neuroscience

Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters (CNiFERs) for Optical Detection of Neurotransmitters In Vivo

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde Fluorescent Merking Tillater målrettet Ekstracellulær Single-enhet Opptak fra Identifiserte Nerveceller<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Funksjonell Bildediagnostiske Bruke Ultrasonic blod-hjerne barrieren avbrudd og mangan forbedret MR

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Tynnet-skull Cortical Window Teknikk for<em&gt; In Vivo</em&gt; Optisk koherens tomografi Imaging

Optical coherence tomography (OCT) is a biomedical imaging technique  with high spatial-temporal resolution. With its minimally invasive approach OCT  has ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Påvisning av Genome og transkripsjoner av en vedvarende DNA virus i nerve vev ved Fluorescent In situ Hybridisering Kombinert med Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Modellering astrocytom Patogenese<em&gt; In Vitro</em&gt; Og<em&gt; In Vivo</em&gt; Bruke kortikal astrocytter eller nevrale stamceller fra Betinget, Genetisk konstruert Mus

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Samtidig<em&gt; Ex vivo</em&gt; Funksjonell testing av to netthinne etter<em&gt; In vivo</em&gt; Elektroretinogrammet System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Optisk overvåkingen av levende Nerve Terminal Merking i hårsekken lansettformede Endings av<em&gt; Ex Vivo</em&gt; Mouse Ear Skin

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Optical Coherence tomografi: Bildebehandling musen Retinal Ganglion celler i Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

Måler i Vivo endringer i ekstracellulære nevrotransmittere under naturlig givende atferd i kvinnelige syrer Hamsters

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or ...

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

I Vivo Overvåking av Circadian klokke genuttrykk i musen Suprachiasmatic kjernen bruker fluorescens journalister

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene ...