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 de 60 mm placa de Petri. </li><li> Transferência de cabeça de peixe até um diâmetro de 35 mm de uma placa de Petri, a metade cheio com solução salina e peixes revestida com Sylgard que tem uma depressão na mesma para posicionar e fixar a cabeça de dissecção cérebro. Posição e garantir a cabeça do lado dorsal com pinos de insetos. </li><li> Corte com cuidado a parte dorsal do crânio em torno do seu perímetro com uma tesoura fina e retire-o delicadamente com uma pinça, expondo a parte dorsal do cérebro, incluindo completamente a parte anterior da medula espinhal. </li> &lt;li&gt; Levante a medula espinhal delicadamente com uma pinça de ponta fina e cortar os nervos conjuntivo bilaterais com uma tesoura de ponta fina: nervos cranianos, nervos espinhais e nervos ópticos no lado ventral do cérebro e dos nervos olfativos na parte anterior do o cérebro. </li><li> Coloque o cérebro totalmente dissecada em uma placa de Petri fresco preenchido com solução salina peixe e verifique cuidadosamente sob o microscópio para garantir todo o cérebro está intacto (incluindo a glândula pituitária), sem quaisquer cortes ou perfurações. Cérebros danificados, não são utilizados para a experimentação. </li></ol> 2. Montagem do cérebro em uma câmara de gravação <ol><li> Colocar uma pequena gota de cianoacrilato ou algum outro tipo de cola de acção rápida com uma agulha no centro da câmara de registo e para espalhar a cola de cerca de 1 cm 2. </li><li> Transferir rapidamente o cérebro e cola-lo em uma posição ventral up-side. </li><li> Adicionar 1 ml de solução salina de peixe de modo que ele preenche a câmara de registo e cobre o cérebro. Perfundir continuamenteo cérebro com salino gaseificado peixe a uma taxa de pelo menos 200 ul / min. </li><li> Usando a ponta fina forceps remover cuidadosamente as meninges sobre a superfície da área de gravação do cérebro. </li></ol> 3. Eletrofisiologia <ol><li> Gravação remendo solto da ação potencial de queima <ol><li> Microeletrodos de vidro (6-10 mohms) são construídos a partir de pipetas de vidro de borosilicato (1B150F-4, Instrumentos de Precisão Mundial), utilizando um extrator de eletrodo (por exemplo, Sutter P-87), e de volta preenchido com a solução de gravação solta-patch no meio do caminho até o eletrodo. </li><li> Usando um microscópio de fluorescência na posição vertical com a charge-coupled device refrigerado (CCD) da câmera, encontrar o GFP-expressando TN-GnRH3 neurônios através de 10x (Figura 1B), então os objetivos de imersão em água 40x (Figura 1C). Eles estão localizados em ou perto da superfície ventral dos bulbos olfativos, anteriormente ao telencéfalo. </li><li> Mudar para um monitor de vídeo que está fornecendoimagens em tempo real a partir do microscópio para localizar o cluster neurônio TN-GnRH3 (Figura 1C). </li><li> Posicionar o microeléctrodo no banho de modo que a ponta do eléctrodo está acima do neurónio alvo. Aplicar constante leve pressão positiva para o microeléctrodo de modo que ela não se torne obstruído, e aproximar-se suavemente o neurónio alvo com a ponta do eléctrodo. </li><li> Controlar e monitorar a resistência do eletrodo no modo tensão-clamp usando software AxoGraph e amplificador Axoclamp 200B com software Axograph (Axon Instruments). Quando você pode ver a partir do monitor de vídeo que a ponta do eletrodo está na superfície do neurônio e da exibição de AxoGraph que a resistência muda um pouco, solte a pressão positiva e aplique uma ligeira pressão negativa se necessário, para fazer um selo de baixa resistência em neurônio (&lt;100 mohms). </li><li> Alternar para o modo atual-clamp, e registrar a tensão de membrana continuamente, sem qualquer entrada de corrente, ajustando a escalada tensão e da velocidade de gravação, se necessário. Os dados são coletados e analisados ​​por software Powerlab (ADInstruments Inc.). </li><li> Actividade da ficha eléctrica da linha de base em solução salina normal para peixes cerca de 5 minutos antes de qualquer tratamento (Figura 2A). Banheira de perfusão de medicamentos, hormônios peptídeos, etc. adicionada a solução salina de peixe continua a uma taxa de 200 ul / min, seguido por um período de lavagem em solução salina normal, peixes 5, 6, 11. </li></ol></li><li> Gravação do potencial de membrana de células inteiras O adesivo de fixação procedimento electrofisiologia célula total é a mesma que a do processo de gravação solta-remendo extracelular acima descrito, a partir das etapas 3.1.1 - 3.1.4. Em seguida, os procedimentos de desviar de acordo: <ol><li> Controlar e monitorar a resistência do eletrodo no modo tensão-clamp. Quando você pode ver a partir do monitor de vídeo que a ponta do eletrodo está na superfície do neurônio e do osciloscópio que a resistência muda um pouco, solte o positivoaplicar pressão e um pouco de pressão negativa para fazer uma vedação de alta resistência no neurónio (~ 3 Giga Ω). Aplicar sucção suave pela boca a ruptura da membrana remendado, você vai ver uma queda brusca da resistência à cerca de 120-250 mohms. </li><li> Alternar para o modo atual-clamp, e registrar a tensão de membrana continuamente, sem qualquer entrada de corrente, ajustando a escala de tensão e taxa de gravação, se necessário. </li><li> Actividade da ficha eléctrica da linha de base em solução salina normal para peixes cerca de 5 minutos antes de qualquer tratamento, como acima descrito no 3.1.7 (Figura 2B). </li></ol></li></ol>

