Agradecemos a Homaira Nawabi, Frederic Moret e Isabelle Sanyas pela ajuda. Este trabalho é apoiado pela CNRS, Association Francaise contre les Myopathies (AFM), ANR YADDLE, Labex DevWeCan, Labex Cortex, ERC YODA to V.C.; C.D-B e A.J são apoiados por uma bolsa la Ligue contre le cancer e Labex DevWeCan, respectivamente.

1. Estratégia de clonagem para marcar receptor plexina1 com pHluorin
<ol><li>Escolha um vetor de expressão apropriado como espinha dorsal (por exemplo, o receptor do camundongo plexinA1 expressando vetor, um presente gentil do Dr. Andreas Puschel11). Nota: Este vetor plexinA1 foi projetado para alcançar uma inserção eficiente do receptor com marca HA ou VSV na membrana plasmática.</li>
	<li>Amplie por PCR a sequência de codificação de pHluorin eclíptica usando o plasmídeo adequado como modelo (por exemplo, receptor GABA A marcado por pHluorin, um presente gentil do Dr. Jacob2). Se necessário, adicione um site de restrição ao final de 5' do primer, a fim de facilitar a etapa de clonagem na espinha dorsal.</li>
	<li>Insira a sequência de pHluorin no quadro entre o peptídeo de sinal e a sequência de codificação do receptor usando locais de restrição (por exemplo, locais de restrição NruI/Kpn2I conforme descrito na Figura 1B). Nota: Como o peptídeo de sinal que garante a mira correta dos receptores é cortado, a pHluorin deve ser colocada depois dele. Isso garante o reconhecimento do peptídeo de sinal e impede o decote da pHLuorin do receptor de interesse.</li>
	<li>Sequencia os construtos obtidos para garantir que nenhuma mutação foi introduzida pelo PCR.</li>
</ol>2. Caracterização do receptor com pHLuorin in vitro em células COS7
A capacidade da proteína de fusão de atingir a membrana plasmática e sua perda reversível de fluorescência à medida que o pH é reduzido pode ser confirmada usando o procedimento a seguir.
<ol><li>Dia 1º. Placa 1,5 x 105 células COS7 em um prato de 35 mm em 2 ml de completo Dulbecco's Modified Eagle Medium (DMEM - 10% Soro Bovino Fetal - 1 mM piruvato de sódio - 25 U/ml penicilina/stretomicina - 2,5 μg/ml Anfotericina B - pH 7.4).</li>
	<li>Dia 2. Células transfectas: Nota: As células devem ser 70-80% confluentes.<ol>
<li>Prepare 200 μl NaCl 150 mM e adicione 3 μg de DNA, ou seja, o receptor de codificação vetorial com pHluorin. Suavemente vórtice e gire brevemente. Nota: Um mapa do vetor pHluorin-plexinA1 usado nesses experimentos é mostrado na Figura 1B.</li>
		<li>Adicione 10 μl de reagente de transfecção (ou a quantidade apropriada do reagente utilizado). Vórtice imediatamente.</li>
		<li>Incubar 10 min na RT.</li>
		<li>Adicione 200 μl da mistura de reagente/DNA de transfecção às células.</li>
		<li>Mova a placa suavemente para obter a repartição da mistura e coloque as células de volta à incubadora de 37 °C.</li>
	</ol>
</li>
	<li>Dia 3. Remova o meio de transfecção e substitua-o por 2 ml de DMEM completo fresco.</li>
	<li>Dia 4. Realizar imagens de células vivas de células transfetugas COS7 pHluorin-plexinA1:<ol>
<li>Prepare duas alíquotas de média completa DMEM e ajuste o pH para 3,5 e 9,5, respectivamente. Nota: Para uma placa de 35 mm, 1,5 ml de cada solução é necessária para realizar o experimento.</li>
		<li>Remova o meio celular e substitua-o por 1 ml de PH médio completo DMEM 7.4.</li>
		<li>Prepare uma seringa de 5 ml com um tipo apropriado de tubulação, a fim de injetar vários componentes diretamente no meio de cultura celular sem abrir a câmara de microscópio.</li>
		<li>Use um módulo que permita a manutenção de uma atmosfera de trabalho úmida de 37 °C e 5% de CO2. Nota: Uma abordagem alternativa ao uso de uma câmara de CO2 é usar meio tamponado hepes (geralmente na faixa de 10-25 mM de acordo com o tipo de célula).</li>
		<li>Coloque as células na câmara e ajuste a tubulação e a seringa. Nota: O microscópio deve ser equilibrado antes de começar a evitar a deriva mecânica durante a gravação.</li>
		<li>Abra o software de imagem e selecione o programa de aquisição multidimensional.</li>
		<li>Encontre células COS7 transfectadas com o objetivo de 40X e marque posição no software para cada uma delas.</li>
		<li>Configure a pilha Z para ter uma aquisição de profundidade de 15 μm (o foco pode mudar ao adicionar mídia na placa).</li>
		<li>Configure a exposição para o filtro GFP e a Fase.</li>
		<li>Configure o tempo de aquisição. Nota: Para todo o experimento com 5 campos de interesse, a aquisição a cada 20 segundos por 10 min deve ser suficiente.</li>
