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

Agradecemos B. Conklin (Universidade da Califórnia, San Francisco) por fornecer os qi5 G e G QS5 cDNAs, A. Schweitzer para assistência com a eletrônica, N. Taylor para a assistência com a seleção de clones, Ian Glaaser e Robert Rifkin para leitura de prova e Olivier Griesbeck para TN-XXL. Este trabalho foi apoiado por bolsas de investigação, através do Instituto Nacional sobre Abuso de Drogas (NIDA) (DA029706; DA037170), o Instituto Nacional de Imagem Biomédica e Bioengenharia (NIBIB) (EB003832), Hoffman-La Roche (88610A) eo &quot;Neuroscience relacionados com a droga de bolsa de formação abuso &quot;por meio de NIDA (DA007315).

Todos os procedimentos com animais realizados neste estudo estão em conformidade com Institutional Animal Care e diretrizes Use Committee (IACUC), e tenham sido aprovados pelas IACUCs no Icahn Escola de Medicina Monte Sinai e da Universidade da Califórnia, San Diego. 1. Gerar GPCR-expressando Lentivírus para transformar células HEK293 <ol><li> Obter o ADNc para um GPCR específico a partir de uma fonte comercial, por exemplo, cdna.org. Alternativamente, amplificar o gene GPCR a partir de uma bibli...

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A criação de CNiFERs proporciona uma estratégia inovadora e original para opticamente medição da libertação de neurotransmissores no cérebro in vivo. CNiFERs são idealmente adequados para a medição da libertação de extrasynaptic, ou seja, o volume de condução, para neurotransmissores. Importante, cada CNiFER possui as propriedades do GPCR nativo, proporcionando uma medição óptica fisiológica das alterações nos níveis de neurotransmissores no cérebro. Até à data, CNiFERs foram c...

Para entender completamente como os neurônios se comunicam no cérebro, é necessário ter um método para medir a liberação de neurotransmissores in vivo. Existem várias técnicas bem estabelecidas para a medição in vivo neurotransmissores. Uma técnica utilizada é de microdiálise, em que uma cânula é inserida no cérebro e um pequeno volume de fluido cerebrospinal são recolhidas e analisadas por cromatografia líquida de elevado desempenho e detecção electroquímica 1. Microdiálise tem uma resolução espacial na ordem de alguns diâmetros da sonda, por exemplo, ~ 0,5 mm para uma microssonda de 200 um de diâmetro. A resolução temporal desta técnica, no entanto, é lento devido a intervalos de amostragem que duram tipicamente ~ 5 minutos ou mais 1. Além disso, as análises não são feitos em tempo real. Uma outra técnica é a pesquisa rápida voltametria cíclica (FSCV), que usa uma sonda de fibra de carbono que é introduzido no cérebro. FSCV tem excelente Tempresolução oral (subsecond), de alta sensibilidade (nanomolar), e resolução espacial com o diâmetro da sonda de 5 a 30 um. No entanto, está limitado a FSCV transmissores, que produzem uma característica de oxidação e redução perfil com tensão em uma sonda potenciométrica de carbono 2. Uma terceira técnica para medir neurotransmissores é diretamente através de neurotransmissores geneticamente codificado (NT) biossensores 3. Com este método, uma proteína de fusão é criada que contém um domínio de ligação ao ligando para um transmissor acoplado a uma transferência de energia de ressonância de fluorescência (FRET) par com base na fluoróforos de 4 ou 5 permutado um GFP. Ao contrário dos dois métodos anteriores, estes biossensores são geneticamente codificado e expresso na superfície de uma célula hospedeira, tal como um neurónio, através da produção de animais transgénicos ou de forma aguda com a utilização de agentes virais para infectar as células. Até à data, biossensores geneticamente codificados só foram desenvolvidos para detecting glutamato e GABA 3-5. Limitações com estas técnicas têm sido a baixa sensibilidade, na gama nM, e a incapacidade para expandir a detecção de um grande número de emissores, por exemplo, neurotransmissores clássicos, neuropéptidos e neuromoduladores, que sinalizam através de receptores acoplados à proteína G (GPCRs). Na verdade, existem cerca de 800 GPCRs no genoma humano. Para lidar com essas deficiências, desenvolvemos uma ferramenta inovadora para liberação medida opticamente de qualquer neurotransmissor que sinaliza através de um GPCR. CNiFERs (com base nas células repórteres fluorescentes modificadas neurotransmissor) são células HEK293 clonais manipuladas para expressar um GPCR específico que, quando estimulados, desencadeia um aumento na intracelular [Ca2 +] que é detectado por um sensor baseado em FRET de Ca 2+ geneticamente codificado, TN-XXL. Assim, obrigatório neurotransmissor CNiFERs transformar-se numa alteração na fluorescência, proporcionando uma óptica R em tempo real e directa do receptoread-out da actividade neurotransmissora local. Ao utilizar o receptor nativo para um determinado neurotransmissor, CNiFERs reter a especificidade química, afinidade e dinâmicas temporais dos receptores expresso endogenamente. Até à data, criámos três tipos de CNiFERs, um para detectar a acetilcolina utilizando o receptor M1, um para a detecção de dopamina utilizando o receptor D2, e um para detectar a norepinefrina usando o receptor de 6,7 α1a. A tecnologia CNiFER é facilmente expansível e escalável, tornando-se passível de qualquer tipo de GPCR. Neste artigo Jove, que descrevem e ilustram a metodologia para conceber, realizar, e teste em CNiFERs vivo para qualquer aplicação.

