We thank Yehui Qin and Mallory Myers for technical support. We also thank Dr. Sigismund Huck for providing us the anti-&#945;4-ECD antibody. This work is supported by National Institutes of Health grant NS22061 to L. W. R.

Todos os experimentos com animais foram realizados de acordo com os Institutos Nacionais de Saúde Guia para o Cuidado e Uso de Animais de Laboratório (NIH Publications No. 80-23, revisado 2012) e estudos foram aprovados pelo Institutional Animal Care e Use para Comitês de Pesquisa em Stony Brook University (# 1618 e # 1792). 1. vHipp-NACC Synaptic Co-culturas <ol><li> Camundongos Sacrifice (pós-natal dias 0-3, a partir do tipo selvagem (WT) ou nAChRs α7 linha de camundongo transgênico) com CO 2. Decapitar cada filhote e salvar a cauda em um tubo de 1,5 ml de centrífuga para post facto genotipagem. Execute as seguintes etapas em uma capa esterilizado. </li><li> Remover o topo do crânio com tesouras e pinças para expor todo o cérebro. Começando na junção onde os córtices cerebrais em parte, de um ao outro caudalmente, obter uma secção coronal que contém os córtices posteriores incluindo o vHipp 5,21 sob um microscópio de dissecação. </li><li> Dissecar vHipp tiras de tecido contendo a região CA1-subículo de acordo com um desenvolvimento atlas de cérebro de ratinho 22 (Figura 1A). Transferir para um prato de cultura de 35 milímetros contendo meio de cultura frio (4 ° C), cortadas em pequenas fatias finas (cerca de 100 mm x 100 mm microslices). </li><li> Pré-incubar 12 milímetros poli-D-lisina / laminina revestidos por lamelas de vidro dentro de 24 placas de cultura de tecidos com meio de cultura (~ 500 mL) durante pelo menos 30 min no incubador a 37 ° C. Remova a mídia antes do plaqueamento microslices. </li><li> A placa com uma pipeta de Pasteur polida ao fogo no centro da lamela com uma quantidade mínima de meio (~ 50 ul, contendo ~ 20 peças de microslices) para facilitar a fixação dos explantes. Placa microslices vHipp provenientes de um único animal (ou a + / + ou - / - genótipo da linha α7 transgênico) em cada lamela. </li><li> Após os explantes resolver sobre a lamela, adicione delicadamente mídia adicional (~ 100 mL) por the parede de modo que o nível dos meios de comunicação é alta o suficiente para cobrir completamente os explantes sobre a periferia, mas não os que estão no centro da lamela. </li><li> Depois de uma O / N de incubação em 37 ° C, 5% de CO2, dispersar NACC neurónios embrionários do dia 18 ao dia pós-natal uma ratinhos WT e adicionar aos explantes. <ol><li> Dissecar tecidos NACC de acordo com um atlas do mouse cérebro em desenvolvimento 20 (Figura 1B). </li><li> Cortar em pequenos pedaços (cerca de 500 mm × 500 mm microslices) e transferir para um tubo de 15 ml. </li><li> Trata-se com 0,25% de tripsina (2 ml) durante 15 min a 37 ° C. Lavar pedaços de tecido três vezes com meio de lavagem frio (5 ml, 4 ° C), seguido por uma lavagem em meio de cultura frio (5 ml, 4 ° C). Permitir suspensão para descansar por 5 minutos em cada etapa de lavagem e, em seguida despeje cuidadosamente o sobrenadante. </li><li> Separar as células por