		<li>Inicie a aquisição e tire 5 imagens de controle no meio DMEM pH 7.4.</li>
		<li>Pausar a aquisição, injetar 1,25 ml de pH 3.5 DMEM completo para alcançar um pH de 5,5 no meio de cultura, marcar evento no software e retomar a aquisição para mais 5 pontos de tempo. Nota: A fluorescência verde deve desaparecer progressivamente.</li>
		<li>Pausar a aquisição, injetar 1,2 ml de pH 9.5 DMEM completo para alcançar um pH 7.4 no meio de cultura, marcar evento no software e retomar a aquisição para mais 5 pontos de tempo. Nota: A fluorescência verde deve reaparecer na membrana plasmática.</li>
		<li>Analise imagens. A Figura 2 mostra imagens representativas obtidas com tal protocolo com a construção pHluorin-plexinA1.
</li>
	</ol>
</li>
</ol>3. Em ovo Eletroporação de pHluorin-plexinA1 Construto
<ol><li>Manuseio de ovos antes da eletroporação:<ol>
<li>Armazene ovos fertilizados em uma geladeira a 14 °C até uma semana antes da incubação.</li>
		<li>Incubar ovos a 38,5 °C (101.3 °F) em uma incubadora com umidade saturada por 50-52 horas até que os embriões cheguem ao estágio HH1412. Nota: Os ovos devem ser colocados horizontalmente durante a incubação para que o embrião esteja devidamente posicionado para eletroporação, flutuando na parte superior da gema. O estágio HH14 é adequado para obter expressão dos plasmídeos em neurônios diferenciados na medula espinhal e no gânglio raiz dorsal com uma taxa de sobrevivência adequada.</li>
	</ol>
</li>
	<li>Embriões eletroporados8-10:<ol>
<li>Prepare a eletroporação:<ol>
<li>Prepare plasmídeos de DNA livres de endotoxinas com uma concentração superior a 2 μg/μl, para poder diluí-lo como a concentração desejada.</li>
			<li>Puxe capilares de vidro suficientes para injetar as diferentes soluções de DNA.</li>
			<li>Prepare PBS estéril (-Ca2+; -Mg2+) - 100 U/ml penicilina/estreptomicina e equilibre a 38,5 °C.</li>
			<li>Esterilize o capô, tesoura curva e fórceps finos.</li>
			<li>Controle o espaçamento do eletrodo. Nota: Um espaço de 4 mm entre os eletrodos é geralmente utilizado.</li>
		</ol>
</li>
		<li>Janela do ovo13 (Figura 3A):<ol>
<li>Use uma tesoura curva para perfurar a casca no lado contundente do ovo.</li>
			<li>Remova 2 ml de albumen usando uma agulha de 0,9 mm x 25 mm e uma seringa de 5 ml. Oriente a agulha verticalmente, a fim de evitar danificar o saco de gema.</li>
			<li>Cubra a parte superior do ovo com fita adesiva para manter a integridade da casca.</li>
			<li>Usando uma tesoura curva, fure a casca no meio da fita para equalizar a pressão ao remover 2 ml de albumen do ovo. Em seguida, corte uma janela grande o suficiente para visualizar o embrião e ser capaz de trabalhar nele.</li>
			<li>Adicione ~2 ml estéril quente PBS (-Ca2+; -Mg2+) - 100 U/ml penicilina/estreptomicina para evitar a desidratação do embrião e torná-lo mais acessível ao manipulador.</li>
		</ol>
</li>
		<li>Injete DNA e eletroporar o embrião<ol>
<li>Diluir plasmídeo em PBS (-Ca2+; -Mg2+) a uma concentração entre 0,5-2 μg/μl e adicionar corante verde rápido para atingir uma concentração final de 0,025%. Carregue a mistura de DNA em um capilar. Recomenda-se o uso de um injetor. Nota: Verifique se a resistência capilar não é muito grande (o que significa que pode haver dificuldades ao injetar embriões) nem muito pequena (o que significa que o capilar pode ser muito grande e pode danificar o embrião). Além disso, a concentração de ácidos nucleicos superiores a 2 μg/μl pode causar efeitos inespecíficos e precisa ser controlada.</li>
			<li>Puna o saco de gema e o tubo neural no lado caudal com o capilar carregado. Digite o tubo neural com um ângulo raso e encha o lúmen da cauda até a cabeça com a mistura de DNA(Figura 3B). Nota: O verde rápido permite controlar a precisão da injeção.</li>
			<li>Coloque rapidamente os eletrodos de platina de 4 mm em ambos os lados do tubo neural no nível que deseja eletroporar e aplique 3 pulsos a 31 V para 50 msec com intervalos de 500 msec(Figura 3C). Nota: Evite colocar os eletrodos no coração ou em vasos grandes e extraembrionários para evitar danificar o embrião em desenvolvimento. Bolhas devem se formar nos eletrodos.</li>
			<li>Com a agulha, remova 2 ml de albumen para diminuir o nível na casca.</li>
			<li>Sele a janela e o lado contundente hermeticamente com fita.</li>
			<li>Coloque os ovos de volta a 38,5 °C na incubadora até chegarem ao estágio desejado.</li>
		</ol>
</li>
	</ol>
</li>
</ol>4. Embriões Incorporando e Cryosectioning