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			<td height="60" style="height:60px;width:387px;">isoflurane chamber/mask</td>
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			<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

Com base nas células repórteres fluorescentes neurotransmissor modificadas (CNiFERs) fornecer uma nova ferramenta para neurocientistas para detectar opticamente a libertação de neurotransmissores no cérebro in vivo. A CNiFER específica é criada a partir de uma célula de rim embrionário humano que expressa estavelmente um receptor específico G acoplado a proteína, que os casais a G q / 11 proteínas G, e uma baseada em FRET Ca 2+ -detector, TN-XXL. A activação do receptor conduz a um aumento no sinal de FRET. CNiFERs tem sensibilidade nM e uma resposta temporal do segundo porque um clone CNiFER utiliza o receptor nativo para um neurotransmissor particular, por exemplo, para D2R dopamina. CNiFERs são directamente implantadas no cérebro, permitindo-lhes sentir a libertação de neurotransmissores, com uma resolução espacial de menos de cem um, tornando-as ideais para medir a transmissão de volume in vivo. CNiFERs também podem ser utilizados para pesquisar outros fármacos para o potencial reactividade cruzada em VIvo. Nós recentemente expandiu a família de CNiFERs para incluir GPCRs que o casal a G i / o G proteínas. CNiFERs estão disponíveis para a detecção de acetilcolina (ACh), dopamina (DA) e norepinefrina (NE). Dado que qualquer GPCR pode ser utilizado para criar um novo CNiFER e que há aproximadamente 800 GPCRs no genoma humano, descrevemos aqui o procedimento geral para conceber, realizar, e testar qualquer tipo de CNiFER.

Um CNiFER é derivada de uma célula de rim embrionário humano (HEK293) que é manipulada para expressar de forma estável pelo menos duas proteínas: uma proteína G específica acoplada do receptor (GPCR) e uma [Ca 2+] Sensor geneticamente codificado, TN-XL. TN-XXL sofre de ressonância de fluorescência de transferência de energia (FRET) entre proteínas ciano e amarelo fluorescente, eCFP e citrino, respectivamente, em resposta ao Ca 2+ 6,15. A activ...

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

Nós apresentamos um protocolo para criar baseada em células neurotransmissor fluorescentes repórteres modificadas (CNiFERs) para a detecção óptica de liberação do neurotransmissor volumétrica.

Construção de-base celular Neurotransmissores fluorescentes Engineered Repórteres (CNiFERs) para detecção óptica de neurotransmissores<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

Labeling fluorescentes retrógrada Permite alvejado Extracelular Gravação Single-unidade dos neurônios identificados<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 ...

Neurociência

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

Neuroimagem Funcional Usando rompimento da barreira hemato-encefálica ultra-sônico e Manganês-RM

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

Diluído crânio-técnica de janela cortical para<em&gt; Em Vivo</em&gt; Tomografia de coerência óptica

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

A detecção do genoma e Transcrições de um vírus DNA persistente em tecidos neuronais por fluorescentes Em situ Hibridização Combinado com Imunocoloração

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

Modelagem astrocitoma Patogênese<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Usando Cortical astrócitos ou células-tronco neurais de condicional, Geneticamente Modificadas Ratos

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

Simultâneo<em&gt; Ex vivo</em&gt; Teste Funcional de Dois retinas por<em&gt; In vivo</em&gt; Sistema Eletrorretinograma

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

Monitoramento óptico do nervo Viver Terminal Etiquetagem na folículo piloso Lanceolate Endings da<em&gt; Ex Vivo</em&gt; Mouse pele da orelha

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 de coerência óptica: Imagens Mouse ganglionares da retina células 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

Medição In Vivo alterações em neurotransmissores extracelulares durante naturalmente recompensando comportamentos em Hamsters sírios femininos

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

Na Vivo Monitoramento de expressão de Gene relógio circadiano no núcleo supraquiasmático do Mouse usando a repórteres de fluorescência

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