Financiado pelo NIH conceder R01HL105635 a SEJ. Os autores gostariam de agradecer a Diego V. Bohórquez para aconselhamento técnico. Agradecemos também R. Ian Cumming para a assistência técnica e realização da citometria de fluxo no Centro de Duke vacina humana Instituto de Pesquisa Citometria de Fluxo de recursos partilhados (Durham, NC). A citometria de fluxo foi realizada no Laboratório de biocontenção Regional na Duke que recebeu o apoio parcial para a construção dos Institutos Nacionais de Saúde, Instituto Nacional de Alergia e Doenças Infecciosas (UC6-AI058607).

Procedimentos envolvendo indivíduos animais foram aprovados pelo Comitê Institucional de Animal Care and Use (IACUC) da Universidade de Duke. 1. Administração Intranasal de Fast Blue Para Fast Blue, administrar o corante, pelo menos, 2 dias antes da eutanásia o mouse. O corante vai persistir por até oito semanas. <ol><li> Anestesiar o rato com anestesia inalatória leve (2,5% sevoflurano) até que começa a respiração lenta. </li><li> Usar uma pipeta de 200 uL com pontas filtrados para instilar-se lentamente 40 ml de solução de corante (0,4 mM Fast Blue, 1% de sulfóxido de dimetilo, solução salina de DMSO, em tampão fosfato, PBS) na narina, parando periodicamente para assegurar o rato é aspiração da solução (Figura 1A). </li><li> Segure o mouse vertical, cabeça para cima e massageie suavemente no peito para garantir o corante se espalha por todo o pulmão. </li></ol> 2. Preparações sobre Dissecção e Dia Análise <ol><li> Preparar 10 ml0; Ganglia dissociação Solução (GADs) por combinação dos ingredientes tal como especificado no Quadro 1. </li><li> Realizar a extracção de ARN em uma área de laboratório dedicado. Antes de começar a experiência, limpar as superfícies com 70% de etanol, 10% de água sanitária, e reagente de descontaminação RNase. Isto inclui a centrifugação de ARN, quaisquer suportes de tubos, e as pipetas. Garantir que não dedicados ponta caixas de filtro para uso apenas RNA. Não utilize este equipamento e área para preparação de ADN, como isso vai contaminar as amostras. </li><li> Mistura fresca de 70% e 80% de etanol com água livre de RNase. Prepare 1 ml para cada amostra a ser usado na extracção de ARN. </li></ol><table fo:keep-together.within-page="1"><tbody><tr><td> Ingrediente </td><td> Quantidade </td></tr><tr><td> Avançada DMEM / F12 </td><td> 9,5 ml </td></tr><tr><td> glutamina </td><td> 100 ul </td></tr><tr><td> HEPES (10 mM) </td><td> 100 ul </td></tr><tr><td> suplemento N2 </td><td> 100 ul </td></tr><tr><td> B27 (sem vitamina A) </td><td> 200 ul </td></tr><tr><td> NGF (50 ug / ml) </td><td> 10 ul </td></tr></tbody></table> Tabela 1. Mistura Reagente para Ganglia dissociação Solution (gads). 3. Dissecção Procedimento <ol><li> Encher tubos de 1,5 ml com 500 ul GADs, uma para cada amostra experimental, um como contraprova positiva de triagem, e uma para um controlo negativo de triagem. tubos de loja no gelo durante todo dissecção. </li><li> Dissecar o gânglio nodoso e fazer dois cortes parciais para facilitar a dissociação, em seguida, colocá-lo no tubo de ensaio experimental. Por favor, consulte o seguinte protocolo JoVE para dissecção dos gânglios nodosos 16 e DRG 17. </li><li> Dissecar e parcialmente em corte gânglios não marcado, tais como gânglio da raiz dorsal (DRG), para os controlos de triagem. Use dois gânglios para o controlo positivo e dois Ganglium para o controlo negativo. </li><li> Para obter neurónios nodosos suficientes para classificar bem sucedida, combinar gânglios a partir de 5 ratos em um tubo de amostra experimental contendo GADs. </li></ol> 4. Sensorial Ganglia dissociação <ol><li> Pipeta 500 GADs ul, deixando gânglios no tubo. Lave gânglios uma vez, adicionando 1 ml PBS, aguarde gânglios para assentar no fundo do tubo, então pipeta PBS. </li><li> Adicionar 1 ml GADs e 22 ul da enzima de digestão de colagenase (I / II e mistura de protease, 2,5 mg / ml em H2O) a cada tubo. </li><li> Adicionar 20 ul azuis Rápido (5,26 mm) para o tubo de controlo positivo. </li><li> Colocar os tubos num banho de água ou bloco de aquecimento já fixada em 37 ° C. Nota: O tempo de digestão gânglios depende da idade da enzima de digestão. No espaço de 1 mês de dissolução do enzima de digestão, digestão durante 30 min, a 2 - 6 meses digerir durante 45 min. Se a enzima é mais de 6 meses de idade, digerir gânglios fou 60 min. </li><li> A cada 15 minutos tubos de agitar brevemente e agite-los para assegurar gânglios são cobertos pela solução. </li><li> Enquanto estão a ser digerido gânglios, preparar gradientes de densidade, utilizando uma solução de partículas (partículas de sílica coloidal revestida com polivinilpirrolidona). <ol><li> Misturar a solução de partículas% 12 em GADs, 500 ul por amostra. </li><li> Misturar a solução de partículas% 28 em GADs, 500 ul por amostra. </li><li> Lentamente e cuidadosamente adicionar solução densidade de 400 ul de 12% na parte superior da solução densidade de 400 ul de 28% em um tubo de 1,5 ml fresco para cada amostra. Não permita que as duas camadas para misturar. Duas camadas distintas deve ser visível no tubo; uma mais escura que a outra. </li></ol></li><li> Enquanto gânglios estão sendo digerida, rotular novos tubos de 1,5 ml de coleta para ordenar (um tubo rápido Azul positivo e um rápido Azul negativo por amostra de classificação). Dependendo dos requisitos da instalação de triagem, estes tubos devem ter tampas amovíveis de modo a não interferirE com o classificador de células. </li><li> Após o tempo de digestão adequada, remova GADs com enzima de digestão e lavar gânglios duas vezes com 1 ml PBS. Adicionar 200 ul de GADs fresco a cada tubo. </li><li> Usar uma pipeta de 200 mL, definido como volume de 100 mL, e gânglios pipeta cima e para baixo várias vezes para separar as células. Repita até que nenhum pedaço intacto do tecido é visto. Evitar a criação de bolhas na solução. </li><li> Passa células dissociadas através de um filtro celular de 70 uM. </li><li> Adicionar mais 100 GADs ul ao tubo de dissociação original para pegar todas as células perdidas restantes esquerda e passar a solução adicional através do filtro de células. Utilizando uma nova ponta de pipeta, recolher qualquer líquido contendo células remanescente que passou através do filtro e agarrou-se à malha. O volume final de células suspensas em GADs é 300 ul. </li><li> camada cuidadosamente os 300 ul de células no topo do gradiente de densidade previamente feita. Evitar bolhas e mistura de cells com as camadas de densidade. </li><li> Centrifugar durante 10 minutos a 2900 xg à temperatura ambiente. </li><li> Enquanto as células são na centrífuga, mistura de tampão de lise (1 ml por amostra experimental) como se segue: 10 ul de 2-mercaptoetanol em 1 ml de tampão de RLT (estojo de extracção de ARN). <ol><li> Adicionar 300 mL de tampão de lise de tubo de coleta positiva Azul Rápido e 600 mL ao tubo rápido Azul negativo de coleta (tubos rotulados a partir do passo 4.7). Esse volume depende do número de células que se espera serem recolhidos. Para &lt;2000 células, adicionar 300 ul, durante&gt; 7.000 células adicionar 600 ul. Isto irá assegurar que o fluido de revestimento de classificação não dilui significativamente o tampão de lise. </li></ol></li><li> Uma vez que a centrifugação estiver concluída, remova cuidadosamente e elimine a camada ul top 700. Esta camada contém a maioria dos resíduos celulares que de outra forma interfira com a separação de células. </li><li> Adicionar 700 ul GADs frescos para a célula restante contendo solução e pipetate para cima e para baixo várias vezes para misturar. </li><li> Centrifugar durante 15 minutos a 2900 xg para sedimentar as células. </li><li> Enquanto as células são na centrífuga, misturar triagem GADs (500 ul por amostra) pela adição de 5 ul de ADNase (10 mg / ml) até 1 ml GADs. </li><li> Depois de centrifugação é feito, cuidadosamente remover e descartar o sobrenadante, deixando o sedimento celular. </li><li> Re-suspender pellet celular em 200 - 300 ml de classificação GADs pipetando cima e para baixo com uma pipeta de 1000 mL ajustado para 200 mL várias vezes. Coloque as amostras em gelo. As células são agora pronto para ser classificado. </li></ol> 5. fluorescência celular ativado (FAC) Classificando Nota: Esta seção requer conhecimento operacional de um classificador FACS ou a assistência de pessoal qualificado. <ol><li> Adicionar uma solução de estoque ul iodeto de propídio (PI) (50 ug / ml) para cada amostra a testar para a viabilidade celular. Divisão de controle negativo em 2 tubos, um com PI e um sem. PI se liga ao ADN apenas em células mortas. E seapós os primeiros tipos não há células mortas, para rotular, não é necessário o uso de PI. </li><li> Células de transporte para citometria de fluxo instrumento, por exemplo., BD FACS AriaII executando o software Diva 8, em gelo. </li><li> Uma vez que o tamanho da célula é variável nesta população, usar um grande bocal (100 uM) para a classificação. Para diminuir a quantidade de tensão da experiência as células e aumentar a viabilidade celular utilizar baixa pressão (20 psi). </li><li> Use o software classificador FACS para configurar as parcelas de análise, de dispersão para a frente, dispersão lateral (SSC), Blue rápido e PI, se necessário. </li><li> Para as células re-suspender, gentilmente vortex brevemente a amostra antes de o colocar na classificador. </li><li> Comece com o controlo negativo, nenhum Fast Blue ou IP, para definir o limite de portão (Figura 2A). Isto vai determinar a quantidade de autofluorescência na população de células. Opcional: Executar a amostra negativa com PI para identificar se existe uma população de células mortas. </li><li> Classificar a amostra de controlo positivo (incubated com Fast Blue), para definir os parâmetros de compensação. </li><li> Uma vez que os portões e parâmetros são definidos, começam a triagem das amostras experimentais. As amostras são classificadas dentro dos tubos de coleta de preparados contendo tampão RNA lise. </li><li> Manter as amostras classificadas em gelo até que a separação de células é completa. </li><li> Comece a etapa de extração de RNA imediatamente após as células são classificadas. </li></ol> 6. Extracção de ARN <ol><li> Vortex células classificadas em tampão de lise durante 1 min para homogeneizar. </li><li> Adicionar 1 volume de etanol a 70% e misture pipetando cima e para baixo várias vezes. </li><li> Transferir para 700 ul da amostra a uma coluna de centrifugação. Centrifugar durante 15 segundos a 8000 x g. Descartar o fluxo de passagem. <ol><li> Repetir até que o volume total da amostra é passada através da coluna de centrifugação. </li></ol></li><li> Adicionar 350 ul de tampão de RW1 e continuar o processo de purificação de ARN, incluindo o passo de tratamento com ADNase, de acordo com Protoco do fabricantel para as células. </li><li> Uma vez que o RNA foi extraído, testar a qualidade de ARN utilizando um sistema de electroforese de microfluidos de acordo com as especificações do fabricante. </li></ol>

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Este protocolo descreve um método para alvejar neurónios das vias aéreas-inervação nos gânglios nodosos do nervo vago. Uma vez marcado, os gânglios são suavemente dissociados para otimizar a preservar o número de células e viabilidade. Estes neurónios são, então, classificados FAC directamente em tampão de lise e o ARN é extraído. A importância deste protocolo é a capacidade de segmentar, isolar e preservar a qualidade de uma população de células sensorial específica. A expressão de genes é descr...

nervos Somatosensoriais transduzir estímulos nocivos causados ​​por ambos os agentes endógenos e ambientais térmica, mecânica, química, e. Os corpos celulares destes nervos aferentes estão localizados em gânglios sensoriais, tais como a raiz dorsal, trigeminal, ou gânglios nodosos. Cada gânglio sensorial inerva regiões específicas do corpo e contém células que inervam órgãos e tecidos separadas dentro dessa região. Por exemplo, os gânglios da raiz dorsal (DRG) são localizados na coluna vertebral e extensão dos processos ao longo do corpo e membros, enquanto que o gânglio trigeminal estão localizados no crânio, contendo neurónios que inervam a face, olhos, meninges ou das vias aéreas superiores 1, 2. A gânglios nodosos do nervo vago está localizado no gargalo abaixo do crânio e contém corpos celulares que se estendem fibras nervosas ao longo do tracto gastrointestinal, no coração e pulmões e vias aéreas inferiores 3. Nos seres humanos o gânglio nodoso está sozinho, no entanto, no ratinho é fundidacom o gânglio jugular, que também inerva os pulmões 4. Este gânglio fundido é muitas vezes chamado a jugular / nodose complexa, gânglio vagal, ou simplesmente nodose gânglio 5. Aqui, é referido como o gânglio nodoso. fibras aferentes do nodosos passar informações de vísceras para o núcleo do tracto solitário (NTS) no tronco cerebral. Entrada sensorial para esta gânglio único controla um conjunto diversificado de funções, tais como a motilidade intestinal 6, 7 da frequência cardíaca, a respiração 8,9, e as respostas respiratórias activado-irritantes 10,11. Com esta diversidade de funções e órgãos inervados, é crítico para alvejar e isolar subpopulações específicas de órgãos do gânglio nodoso, a fim de estudar vias neuronais individuais. No entanto, dado o pequeno tamanho do nodosos e ao limitado número de neurónios que contém esta não é uma tarefa trivial. Cada rato nodose gânglio contém cerca de 5.000 neurônios 12em adição a uma grande população de células de suporte satélite. Dos 5.000 neurónios nodosos, apenas 3 - 5% inervam as vias aéreas. Portanto, quaisquer alterações funcionais, morfológicos ou moleculares dentro dos neurônios das vias aéreas-inervação, devido a estimulação respiratória ou patologias, serão perdidos no gânglio nodoso densamente. Para resolver este problema, foi desenvolvido um método para identificar e isolar neurónios que inervam as vias aéreas. As vias aéreas foram expostos a um corante marcador fluorescente para identificar os neurónios nodosos inervação subsequentes. Azul rápida foi apanhada por neurônios e viaja rapidamente para seus corpos celulares, onde é retido por até oito semanas 13 - 15. Uma vez identificados, um protocolo de dissociação suave, mas eficiente foi usado para preservar rotulagem corante e viabilidade celular para celular ativado fluorescente (FAC) de classificação. As células seleccionadas são usadas para extrair o ácido ribonucleico de alta qualidade (ARN) para determinar a expressão do gene ou fou outra análise molecular a jusante. Este protocolo proporciona uma técnica útil e robusto para isolar neurónios sensoriais que inervam um tecido de interesse.

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			<td height="38" style="height:39px;width:405px;">Fast Blue</td>
			<td style="width:157px;">Polysciences, Inc.</td>
			<td style="width:132px;">17740-2</td>
			<td style="width:248px;">stock 2 mg/ml in water</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">NeuroTrace 530/615 red Nissle stain</td>
			<td>Life Technologies</td>
			<td>N21482</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Dimethyl Sulfoxide (DMSO)</td>
			<td>Fisher Scientific</td>
			<td>D128-500</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:405px;">Dulbecco&#39;s Phosphate Buffered Saline (PBS) Ca and Mg free</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Advanced DMEM/F12</td>
			<td>Gibco</td>
			<td>12634-010</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">glutamine (Glutamax)</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">HEPES</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">N2</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">B27 (no vitamin A)</td>
			<td>Gibco</td>
			<td>12587-010</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Nerve Growth Factor (NGF)</td>
			<td>Sigma</td>
			<td>N6009</td>
			<td>stock 50 &#181;g/ml in PBS/10% FBS</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">digestion enzyme, Liberase DH Research Grade</td>
			<td>Roche</td>
			<td>5401054001</td>
			<td>stock 2.5 mg/ml in water</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">particle solution (Percoll)</td>
			<td>Sigma</td>
			<td>P1644-25ML</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Heating block</td>
			<td>LabNet</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">70 um cell strainer</td>
			<td>Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Absolute Ethanol (200 proof)</td>
			<td>Fisher Scientific</td>
			<td>BP2818-500</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">RNase free water</td>
			<td>Fisher Scientific</td>
			<td>BP2484-100</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">RNase decontamination reagent, RNase AWAY</td>
			<td>invitrogen</td>
			<td>10328-011</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">2-mercaptoethanol</td>
			<td>VWR</td>
			<td>EM-6010</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">RNA extraction kit, RNeasy Plus Micro Kit</td>
			<td>Qiagen</td>
			<td>74034</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">DNase kit, RNase-Free DNase Set</td>
			<td>Qiagen</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">DNase</td>
			<td>Sigma</td>
			<td>D5025-15KU</td>
			<td>stock 10 mg/ml in 0.15 M NaCl</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Propidium Iodide</td>
			<td>Sigma</td>
			<td>P4170-10MG</td>
			<td>stock 10 &#181;g/ml in PBS</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Microfluidic electrophoresis system (TapeStation 2200)</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a method to target and isolate airway-innervating sensory neurons in mice