<ol><li>48 horas após a eletroporação, colhe cuidadosamente embriões eletroporados (estágio HH24). Corte a fita e metade da membrana corioallantóide. Para evitar que os embriões afundem na gema, posicione um coador sob o embrião e corte a segunda metade da membrana corioallantóide.</li>
	<li>Transfira o embrião para um prato de dissecção cheio de PBS gelado.</li>
	<li>Verifique a eficiência da eletroporação procurando fluorescência no tubo neural com um microscópio estéreo de fluorescência. Nota : A coeletrificação de uma codificação plasmida de controle do RFP pode ajudar a visualizar a área eletroporada.</li>
	<li>Dissecar embriões usando uma microscalpela a fim de selecionar a área eletroporada da medula espinhal.</li>
	<li>Transfira embriões dissecados para uma placa de 24 poços e fixe em pH 7,4 4% Paraformaldeído (PFA) - Salina tampão fosfato (PBS), O/N a 4 °C. Nota: A etapa de fixação é crucial para permitir a estabilização da pHLuorina em sua conformação "viva" e, assim, ser capaz de usar pHluorin em tecido fixo/permeabilized. Embora a fixação desacelere drasticamente a mudança dependente do pH da fluorescência, deve-se levar em conta que mudanças conformais/de protonação ainda podem ocorrer após a fixação. Assim, o seguinte protocolo (incorporação, criosecções e observação) deve ser realizado no prazo de 3 dias após a etapa de fixação, com todos os buffers em pH 7. Se a fixação não for necessária, recomenda-se a realização de observação em seções de tecido vivo.</li>
	<li>Remova 4% de PFA e lave embriões em pH 7,4 PBS.</li>
	<li>Incubar embriões em PBS-15% de sacarose e manter a 4 °C até que os embriões afundem.</li>
	<li>Incubar embriões fixos em pH 7,4 7,5% de gelatina- 15% de sacarose por 45 min a 37 °C para que os embriões estejam completamente incorporados.</li>
	<li>Coloque moldes de incorporação no gelo e adicione 400 μl de pH 7,4 7,5% de gelatina- 15% de sacarose para alcançar uma base sólida de 2 mm.</li>
	<li>Aspire o embrião embutido com uma ponta de corte e coloque o embrião na base de gelatina sólida.</li>
	<li>Cubra com pH 7,4 7,5% de gelatina- 15% de sacarose e posicione o embrião com fórceps antes que a gelatina se solidifique.</li>
	<li>Uma vez que a gelatina esteja sólida, prepare um banho isopropanol de -40 °C (use gelo seco ou nitrogênio líquido) e congele o bloco de gelatina por 5 minutos.</li>
	<li>Mantenha os blocos congelados a -80 °C.</li>
	<li>Coloque o bloco congelado a -20 °C por 1 hora.</li>
	<li>Remova o molde e fixe o bloco em um mandril com um meio de polietileno glicol.</li>
	<li>Uma vez que o bloco esteja bem fixo, coloque o mandril no sistema criostat. Nota: Use lâminas revestidas para evitar a perda de tecido durante a coloração.</li>
	<li>Realize criosections seriais (20 μm criosections são geralmente realizadas).</li>
	<li>Deixe as criosecções secarem por 15 minutos no RT. Nota: as crioseções devem ser protegidas contra exposição desnecessária à luz, a fim de evitar o branqueamento da fluorescência GFP.</li>
</ol>5. Análise microscópica das criogenias
<ol><li>Reidratar crioseções em pH 7.4 PBS em RT por 10 min. Nota: Se necessário, os núcleos podem ser manchados com Hoechst.</li>
	<li>Use uma solução Hoechst de 0,5 μg/ml em PBS e incubar criosections por 15 minutos.</li>
	<li>Enxágüe os slides 3x com pH 7,4 PBS por 5 min.</li>
	<li>Prossiga para a montagem de slides. Uma solução de montagem de álcool polivinyl pH 7.4 (ou mais básica) que endurece O/N pode ser usada: posicione cuidadosamente o deslizamento para evitar a formação de bolhas de ar entre o slide e o deslizamento de tampas.</li>
	<li>Deixe o meio de montagem endurecer O/N a 4 °C no escuro.</li>
	<li>Use um microscópio confocal invertido para visualizar com precisão a proteína de fusão pHluorin-plexinA1: realize z-stack no orifício ideal e resolução óptica e use uma lente de 20X (NA 0,75) ou 40X (NA 1.3). Nota: a pHluorina é detectável com os mesmos parâmetros utilizados para detectar GFP (ou seja, pico de emissões a 509 nm). As configurações do filtro de excitação e detecção do comprimento de onda são definidas de forma ideal pelo software de imagem. Hoechst é detectado entre 425-460 nm (excitação é de 405 nm), GFP ou pHluorin é detectado entre 485-545 nm (excitação é de 473 nm) e RFP é detectado entre 575-675 nm (excitação é de 559 nm).</li>
</ol>Imagens representativas da expressão pHluorin-plexinA1 e eGFP na medula espinhal do embrião do filhote são mostradas na Figura 4.

