Name: recording electrical activity from identified neurons in the intact brain of transgenic fish
Uploaded: 2013-04-30T13:00:00
Description: Watch this Scientific Journal Video about Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish at JoVE.com

Agradecemos ao Dr. Meng-Lin Chin e Sra. Yuan Dong para assistência técnica. Este trabalho foi apoiado por uma bolsa da National Institutes of Health HD053767 (subcontrato para NLW) e por fundos do Departamento de Fisiologia e Gabinete do Vice-Reitor de Pesquisa, Universidade da Califórnia-Los Angeles (NLW).

1. Dissecação de cérebros de adultos Medaka <ol><li> Anestesiar adulto do sexo masculino ou do sexo feminino (Figura 1A) por imersão em 5 ml MS-222 (150 mg / L, pH 7,4), aguarde alguns minutos após movimentos brânquias cessaram antes de decapitar. Todos os procedimentos foram aprovados pelo Institutional Animal Care e do Comitê Use da Universidade da Califórnia-Los Angeles. </li><li> Decapitar o peixe em solução salina peixe na extremidade caudal do opérculo com uma tesoura em um diâmetro ...

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GnRH 3: GFP peixes transgênicos oferecem modelos exclusivos para estudar os mecanismos neurofisiológicos subjacentes à integração e regulamentação de controle central de comportamentos que estão direta e indiretamente envolvidos na reprodução 3, 8-10 neuronal. Uma das vantagens significativas deste sistema modelo é que muitas GnRH3 neurónios expressando GFP estão perto da superfície ventral do cérebro, o que permite um acesso relativamente fácil para os neurónios para o registo elect...

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Microscope</td>
 <td>Olympus</td>
 <td></td>
 <td>BX50W (Upright)</td>
 </tr>
 <tr>
 <td>Amplifier</td>
 <td>Axon Instruments</td>
 <td></td>
 <td>Axoclamp 200B </td>
 </tr>
 <tr>
 <td>A-D converter</td>
 <td>Computer Interference Corp.</td>
 <td></td>
 <td>Digidata ITC-18 </td>
 </tr>
 <tr>
 <td>Cooled CCD camera</td>
 <td>PCO Computer Optics </td>
 <td></td>
 <td>Sensicam</td>
 </tr>
 <tr>
 <td>Xenon lamp</td>
 <td>Sutter Instruments Co.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>GFP filter set</td>
 <td>Chroma Technologies</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Imaging Software</td>
 <td>Intelligent Imaging Innovations</td>
 <td></td>
 <td>Slidebook software </td>
 </tr>
 <tr>
 <td>Electrophysiology Data Acquisition Software</td>
 <td>Axon Instruments</td>
 <td></td>
 <td>Axograph software </td>
 </tr>
 <tr>
 <td>Electrophysiology Data Acquisition Software</td>
 <td>AD Instruments Inc.</td>
 <td></td>
 <td>PowerLab </td>
 </tr>
 <tr>
 <td>Headstage for electrophysiology</td>
 <td>Axon Instruments</td>
 <td></td>
 <td>CV 203BU </td>
 </tr>
 <tr>
 <td>Micromanipulator</td>
 <td>Sutter Instrument Co </td>
 <td>MP-285 </td>
 <td></td>
 </tr>
 <tr>
 <td>Recording Chamber Platform</td>
 <td>Warner Instrument Corp.</td>
 <td></td>
 <td>P1 </td>
 </tr>
 <tr>
 <td>Recording Chamber</td>
 <td>Warner Instrument Corp.</td>
 <td></td>
 <td>RC-26G </td>
 </tr>
 <tr>
 <td>Electrode Puller</td>
 <td>Sutter instruments</td>
 <td></td>
 <td>P87 </td>
 </tr>
 <tr>
 <td>Filament for electrode puller</td>
 <td>Sutter Instruments</td>
 <td>FB330B</td>
 <td>3.0 mm wide trough filament</td>
 </tr>
 <tr>
 <td>1.5 mm glass capillaries</td>
 <td>World Precision Instruments</td>
 <td>1B150-4</td>
 <td>Microelectrode for recording</td>
 </tr>
 <tr>
 <td>Syringe</td>
 <td>Becton Dickinson</td>
 <td>309586</td>
 <td>3 ml</td>
 </tr>
 <tr>
 <td>MS-222 </td>
 <td>Sigma</td>
 <td>E10521-10G</td>
 <td>Ethyl 3-aminobenzoate methanesulfonate salt</td>
 </tr>
 <tr>
 <td>Fish saline</td>
 <td></td>
 <td></td>
 <td>mM: 134 NaCl; 2.9 KCl; 2.1 CaCl2; 1.2 MgCl2; 10 HEPES </td>
 </tr>
 <tr>
 <td>Electrode solution (loose-patch) </td>
 <td></td>
 <td></td>
 <td>mM: 150 NaCl; 3.5 KCl; 2.5 CaCl2; 1.3 MgCl2; 10 HEPES; 10 glucose </td>
 </tr>
 <tr>
 <td>Electrode solution (whole-cell patch) </td>
 <td></td>
 <td></td>
 <td>mM: 112.5 K-gluconate; NaCl; 17.5 KCl; 0.5 CaCl2; 1 MgCl2; 5 MgATP; 1 EGTA; 10 HEPES; 1 GTP; 0.1 leupeptin;10 phospho-creatine </td>
 </tr>
 </tbody>
</table>

