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

Ringraziamo B. Conklin (University of California, San Francisco) per fornire i qi5 G e G QS5 cDNA, A. Schweitzer per l&#39;assistenza con l&#39;elettronica, N. Taylor per l&#39;assistenza con lo screening di cloni, Ian Glaaser e Robert Rifkin per correzione di bozze , e Olivier Griesbeck per TN-XXL. Questo lavoro è stato sostenuto da borse di ricerca attraverso l&#39;Istituto nazionale degli Stati Uniti on Drug Abuse (NIDA) (DA029706; DA037170), l&#39;Istituto Nazionale di Biomedical Imaging e Bioingegneria (NIBIB) (EB003832), Hoffman-La Roche (88610A) e il &quot;Neuroscience correlati alla droga di concessione di abuso &quot;formazione attraverso NIDA (DA007315).

Tutte le procedure sugli animali eseguiti in questo studio sono conformi Istituzionale cura degli animali e le linee guida Comitato Usa (IACUC), e sono stati approvati dai IACUCs presso la Scuola di Medicina Icahn al Monte Sinai e la University of California, San Diego. 1. Generare GPCR che esprimono Lentivirus per trasformare cellule HEK293 <ol><li> Ottenere il cDNA per un GPCR specifico da una fonte commerciale, ad esempio, cdna.org. In alternativa, amplificano il gene GPCR da una libreria di cDNA m...

<ol>
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J., Tian, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Chemical+Neurotransmission+with+Genetically+Encoded+Fluorescent+Sensors.">Imaging Chemical Neurotransmission with Genetically Encoded Fluorescent Sensors.</a> ACS Chem Neurosci. , (2015).</li><li>Okubo, Y., 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=Imaging+extrasynaptic+glutamate+dynamics+in+the+brain.">Imaging extrasynaptic glutamate dynamics in the brain.</a> Proc. Natl. Acad. Sci. USA. 107, 6526-6531 (2010).</li><li>Marvin, J. 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A., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-based+reporters+reveal+in+vivo+dynamics+of+dopamine+and+norepinephrine+release+in+murine+cortex.">Cell-based reporters reveal in vivo dynamics of dopamine and norepinephrine release in murine cortex.</a> Nat Methods. 11, 1245-1252 (2014).</li><li>Lorenz, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+chain+reaction:+basic+protocol+plus+troubleshooting+and+optimization+strategies.">Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies.</a> J Vis Exp. , e3998 (2012).</li><li>Wang, X., McManus, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus+production.">Lentivirus production.</a> J Vis Exp. , (2009).</li><li>Conklin, B. R., Farfel, Z., Lustig, K. D., Julius, D., Bourne, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substitution+of+three+amino+acids+switches+receptor+specificity+of+Gq&#945;+to+that+of+Gj&#945;.">Substitution of three amino acids switches receptor specificity of Gq&#945; to that of Gj&#945;.</a> Nature. 363, 274-276 (1993).</li><li>Cetin, A., Komai, S., Eliava, M., Seeburg, P. H., Osten, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotaxic+gene+delivery+in+the+rodent+brain.">Stereotaxic gene delivery in the rodent brain.</a> Nat Protoc. 1, 3166-3173 (2006).</li><li>Shih, A. Y., Mateo, C., Drew, P. J., Tsai, P. 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Y., 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=Two-photon+microscopy+as+a+tool+to+study+blood+flow+and+neurovascular+coupling+in+the+rodent+brain.">Two-photon microscopy as a tool to study blood flow and neurovascular coupling in the rodent brain.</a> J Cereb Blood Flow Metab. 32, 1277-1309 (2012).</li><li>Yamauchi, J. 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La creazione di CNiFERs fornisce una strategia innovativa e unica per misurare otticamente rilascio di neurotrasmettitori nel cervello in vivo. CNiFERs sono ideali per misurare il rilascio extrasinaptica, vale a dire, il volume di conduzione, per neurotrasmettitori. È importante sottolineare che ogni CNiFER possiede le proprietà del GPCR nativa, fornendo una misurazione ottica fisiologica dei cambiamenti nei livelli di neurotrasmettitori nel cervello. Fino ad oggi, sono stati creati per la rilevazion...