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+measurements+of+vesicle-SNARE+recycling+in+synapses+of+the+central+nervous+system.">Real-time measurements of vesicle-SNARE recycling in synapses of the central nervous system.</a> Nat. Cell. Biol. 2, 197-204 (2000).</li><li>Stark, D. A., Kasemeier-Kulesa, J. C., Kulesa, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivation+cell+labeling+for+cell+tracing+in+avian+development.">Photoactivation cell labeling for cell tracing in avian development.</a> CSH Protoc.. 2008, (2008).</li><li>Hildick, K. L., Gonzalez-Gonzalez, I. M., Jaskolski, F., Henley, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+diffusion+and+exocytosis+of+membrane+proteins+in+cultured+neurons+assessed+using+fluorescence+recovery+and+fluorescence-loss+photobleaching.">Lateral diffusion and exocytosis of membrane proteins in cultured neurons assessed using fluorescence recovery and fluorescence-loss photobleaching.</a> J. Vis. Exp. (60), (2012).</li><li>Hanson, G. T., 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=Green+fluorescent+protein+variants+as+ratiometric+dual+emission+pH+sensors.+1.+Structural+characterization+and+preliminary+application.">Green fluorescent protein variants as ratiometric dual emission pH sensors. 1. Structural characterization and preliminary application.</a> Biochemistry. 41, 15477-15488 (2002).</li><li>Rose, T., Schoenenberger, P., Jezek, K., Oertner, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+refinement+of+vesicle+cycling+at+schaffer+collateral+synapses.">Developmental refinement of vesicle cycling at schaffer collateral synapses.</a> Neuron. 77, 1109-1121 (2013).</li><li>Li, Y., Tsien, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pHTomato,+a+red,+genetically+encoded+indicator+that+enables+multiplex+interrogation+of+synaptic+activity.">pHTomato, a red, genetically encoded indicator that enables multiplex interrogation of synaptic activity.</a> Nat. Neurosci. 15, 1047-1053 (2012).</li><li>de Wit, J., Toonen, R. 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Os autores não têm nada a revelar.