nervos Somatosensoriais transduzir estímulos nocivos causados ​​por ambos os agentes endógenos e ambientais térmica, mecânica, química, e. Os corpos celulares destes neurónios aferentes estão localizados dentro do gânglio sensorial. gânglios sensoriais que inervam um órgão específico ou porção do corpo. Por exemplo, o gânglio da raiz dorsal (DRG) são localizados na coluna vertebral e extensão dos processos ao longo do corpo e membros. O gânglio trigeminal estão localizados no crânio e inervam a face, e das vias aéreas superiores. aferentes vagais dos gânglios nodosos se estender por todo o intestino, coração e pulmões. Os neurónios nodosos controlar um conjunto diversificado de funções, tais como: freqüência respiratória, irritação das vias aéreas, e reflexo de tosse. Assim, entender e manipular a sua função, é crítica para identificar e isolar sub-populações neuronais específicas das vias aéreas. No rato, as vias aéreas estão expostas a um corante marcador fluorescente, azul rápido, para rastreamento retrógrado de neurônio nodose específica das vias aéreass. Os gânglios nodosos são dissociados e de células activadas por fluorescência (FAC) de classificação é usado para coletar células positivas de corante. Em seguida, o ácido ribonucleico, de alta qualidade (ARN) é extraído a partir de células positivas de corante para a próxima geração de sequenciação. Usando este método de expressão do gene de vias aéreas neuronal específica é determinada.

Usando este método, os neurónios da via aérea-inervação são rotulados por via intranasal incutir Fast Blue (Figura 1A). Após dois dias, as células marcadas azul depressa aparecem nos gânglios nodosos (Figura 1C). Essas células formam 3-5% da população neuronal total do gânglios nodosos. Outros corantes retrógrada, que têm sido utilizados para este fim incluem DiI (1,1&#39;-dioctadecil-3,3,3 &#39;, 3&#39;-perclorato tetramethylindocarbocyan...

Watch this Scientific Journal Video about A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice at JoVE.com

A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice

Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined.

Um método para alvejar e Isolar Airway-inervação neurônios sensoriais em Ratos

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of ...

a-method-to-target-isolate-airway-innervating-sensory-neurons

Research

JoVE Journal

Neuroscience

13.1K visualizações.  Yale University. Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined.

Vídeo: A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (&alpha;, &beta;I, &beta;II and &gamma;), novel type I PKCs (&#949; and &eta;), novel type II PKCs (&delta; and &theta;), and atypical PKCs (&zeta; and &iota;). The classical PKCs are activated by Ca2+ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca2+-independent. The atypical PKCs are activated by neither Ca2+ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation 1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail 3. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article 4, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

Live-imagem de translocação da PKC em células Sf9 e em neurônios sensoriais Aplysia

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

Neurociência

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

Lentivírus mediada Manipulação Genética e Visualização de Olfactory neurônios sensoriais In vivo</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

Flash Fotólise de Compostos Caged no Cilia de Olfactory neurônios sensoriais

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

Isolamento de neurônios sensoriais de<em&gt; Aplysia californica</em&gt; Para Remendo Recordings Grampo de Correntes glutamatérgicos

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

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 ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Monte todo imunomarcação de Neurônios Receptores Olfatórios no<em&gt; Drosophila</em&gt; Antena

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

Produção e Isolamento de axônios de neurônios sensoriais para análise bioquímica Usando filtros porosos

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

Isolamento e Cultura de Dissociated neurônios sensoriais de Chick Embriões

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Um método simples e barato para a determinação da sensibilidade Fria e Adaptação no Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Dynamic Quantitative Sensory Testing to Characterize Central Pain Processing

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain processing is present in many chronic pain conditions and can influence responses to specific therapies. Thus, the ability to precisely describe the state of central pain processing has profound clinical significance in both prognosis and prediction. Because it is not practical to record neuronal firings directly in the human spinal cord, surrogate behavior tests become an important tool to assess the state of central pain processing. Dynamic QST is one such test, and can probe both the ascending facilitation and descending modulation of incoming nociceptive signals via TS and CPM, respectively. Due to the large between-individual variability in the sensitivity to noxious signals, standardized TS and CPM tests may not yield any meaningful data in up to 50% of the population due to floor or ceiling effects. We present methodologies to individualize TS and CPM so we can capture these measures in a broader range of individuals than previously possible. We have used these methods successfully in several studies at the lab, and data from one ongoing study will be presented to demonstrate feasibility and potential applications of the methods.

By assessing pain in response to repetitive or different types of standardized stimuli, dynamic quantitative sensory testing (QST) can reveal changes in the central processing of pain. We present methods to optimize and individualize two dynamic QST measures: temporal summation (TS) and conditioned pain modulation (CPM).