Per comprendere appieno come i neuroni comunicano nel cervello, è necessario disporre di un metodo per misurare il rilascio di neurotrasmettitori in vivo. Ci sono diverse tecniche consolidate per la misurazione neurotrasmettitori in vivo. Una tecnica comunemente usata è microdialisi, in cui una cannula viene inserita nel cervello e un piccolo volume di liquido cerebrospinale viene raccolto ed analizzato mediante cromatografia liquida ad alte prestazioni e rilevazione elettrochimica 1. Microdialisi ha una risoluzione spaziale dell&#39;ordine di pochi diametri della sonda, ad esempio, ~ 0,5 mm per una microsonda 200 micron di diametro. La risoluzione temporale di questa tecnica, tuttavia, è lento a causa di intervalli di campionamento che tipicamente durano ~ 5 minuti o più 1. Inoltre, le analisi non sono fatti in tempo reale. Un&#39;altra tecnica è scansione veloce voltammetria ciclica (FSCV), che utilizza una sonda in fibra di carbonio che viene inserito nel cervello. FSCV ha eccellenti temperaturaRisoluzione orale (in frazioni), alta sensibilità (nanomolari), e la risoluzione spaziale con diametri della sonda da 5 a 30 micron. Tuttavia, FSCV è limitato ai trasmettitori che producono ossidazione caratteristico profilo riduzione con tensione su una sonda potenziometrica carbonio 2. Una terza tecnica per misurare neurotrasmettitori è direttamente attraverso neurotrasmettitori geneticamente codificato biosensori (NT) 3. Con questo metodo, una proteina di fusione viene creato che contiene un dominio ligando vincolante per un trasmettitore accoppiato ad un trasferimento di energia di risonanza di fluorescenza (FRET) accoppiamento sede di fluorofori 4 o un GFP permutati 5. A differenza delle precedenti due metodi, questi biosensori sono geneticamente codificati ed espressi sulla superficie di una cellula ospite, come un neurone, attraverso la produzione di animali transgenici o acutamente con l&#39;uso di agenti virali per infettare le cellule. Fino ad oggi, biosensori geneticamente codificati sono stati sviluppati solo per detecting glutammato e GABA 3-5. Limitazioni con queste tecniche sono la bassa sensibilità, nell&#39;intervallo nM, e l&#39;incapacità di espandere il rilevamento del gran numero di trasmettitori, ad esempio, neurotrasmettitori classici, neuropeptidi e neuromodulatori, che segnalano attraverso recettori accoppiati alla proteina G (GPCR). In realtà, ci sono quasi 800 GPCR nel genoma umano. Per far fronte a queste carenze, abbiamo sviluppato uno strumento innovativo per misura rilascio otticamente di qualsiasi neurotrasmettitore che segnala attraverso un GPCR. CNiFERs (neurotrasmettitore fluorescenti a base di cellule reporter ingegnerizzati) sono cellule HEK293 clonali ingegnerizzate per esprimere un GPCR specifico che, quando stimolato, provoca un aumento intracellulare [Ca 2+] che viene rilevata da un sensore geneticamente codificato FRET-based Ca 2+, TN-XXL. Così, CNiFERs trasformare recettore neurotrasmettitore vincolante in un cambiamento della fluorescenza, fornendo un r ottica tempo reale diretta eead-out di attività neurotrasmettitore locale. Utilizzando il recettore nativo per un determinato neurotrasmettitore, CNiFERs conservano la specificità chimica, affinità e dinamiche temporali dei recettori endogenamente espressi. Ad oggi, abbiamo creato tre tipi di CNiFERs, uno per rilevare acetilcolina usando il recettore M1, uno per rilevare dopamina utilizzando il recettore D2, e uno per rilevare noradrenalina usando l&#39;6,7 recettore tipo a1A. La tecnologia CNiFER è facilmente espandibile e scalabile, rendendolo suscettibile a qualsiasi tipo di GPCR. In questo articolo Giove, descriviamo e illustrare la metodologia per la progettazione, la realizzazione, e la prova CNiFERs vivo per qualsiasi applicazione.