Este protocolo fornece um procedimento passo-a-passo para acompanhar a dinâmica de um receptor de orientação de axônio tanto na cultura celular quanto no contexto de desenvolvimento da medula espinhal do embrião de filhotes.
Para projetar uma proteína marcada por novo pHluorin, dois pontos precisam ser considerados em relação à estratégia de clonagem. Primeiro, a etiqueta de pHluorin deve ser exposta ao lúmen dos endosários ácidos e, consequentemente, ao compartimento ext...

Durante sua navegação, os axônios integram várias pistas ambientais que os guiam em direção ao seu alvo. Essas pistas ativam receptores de orientação na superfície dos terminais de axônio, os cones de crescimento, que por sua vez iniciam uma via de sinalização apropriada. Assim, a regulação temporal e espacial da distribuição da superfície celular dos receptores é fundamental para definir a sensibilidade do cone de crescimento1. Neste contexto, a travessia midline por axônios comissural é um excelente modelo para investigar a regulação dos níveis de superfície das células receptoras. Na medula espinhal em desenvolvimento, os axônios comissural são inicialmente atraídos para a placa do piso ventral onde cruzam a linha média. Após a travessia, eles perdem sua capacidade de resposta aos atrativos da placa de piso e ganham resposta aos repelentes da placa de piso para que possam sair da placa do chão e navegar em direção ao seu destino final no lado contralateral do sistema nervoso2,3. A regulação da disponibilidade do receptor na superfície do cone de crescimento é um dos mecanismos subjacentes à mudança de responsividade para as pistas de linha média4,5. Assim, o monitoramento seletivo dos receptores presentes na membrana plasmática dos cones de crescimento é de grande importância. Descrevemos aqui um método baseado na propriedade de fluorescência dependente do pH de uma variante de proteína fluorescente verde (GFP) para visualizar especificamente os receptores de orientação do axônio que são abordados à membrana plasmática in vitro e in vivo,na medula espinhal do pintinho em desenvolvimento.
Rothman e colegas projetados por mutações pontuais de variantes sensíveis ao PH do GFP, incluindo o pHluorin eclíptico6. A pHLuorina eclíptica tem a propriedade de não ser fluorescente quando exposta ao pH ácido (<6), enquanto é fluorescente em pH neutro. Isso permite distinguir receptores não fluorescentes localizados em compartimentos ácidos intracelulares (ou seja, endosários, vesículas de tráfico) de receptores fluorescentes incorporados à membrana plasmática e, assim, expostos ao pH neutro extracelular7. Aproveitamos isso para monitorar a localização da membrana plasmática do plexinA1, um receptor de orientação de axônio mediando a resposta do cone de crescimento à semaforina repelente midline 3B5 (Figura 1A). Descrevemos aqui a caracterização in vitro de uma construção pHluorin-plexinA1, juntamente com a eletroporação ovo8-10 deste construto na medula espinhal do filhote em desenvolvimento seguido pela análise microscópica de criosections que permitem acompanhar a dinâmica do receptor de orientação de axônio in vivo com resoluções espaciais e temporais.