recording electrical activity from identified neurons in the intact brain of transgenic fish

Compreendendo a fisiologia da célula de circuitos neurais que regulam comportamentos complexos é muito maior usando sistemas de modelo em que este trabalho podem ser realizados em uma preparação cérebro intacto, onde o circuito neural do SNC permanece intacta. Usamos peixe transgênico no qual hormônio liberador de gonadotrofinas (GnRH) neurônios são geneticamente marcados com proteína fluorescente verde para identificação no cérebro intacto. Os peixes têm múltiplas populações de neurónios da GnRH, e as suas funções são dependentes da sua localização no cérebro e o gene de GnRH que expressam um. Nós nos concentramos em nossa demonstração GnRH3 neurônios localizados nos nervos terminais (TN) associados aos bulbos olfatórios usando o cérebro intacto de transgênicos medaka peixe (Figura 1B e C). Estudos sugerem que medaka TN-GnRH3 neurónios são neuromodulador, agindo como um transmissor de informação a partir do ambiente externo para o sistema nervoso central; they não desempenham um papel direto na regulação das funções hipofisário-gonadal, assim como o bem conhecido hipotálamo GNRH1 neurônios 2, 3. padrão A tônica do potencial de ação espontâneo disparo de TN-GnRH3 neurônios é uma propriedade intrínseca 4-6, a freqüência de que é modulada por pistas visuais de dois membros da mesma espécie e do neuropeptídeo Kisspeptin 1 5. Neste vídeo, usamos uma linha estável de transgênicos medaka em que TN-GnRH3 neurônios expressam um transgene contendo a região promotora de Gnrh3 ligado ao aumento da proteína verde fluorescente 7 para mostrar a você como identificar neurônios e monitorar a atividade elétrica em todo o cérebro preparação 6.

Um exemplo de grupos bilaterais de GFP marcadas com TN-GnRH3 neurónios do cérebro excisada de medaka peixes são apresentados nas Figuras 1B e 1C. Cada cluster contém cerca de 8-10 neurônios GnRH. As actividades neuronais espontâneas do destino TN-GnRH3 foram registrados no modo de corrente-braçadeira (I = 0), com taxas de queima típicas de 0,5-6 Hz. O padrão de ação potencial de queima é tipicamente um padrão tônico ou batendo, com um intervalo bastante regular interspike....

Watch this Scientific Journal Video about Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish at JoVE.com

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Neste vídeo, vamos demonstrar como para registrar a atividade elétrica dos neurônios individuais identificados em uma preparação do cérebro inteiro, o que preserva os circuitos neurais complexos. Usamos peixe transgênico no qual hormônio liberador de gonadotrofinas (GnRH) neurônios são geneticamente marcadas com uma proteína fluorescente para identificação na preparação cérebro intacto.

Registro da atividade elétrica dos neurônios identificados no cérebro intacto de peixes transgénicos

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