trituração suave em 2 ml de meio de cultura com uma pipeta de Pasteur polida ao fogo levemente. </li><li> Transsuspensão de células fer para outro tubo de 15 ml e centrifugar a 2000 x rpm durante 5 min. Remover o sobrenadante e ressuspender as células em meios de cultura em 1 ml por 6 filhotes. Dispersar as células com cuidado pipetando em mídia várias vezes com uma pipeta Pasteur polida-fogo. </li><li> Adicionar 0,25 ml de células NACC dispersos para cada lamela com explantes vHipp. </li><li> Manter as culturas numa atmosfera humidificada a 37 ° C, 5% de CO2. Coloque placas de cultura de compressas de gaze estéril umedecido para estabilizar mecanicamente e manter a umidade, facilitando a formação de sinapses. </li></ol></li></ol> 2. Immunocytochemistry <ol><li> Fix culturas (5-7 dias in vitro) em paraformaldeído a 4% / 4% de sacarose / PBS (~ 500 ul por lamela, 20 min, RT). </li><li> Permeabilizar as culturas com 0,25% de Triton X-100 / PBS (~ 500 ul por lamela, 5 min, RT). </li><li> Culturas em bloco com 10% de soro de burro normal em PBS (~ 500 mL por lamela, 30 min, RT) antes de Antibody coloração. Incubar com anticorpos primários o / n a 4 ° C. Use os seguintes anticorpos primários: anticorpo anti- (nAChRs α 4 -ECD 1: 500; anti-α 5 subunidades, 1: 500), anti-vesicular transportador de glutamato 1 (1: 250), anti-GAD65 (1: 100). </li><li> Lavar anticorpos primários com PBS (3 vezes / ~ 500 ul por lamela, 5 min) e depois incubar as culturas em anticorpos secundários conjugados com Alexa 488 (~ 500 mL por lamela, 1: 500) ou Alexa 594 (~ 500 mL por lamela , 1: 500) durante 1 hora a 37 ° C. </li><li> Incubar em culturas αBgTx conjugados com Alexa 594 (~ 500 mL por lamela, 1: 1000 em meio de cultura) durante 45 min a 37 ° C, antes da fixação para rotulagem superfície α7 * nAChRs. </li><li> Montagem e vedação lamelas. </li><li> Capturar imagens com um microscópio de fluorescência equipado com objectivos Plan-Apocromática (20X com 0,8 NA ou 63X de petróleo com 1,4 NA) e uma câmera CCD. </li></ol> 3. FM1-43 Based vesicular Fusão <ol><li> Manter culturas (5-7 dias in vitro) em uma câmara de imagem, montar a câmara no disco giratório microscópio confocal. Continuamente perfundir (1 ml / min) com HBS cocktail à TA. </li><li> Parar de perfusão e de carga culturas com 10 uM FM1-43 em 56 mM de K + ACSF (~ 500 mL) durante 90 segundos (coloração), lavar corante externo afastado durante 15 min com Ca 2+ livre HBS contendo ADVASEP-7 (0,1 mM) para eliminar FM1-43 ligada à membrana. </li><li> Estimular as culturas com 56 mM de K + ACSF (~ 500 mL) sem FM1-43, durante 120 segundos (descoloração), recarregar as culturas com FM1-43 utilizando as mesmas condições, e lava-se novamente com o Ca2 + livre de HBS contendo ADVASEP-7 para 15 min. </li><li> Adquirir FM1-43 imagens de fluorescência com disco giratório microscópio confocal equipado com uma objectiva Plan-Apochromat (60X de água com 1,4 NA, excitação 488 nm, emissão 530 nm) e capturar imagens com uma câmera CCD. </li><li> Coletar imagens FM1-43 fluorescência de base (a cada 2 s por 1 min) Como o controlo de pré-nicotina; aplicam-se