<table border="0" cellpadding="0" cellspacing="0" width="1007">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">COS7 cells</td>
			<td style="width:147px;">ATCC</td>
			<td style="width:219px;">CRL-1651</td>
			<td style="width:379px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">DMEM GlutaMAX</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">61965-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Sodium pyruvate</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">11360-039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Amphotericin B</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:219px;">A2942</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Fetal bovine serum</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">10270-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Penicillin/Streptomycin</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:264px;">Exgen500 reagent</td>
			<td style="width:147px;">Euromedex Fermentas</td>
			<td style="width:219px;">ET0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">PBS -Ca2+ -Mg2+</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Fast green dye</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:219px;">F7252</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:264px;">32% Paraformaldehyde aqueous solution</td>
			<td style="width:147px;">Electron Microscopy</td>
			<td style="width:219px;">15714-S</td>
			<td>Dilute extemporaneously in PBS to achieve a 4% solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Gelatin from cold water fish skin</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:219px;">G7041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Sucrose</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:219px;">S0389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Cryomount</td>
			<td style="width:147px;">Histolab</td>
			<td style="width:219px;">00890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Hoechst 34580</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:219px;">H21486</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Mowiol 4-88</td>
			<td style="width:147px;">Fluka</td>
			<td style="width:219px;">81381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1008px;">Consumables</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Bottom-glass 35 mm dish</td>
			<td style="width:147px;">MatTek</td>
			<td style="width:219px;">P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">5 ml Syringe</td>
			<td style="width:147px;">Terumo</td>
			<td style="width:219px;">SS-05S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Needles 0.9 mm x 25 mm</td>
			<td style="width:147px;">Terumo</td>
			<td style="width:219px;">NN-2025R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Capillaries</td>
			<td style="width:147px;">CML</td>
			<td style="width:219px;">PP230PO</td>
			<td>capillaries are stretched manually in the flame</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Superfrost Plus Slides</td>
			<td style="width:147px;">Thermo Scientific</td>
			<td>4951PLUS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1008px;">Material</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Curved scissors</td>
			<td style="width:147px;">FST</td>
			<td style="width:219px;">129-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Microscalpel</td>
			<td style="width:147px;">FST</td>
			<td style="width:219px;">10316-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Forceps</td>
			<td style="width:147px;">FST</td>
			<td style="width:219px;">Dumont #5 REF#11254</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1008px;">Equipment/software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Time lapse microscope</td>
			<td style="width:147px;">Zeiss</td>
			<td style="width:219px;">Observer 1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:264px;">Temp module S</td>
			<td style="width:147px;">PECON for Zeiss</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">CO2 module S</td>
			<td style="width:147px;">PECON for Zeiss</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Metamorph software</td>
			<td style="width:147px;">Metamorph</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:264px;">Eggs incubator</td>
			<td style="width:147px;">Sanyo</td>
			<td style="width:219px;">MIR154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Electroporator apparatus</td>
			<td style="width:147px;">Nepa Gene CO., LTD</td>
			<td style="width:219px;">CUY21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Electrodes</td>
			<td style="width:147px;">Nepa Gene CO., LTD</td>
			<td style="width:219px;">CUY611P7-4</td>
			<td>4 mm platinum electrodes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Fluorescence stereomicroscope</td>
			<td style="width:147px;">LEICA</td>
			<td style="width:219px;">MZ10F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Cryostat</td>
			<td style="width:147px;">MICROM</td>
			<td style="width:219px;">HM550</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Confocal microscope</td>
			<td style="width:147px;">Olympus</td>
			<td style="width:219px;">FV1000, X81</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Fluoview software</td>
			<td style="width:147px;">Olympus</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CLC Main Workbench software</td>
			<td style="width:147px;">CLC Bio</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