<ol>
	<li>Kah, O., Lethimonier, C., Lareyre, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gonadotrophin-releasing+hormone+(GnRH)+in+the+animal+kingdom.">Gonadotrophin-releasing hormone (GnRH) in the animal kingdom.</a> J. Soc. Biol. 198 (1), 53-60 (2004).</li><li>Ramakrishnan, S., Wayne, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+cues+from+conspecifics+alter+electrical+activity+of+gonadotropin-releasing+hormone+neurons+in+the+terminal+nerve+via+visual+signals.">Social cues from conspecifics alter electrical activity of gonadotropin-releasing hormone neurons in the terminal nerve via visual signals.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 297 (1), R135-R141 (2009).</li><li>Abe, H., Oka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+neuromodulation+by+a+nonhypophysiotropic+GnRH+system+controlling+motivation+of+reproductive+behavior+in+the+teleost.">Mechanisms of neuromodulation by a nonhypophysiotropic GnRH system controlling motivation of reproductive behavior in the teleost.</a> 57 (6), 665-674 (2011).</li><li>Oka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrodotoxin-resistant+persistent+Na++current+underlying+pacemaker+potentials+of+fish+gonadotrophin-releasing+hormone+neurones.">Tetrodotoxin-resistant persistent Na+ current underlying pacemaker potentials of fish gonadotrophin-releasing hormone neurones.</a> J. Physiol. 482 (Pt. 1), 1-6 (1995).</li><li>Zhao, Y., Wayne, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Kisspeptin1+on+Electrical+Activity+of+an+Extrahypothalamic+Population+of+Gonadotropin-Releasing+Hormone+Neurons+in+Medaka.">Effects of Kisspeptin1 on Electrical Activity of an Extrahypothalamic Population of Gonadotropin-Releasing Hormone Neurons in Medaka.</a> PLoS One. 7 (5), e37909 (2012).</li><li>Wayne, N. L., 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=Whole-cell+electrophysiology+of+gonadotropin-releasing+hormone+neurons+that+express+green+fluorescent+protein+in+the+terminal+nerve+of+transgenic+medaka+(Oryzias+latipes).">Whole-cell electrophysiology of gonadotropin-releasing hormone neurons that express green fluorescent protein in the terminal nerve of transgenic medaka (Oryzias latipes).</a> Biol. Reprod. 73 (6), 1228-1234 (2005).</li><li>Okubo, K., 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=Forebrain+gonadotropin-releasing+hormone+neuronal+development:+insights+from+transgenic+medaka+and+the+relevance+to+X-linked+Kallmann+syndrome.">Forebrain gonadotropin-releasing hormone neuronal development: insights from transgenic medaka and the relevance to X-linked Kallmann syndrome.</a> Endocrinology. 147 (3), 1076-1084 (2006).</li><li>Okubo, K., 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=A+novel+form+of+gonadotropin-releasing+hormone+in+the+medaka,+Oryzias+latipes.">A novel form of gonadotropin-releasing hormone in the medaka, Oryzias latipes.</a> Biochem. Biophys. Res. Commun. 276 (1), 298-303 (2000).</li><li>Ramakrishnan, S., 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=Acquisition+of+spontaneous+electrical+activity+during+embryonic+development+of+gonadotropin-releasing+hormone-3+neurons+located+in+the+terminal+nerve+of+transgenic+zebrafish+(Danio+rerio).">Acquisition of spontaneous electrical activity during embryonic development of gonadotropin-releasing hormone-3 neurons located in the terminal nerve of transgenic zebrafish (Danio rerio).</a> Gen. Comp. Endocrinol. 168 (3), 401-407 (2010).</li><li>Abraham, E., 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=Targeted+gonadotropin-releasing+hormone-3+neuron+ablation+in+zebrafish:+effects+on+neurogenesis,+neuronal+migration,+and+reproduction.">Targeted gonadotropin-releasing hormone-3 neuron ablation in zebrafish: effects on neurogenesis, neuronal migration, and reproduction.</a> Endocrinology. 151 (1), 332-340 (2010).</li><li>Wayne, N. L., Kuwahara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-endorphin+alters+electrical+activity+of+gonadotropin+releasing+hormone+neurons+located+in+the+terminal+nerve+of+the+teleost+medaka+(Oryzias+latipes.">Beta-endorphin alters electrical activity of gonadotropin releasing hormone neurons located in the terminal nerve of the teleost medaka (Oryzias latipes.</a> Gen. Comp. Endocrinol. 150 (1), 41-47 (2007).</li><li>Oka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+types+of+gonadotrophin-releasing+hormone+neurones+and+steroid-sensitive+sexually+dimorphic+kisspeptin+neurones+in+teleosts.">Three types of gonadotrophin-releasing hormone neurones and steroid-sensitive sexually dimorphic kisspeptin neurones in teleosts.</a> J. Neuroendocrinol. 21 (4), 334-338 (2009).</li><li>Molleman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Patch Clamping: An Introductory Guide To Patch Clamp Electrophysiology. , (2003).</li></ol>