a nicotina (1 uM) por perfusão rápida (2 ml / min) durante 1 min, e em seguida lavar com nicotina para fora HBS coquetel. Mantenha lapso de tempo imagens de captura antes, durante e após a aplicação de nicotina cada 2 s por 5 min. </li><li> Quantificar FM1-43 intensidade de fluorescência antes (F coloração) e depois (F descoloração) aplicação de nicotina em cada bouton sináptica. </li><li> Calcular quantidade total de fluorescência FM1-43 libertado ao longo dos axónios vHipp pela quantificação da diferença de FM1-43 intensidade de fluorescência antes e após a aplicação de nicotina (Af = F coloração -F descoloração). Analisar fração de diminuição da intensidade de fluorescência após a nicotina com a seguinte equação: F diminuição% = Af / F coloração. </li></ol> Imagem 4. Cálcio <ol><li> Enxágüe culturas (5-7 dias in vitro) rapidamente com HBS normal, em seguida, carregar culturas com 5 Indicador de Fluo-4 Ca 2+ (Éster AM) e 0,02% de Pluronic F-127 em HBS (2 ml) durante 30 min a 37 ° C e 5% de CO 2. </li><li> Lave a Fluo-4 com solução HBS (3 vezes / 5 min). Retorno culturas para incubadora (37 ° C / 5% de CO 2) durante pelo menos 30 min. </li><li> Manter as culturas na mesma câmara de imagem e mesmas condições que as descritas na secção de fusão vesicular com base FM1-43 (veja o passo 3.1). </li><li> Coletar imagens Fluo-4 fluorescência de projeções axonais dos micro-fatias vHipp com um objectivo Plan-Apochromat (60X de água com 1,4 NA, excitação 488 nm, emissão 530 nm) e uma câmera CCD. </li><li> Recolha de linha de base de Fluo-4 imagens de fluorescência (a cada 10 segundos durante 2 minutos) como o controlo de pré-nicotina, nicotina aplica (1 uM) por perfusão rápida (2 ml / min) durante 1 min, e em seguida lavar com nicotina para fora HBS coquetel. Manter lapso de tempo de captura de imagens, antes, durante e após a aplicação da nicotina a cada 10 segundos durante 30 minutos. </li><li> Salve todas as molduras dos fluo-4 imagens de fluorescência-primas, a exportação como uma série deImagens em formato TIFF para posterior análise. </li><li> Recolha a intensidade integrada de fluo-4 fluorescência ao longo vHipp axónios antes e após a aplicação de nicotina. </li><li> Normalizar a intensidade de fluorescência integrada com a seguinte equação: Af / F 0 = (FF 0) / 0 F, em que F representa a 0 pré-nicotina intensidade de fluorescência integrada corrigida-fundo e F é a intensidade de fluorescência integrada ao longo dos axónios vHipp em cada ponto temporal após a aplicação de nicotina. Analisar e traçar normalizada intensidade de fluorescência integrada. </li></ol>

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A preparação de co-cultura descrito re-circuitos do hipocampo ventral cede-accumbens in vitro. Esta análise permite preparação relativamente simples e de confiança dos perfis espaciais e temporais pelo qual a activação de nAChRs pré-sinápticos eliciar melhoradas transmissão glutamatérgica 5, 21. Co-culturas são definidos como o crescimento de diferentes tipos de células específicas em um prato que pode proporcionar condições fisiológicas in vitro par...