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			<td height="20" style="height:20px;">BD 40 um Falcon cell strainers</td>
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			<td>352340</td>
			<td>FACS</td>
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			<td height="20" style="height:20px;">0.05% Trypsin EDTA&#160;</td>
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			<td>25200056</td>
			<td>FACS</td>
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			<td height="20" style="height:20px;">96 Well Plate, flat bottom, clear&#160;</td>
			<td>Corning&#160;</td>
			<td>3596</td>
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			<td></td>
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			<td>CLS3997</td>
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			<td height="20" style="height:20px;">Optilux black clear bottom&#160;</td>
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			<td>3603</td>
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			<td height="20" style="height:20px;">Flexstation pipet tips</td>
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			<td>9000-0911</td>
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			<td>Flexstation</td>
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			<td style="width:191px;">SimgaAldrich</td>
			<td style="width:429px;">PHR1090</td>
			<td>Flexstation</td>
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			<td style="width:191px;">SimgaAldrich</td>
			<td style="width:429px;">A2129</td>
			<td>Flexstation</td>
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			<td style="width:191px;">SimgaAldrich</td>
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			<td>Flexstation</td>
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			<td>Flexstation</td>
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			<td style="width:429px;">S6883</td>
			<td>Flexstation</td>
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			<td height="20" style="height:20px;">Adenosine</td>
			<td style="width:191px;">SimgaAldrich</td>
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			<td>Flexstation</td>
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			<td style="width:429px;">Flexstation 3</td>
			<td>Flexstation</td>
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			<td height="20" style="height:20px;width:387px;"></td>
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			<td style="width:191px;">Life Technologies</td>
			<td style="width:429px;">10569-010</td>
			<td>Tissue culture</td>
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			<td height="20" style="height:20px;width:387px;">&#160;Fetal bovine serum</td>
			<td style="width:191px;">Life Technologies</td>
			<td style="width:429px;">10082-139</td>
			<td>Tissue culture</td>
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			<td style="width:191px;">Life Technologies</td>
			<td style="width:429px;">15140-122</td>
			<td>Tissue culture</td>
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			<td style="width:191px;">InvivoGen</td>
			<td style="width:429px;">ant-pr-1</td>
			<td>Tissue culture</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:387px;">&#160;Fibronectin&#160;</td>
			<td style="width:191px;">SimgaAldrich</td>
			<td style="width:429px;">F0895</td>
			<td>Tissue culture</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:387px;">CoolCell LX Alcohol-free controlled-rate cell freezing box</td>
			<td style="width:191px;">Bioexpress</td>
			<td style="width:429px;">D-3508)</td>
			<td>Tissue culture</td>
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			<td height="20" style="height:20px;width:387px;"></td>
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			<td height="20" style="height:20px;width:387px;">cyanoacrylate glue&#160;</td>
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			<td style="width:429px;">Loctite no. 495</td>
			<td>surgery and stereotaxic injection</td>
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			<td>surgery and stereotaxic injection</td>
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			<td height="20" style="height:20px;">NANOINJECTOR</td>
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			<td style="width:429px;">3-000-204</td>
			<td>surgery and stereotaxic injection</td>
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			<td height="20" style="height:20px;">GLASS ELECTRODES</td>
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			<td style="width:429px;">3-000-203G</td>
			<td>surgery and stereotaxic injection</td>
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			<td style="width:429px;">Exl-M40</td>
			<td>surgery and stereotaxic injection</td>
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			<td style="width:191px;">Fine Scientific</td>
			<td style="width:429px;">19007-05; 19007-07)</td>
			<td>surgery and stereotaxic injection</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:387px;">Sterilizing bath</td>
			<td style="width:191px;">FST</td>
			<td style="width:429px;">18000-45, Hot Bead Sterilizer</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="60">
			<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>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:387px;">3D scope with arm</td>
			<td style="width:191px;">Zeiss</td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Betadine&#160;</td>
			<td style="width:191px;"></td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl 0.9% (INJECTION, USP, 918610)</td>
			<td style="width:191px;"></td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CYCLOSPORINE (INJECTION, USP)</td>
			<td style="width:191px;"></td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgifoam</td>
			<td style="width:191px;">Ethicon</td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<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>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Instant SuperGlue&#160;</td>
			<td style="width:191px;">NDindustries</td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LOCTITE 4041</td>
			<td style="width:191px;"></td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">METABOND</td>
			<td style="width:191px;">C&#38;B</td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">no. 0 cover glass</td>
			<td style="width:191px;">Fisher</td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">stereotaxic frame&#160;</td>
			<td style="width:191px;">Kopf</td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Rectal probe and heating pad</td>
			<td style="width:191px;">FHC</td>
			<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>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">optical breadboard for imaging</td>
			<td style="width:191px;">Thorlabs</td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mineral oil</td>
			<td style="width:191px;">Fisher</td>
			<td style="width:429px;">S55667</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Kwik-Cast (Silicone elastomer)</td>
			<td style="width:191px;">World Precision Instruments</td>
			<td style="width:429px;"></td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Suture&#160;&#160;</td>
			<td style="width:191px;">Ethicon</td>
			<td style="width:429px;">18&#8217;&#8217;, 1667, 4-0</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scissors</td>
			<td style="width:191px;">Fine Scientific Tools</td>
			<td style="width:429px;">91500-09, 15018-10</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Forcepts</td>
			<td style="width:191px;">Fine Scientific Tools</td>
			<td style="width:429px;">11252-30; #55, 11295-51; Grafe, 11050-10</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Student Halsted-Mosquito Hemostats</td>
			<td style="width:191px;">Fine Scientific Tools</td>
			<td style="width:429px;">91308-12</td>
			<td>surgery and stereotaxic injection</td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="20">
			<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>
		</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="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