use of phluorin to assess the dynamics of axon guidance receptors in cell culture and in the chick embryo

Durante o desenvolvimento, os receptores de orientação do axônio desempenham um papel crucial na regulação da sensibilidade dos axônios a pistas atraentes e repulsivas. De fato, a ativação dos receptores de orientação é o primeiro passo dos mecanismos de sinalização que permitem pontas de axônio, os cones de crescimento, para responder aos ligantes. Como tal, a modulação de sua disponibilidade na superfície celular é um dos mecanismos que participam na definição da sensibilidade do cone de crescimento. Descrevemos aqui um método para visualizar precisamente a dinâmica da superfície celular espócica de um receptor de orientação de axônio tanto in vitro quanto in vivo na medula espinhal do pintinho em desenvolvimento. Aproveitamos a propriedade de fluorescência dependente do pH de uma variante de proteína fluorescente verde (GFP) para detectar especificamente a fração do receptor de orientação do axônio que é endereçado à membrana plasmática. Primeiro descrevemos a validação in vitro de tais construtos dependentes de pH e detalhamos ainda mais seu uso in vivo, na corda vertebral do pintinho, para avaliar a dinâmica espátula-temporal do receptor de orientação do axônio de interesse.

<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50883/50883fig1highres.jpg" src="/files/ftp_upload/50883/50883fig1.jpg"> Figura 1. A. Esquema das propriedades de fluorescência pHluorin-plexinA1 em um contexto celular. A phluorina é não fluorescente em compartimentos intracelulares onde o pH é ácido (<6) como em vesicules de tráfico ou em endosários e é fluorescente quando exposto ao meio extracelular onde o pH é neutr...

Watch this Scientific Journal Video about Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo at JoVE.com

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

Descrevemos aqui o uso de uma variante de proteína fluorescente verde sensível ao pH, pHluorin, para estudar a dinâmica espátula-temporal dos receptores de orientação de axônio que traficam na superfície celular. O receptor com marca de phluorina é expresso tanto na cultura celular quanto in vivo,usando eletroporação do embrião de filhote.

Uso de pHluorin para avaliar a dinâmica dos receptores de orientação de axônio na cultura celular e no embrião de filhotes

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

use-phluorin-to-assess-dynamics-axon-guidance-receptors-cell-culture

Research

JoVE Journal

Neuroscience

8.1K visualizações.  University of Lyon. Descrevemos aqui o uso de uma variante de proteína fluorescente verde sensível ao pH, pHluorin, para estudar a dinâmica espátula-temporal dos receptores de orientação de axônio que traficam na superfície celular. O receptor com marca de phluorina é expresso tanto na cultura celular quanto in vivo,usando eletroporação do embrião de filhote.