Os autores não têm nada a revelar.

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

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be ...

recording-electrical-activity-from-identified-neurons-intact-brain

Research

JoVE Journal

Neuroscience

13.4K visualizações.  University of California, Los Angeles. 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.

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

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

De gravação simultânea de sinais de cálcio dos neurônios identificados e comportamento alimentar de Drosophila melanogaster</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Neurociência

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

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

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

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

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 ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

Tensão sensível Gravação Dye de axônios, dendritos e espinhas dendríticas de neurônios individuais em fatias de cérebro

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

This article describes three nervous system preparations using leeches: intracellular recording from neurons in ventral ganglia, culturing neurons from ventral ganglia, and recording from a patch of innervated skin to map sensory fields.

Intracelular de gravação, Sensorial Campo Mapping, e cultivando neurônios identificados nos Sanguessuga, Hirudo medicinalis</i

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

Gravação eletrofisiológica no cérebro de Intacta Zebrafish Adulto

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

Um Guia para<em&gt; In vivo</em&gt; Gravação única unidade de Optogenetically Identificado Cortical Inibitória Interneurônios

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Perfurado gravação patch-clamp de rato Olfactory neurônios sensoriais na Intact neuroepitélio: Análise Funcional do Neurônios Expressando uma Odorant Receptor Identificados

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

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

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

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

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

Gravação induzida pela temperatura actividade neuronal através de monitoramento das alterações de cálcio no bulbo olfativo de<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. ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Imagem de atividade Neural no córtex somatossensorial primário usando Thy1-GCaMP6s de ratos transgênicos

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

Gravação eletrofisiológica sem fio dos neurônios por tetrodes móveis em peixes livremente da natação

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...