Basate sulle cellule neurotrasmettitore fluorescenti reporter ingegnerizzati (CNiFERs) fornire un nuovo strumento per i neuroscienziati di rilevare visivamente il rilascio di neurotrasmettitori nel cervello in vivo. Una specifica CNiFER è creata da una cellula embrionali renali umane che esprime stabilmente uno specifico recettore accoppiato alla proteina G, che le coppie di G q / 11 proteine ​​G, e un FRET-based Ca 2+ -detector, TN-XXL. L&#39;attivazione del recettore porta ad un aumento nel segnale FRET. CNiFERs hanno una sensibilità nm e una risposta temporale di secondi perché un clone CNiFER utilizza il recettore nativo per un particolare neurotrasmettitore, ad esempio, D2R per la dopamina. CNiFERs sono direttamente impiantati nel cervello, consentendo loro di percepire il rilascio di neurotrasmettitore con una risoluzione spaziale di meno di cento micron, che li rende ideali per misurare il volume di trasmissione in vivo. CNiFERs può anche essere usata per selezionare altri farmaci per il potenziale reattività incrociata in vivo. Recentemente abbiamo ampliato la famiglia di CNiFERs per includere GPCR quella coppia a G I / O G proteine. CNiFERs sono disponibili per rilevare acetilcolina (ACh), dopamina (DA) e noradrenalina (NE). Dato che ogni GPCR può essere utilizzato per creare un romanzo CNiFER e che ci sono circa 800 GPCR nel genoma umano, che descriviamo qui la procedura generale per progettare, realizzare e testare qualsiasi tipo di CNiFER.

Un CNiFER è derivato da una cellula umana embrionali renali (HEK293) che è progettato per esprimere stabilmente almeno due proteine: una specifica proteina G recettori accoppiati (GPCR) e geneticamente codificato sensore [Ca 2+], TN-XXL. TN-XXL subisce Fluorescence Resonance Energy Transfer (FRET) tra il ciano e giallo fluorescenti proteine, rispettivamente ECFP e citrino,, in risposta a Ca 2+ ioni 6,15. Attivazione GPCRs che coppia a endogeno G q<...

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 presentiamo un protocollo per creare celle a base di neurotrasmettitore fluorescenti reporter ingegnerizzati (CNiFERs) per la rilevazione ottica del rilascio di neurotrasmettitore volumetrico.

Costruzione di Cell-based neurotrasmettitore fluorescenti Engineered Reporters (CNiFERs) per il rilevamento ottico dei neurotrasmettitori<em&gt; In 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

Retrograda Etichettatura fluorescente Consente mirata extracellulare singola unità di registrazione da parte dei neuroni identificati<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 ...

Ricerca

Neuroscienze

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

Neuroimaging funzionale con l&#39;impiego dell&#39;ecografia emato-encefalica e la barriera di Turbativa del Manganese-enhanced MRI

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

Diluito-cranio Tecnica finestra corticale per<em&gt; In Vivo</em&gt; Tomografia a coerenza ottica 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

La rilevazione del genoma e Trascrizioni di un virus DNA persistente in tessuti neuronali da fluorescente In situ Ibridazione In combinazione con 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

Modellazione Astrocitoma Patogenesi<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Utilizzo corticale astrociti o cellule staminali neurali da condizionale, topi geneticamente ingegnerizzati

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

Simultaneo<em&gt; Ex vivo</em&gt; Test funzionale di due retine per<em&gt; In vivo</em&gt; Sistema Elettroretinogramma

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

Il monitoraggio ottico di Living nervo terminale Etichettatura nel follicolo pilifero lanceolate Endings del<em&gt; Ex Vivo</em&gt; Mouse Ear pelle

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

Tomografia a coerenza ottica: Imaging Mouse ganglionari cellule In 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

Misurare In Vivo i cambiamenti in neurotrasmettitori extracellulare durante naturalmente Premiando i comportamenti in criceti siriani femminili

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

In Vivo Monitoraggio dell'espressione genica orologio circadiano nel nucleo soprachiasmatico Mouse mediante fluorescenza reporter

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