Vídeo: Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Visualização de Propriocepção em<em&gt; Drosophila</em&gt; As larvas e pupas

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Neurociência

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

Imagiologia pHluorin-tagged Inserção receptor para a membrana plasmática em primários de rato cultivadas Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

Eletroporação do rombencéfalo rastrear Trajetórias axonal e Metas Synaptic no embrião de galinha

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Induzir Plasticidade de astrocítica Receptores de Manipulação de Neuronal queima de moeda

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

Todo-mount imagem de embrião de rato Projeções Sensorial Axon

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Imagem Ca<sup&gt; 2+</sup&gt; Dynamics nos terminais Cone fotorreceptores Axon do mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

Utilizando metodologias combinadas para definir o papel do Plasma Membrane entrega durante Axon Ramificação e Neuronal morfogênese

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

O uso do condicionamento de rastreamento Eyeblink clássico para avaliar disfunção Hippocampal em um modelo do rato de transtornos do espectro do álcool Fetal

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, assembly and activity-dependent plasticity. Giant ankyrin-G, the master organizer of AIS, directly associates with membrane-spanning voltage gated sodium (VSVG) and potassium channels (KCNQ2/3), as well as 186 kDa neurofascin, a L1CAM cell adhesion molecule. Giant ankyrin-G also binds to and recruits cytoplasmic AIS molecules including beta-4-spectrin, and the microtubule-binding proteins, EB1/EB3 and Ndel1. Giant ankyrin-G is sufficient to rescue AIS formation in ankyrin-G deficient neurons. Ankyrin-G also includes a smaller 190 kDa isoform located at dendritic spines instead of the AIS, which is incapable of targeting to the AIS or rescuing the AIS in ankyrin-G-deficient neurons. Here, we described a protocol using cultured hippocampal neurons from ANK3-E22/23-flox mice, which, when transfected with Cre-BFP exhibit loss of all isoform of ankyrin-G and impair the formation of AIS. Combined a modified Banker glia/neuron co-culture system, we developed a method to transfect ankyrin-G null neurons with a 480 kDa ankyrin-G-GFP plasmid, which is sufficient to rescue the formation of AIS. We further employ a quantification method, developed by Salzer and colleagues to deal with variation in AIS distance from the neuronal cell bodies that occurs in hippocampal neuron cultures. This protocol allows quantitative studies of the de novo assembly and dynamic behavior of AIS.

Here, we described a protocol to quantitatively study the assembly and structure of the axon initial segments (AIS) of hippocampal neurons that lack pre-assembled AIS due to the absence of a giant ankyrin-G.

Uso de neurônios hipocampais primários para estudar a montagem de segmentos iniciais de Axon

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, ...

Isolation and Culture of Chick Ciliary Ganglion Neurons

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the eye. This ganglion is constituted by a homogenous population of ciliary and choroidal neurons that innervate striated and smooth muscle fibers, respectively. Each of these neuronal types regulate specific eye structures and functions. Over the years, neuronal cultures of the chick ciliary ganglia were shown to be effective cell models in the study of muscle-nervous system interactions, which communicate through cholinergic synapses. Ciliary ganglion neurons are, in its majority, cholinergic. This cell model has been shown to be useful comparatively to previously used heterogeneous cell models that comprise several neuronal types, besides cholinergic. Anatomically, the ciliary ganglion is localized between the optic nerve (ON) and the choroid fissure (CF). Here, we describe a detailed procedure for the dissection, dissociation and in vitro culture of ciliary ganglia neurons from chick embryos. We provide a step-by-step protocol in order to obtain highly pure and stable cellular cultures of CG neurons, highlighting key steps of the process. These cultures can be maintained in vitro for 15 days and, hereby, we show the normal development of CG cultures. The results also show that these neurons can interact with muscle fibers through neuro-muscular cholinergic synapses.

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system. Neuronal cultures of chick CG neurons were shown to be effective cell models in the study of nerve muscle interactions. We describe a detailed protocol for the dissection, dissociation and in vitro culture of CG neurons from chick embryos.

Isolamento e Cultura de Chick Ciliary Ganglion Neurons

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the ...