Modulação do circuito colinérgico excitabilidade contribui para os aspectos fundamentais de cognição, e modulação colinérgica alterada é uma característica de distúrbios neurodegenerativos e neuropsiquiátricas incluindo a doença de Alzheimer, doença de Parkinson, esquizofrenia e dependência 1-4. Um mecanismo estabelecido de facilitação de transmissão sináptica colinérgica no sistema nervoso central é, por activação directa de nAChRs localizadas em locais pré-sinápticos. A activação destes receptores pré-sinápticos leva a um aumento de Ca2 + intracelular ([Ca 2+] i) nos terminais pré-sinápticos - tanto directamente, devido à relativamente elevada condutância de cálcio de certos subtipos de nAChR, e indirectamente, por cascatas de sinalização intracelular 5, aumentando, assim, a libertação de neurotransmissores. De facto, a activação dos nAChRs pré-sinápticos tem sido associada com mudanças na libertação de uma grande variedade de neurotransmissores, incluindo glutamato, GABA, acetilcolina, umND dopamina 6-10. Embora este processo tem sido estudada usando métodos electrofisiológicos indirectamente em diversas sinapses, indicadores ópticos de [Ca2 +] i e a reciclagem das vesículas sinápticas permitir uma medição mais directa e temporalmente precisa dos fenómenos pré-sinápticos. Localização pré-sináptica dos nAChRs foi demonstrada de forma convincente com rotulagem imuno-ouro direta de nAChRs na microscopia eletrônica (EM) nível 11,12. Várias outras técnicas também têm sido utilizados para tratar a localização do nAChR indirectamente, incluindo a detecção de locais de nAChRs subunit- quimeras de proteína fluorescente em neurónios cultivados 13,14, electrofisiológico gravação de correntes de nAChR em terminais sinápticos 15,16, monitorização de nicotina induziu alterações na [Ca 2 +] i em terminais nervosos sinápticas por imagens ao vivo de células 17 e monitoramento indireto da liberação do neurotransmissor no terminal sináptico portécnicas de imagem de células vivas com indicadores fluorescentes, incluindo exocitose das vesículas sinápticas vistos por estirilo corantes anfipï¿½icos FM (FM1-43 e FM4-64) e / ou synapto-pHluorin e por repórteres fluorescentes neurotransmissores específicos, como CNiFERs para ACh e iGluSnFr para glutamato 18-20. No geral, essas abordagens atuais para identificar a localização pré-sináptica dos nAChRs são complicadas e exigem sistemas e técnicas especiais para permitir a identificação confiável e monitorização fisiológica da atividade pré-sináptica. Aqui nós descrevemos protocolos e equipamentos para um sistema in vitro de co-cultura de um hipocampo ventral (vHipp) - núcleo accumbens (NACC) circuito que fornece acesso direto para identificar e analisar os dois componentes pré e pós-sinápticos de transmissão sináptica. Mostramos exemplos de localização pré-sináptica dos nAChRs ea imagens de células vivas de nAChR mediada sinalização de Ca2 + e liberação de neurotransmissores aloaxônios ng vHipp. Uma extensão natural (e direta) do protocolo apresentado aqui é a preparação de contatos pré e pós-sinápticos composta de neurônios de diferentes genótipos. Deste modo, a contribuição de um produto de gene em particular para o pré e / ou de mecanismos pós-sinápticos de modulação pode ser avaliada directamente.

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	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>1, Culture Media (50 ml)</td>
		</tr>
		<tr>
			<td>Neurobasal&#160;</td>
			<td>GIBCO</td>
			<td>10888022</td>
			<td>48 ml</td>
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		<tr>
			<td>B-27 Supplements</td>
			<td>GIBCO</td>
			<td>0080085-SA</td>
			<td>1 ml</td>
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		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>GIBCO</td>
			<td>10908-010</td>
			<td>0.5 ml</td>
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		<tr>
			<td>GlutaMAX Supplement</td>
			<td>GIBCO</td>
			<td>35050-061</td>
			<td>0.5 ml</td>
		</tr>
		<tr>
			<td>Brain-derived neurotrophic factor (BDNF)</td>
			<td>GIBCO</td>
			<td>15140-122</td>
			<td>20 ng/ml</td>
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		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>2, washing media (HBSS, 100 ml)</td>
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			<td>HBSS, no calcium, no magnesium, no phenol red&#160;</td>
			<td>GIBCO</td>
			<td>14175-095</td>
			<td>99 ml</td>
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			<td>HEPES ( 1M)</td>
			<td>GIBCO</td>
			<td>15630-130</td>
			<td>1 ml</td>
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			<td>3, HEPES buffered saline&#160; (HBS)&#160;&#160; pH=7.3</td>
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			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888&#160;</td>
			<td>135 mM</td>
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			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333&#160;</td>
			<td>5 mM</td>
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			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266&#160;</td>
			<td>1 mM</td>
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			<td>CaCl2,</td>
			<td>Sigma</td>
			<td>C1016&#160;</td>
			<td>2 mM</td>
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		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375&#160;</td>
			<td>10 mM</td>
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			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
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		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>4, HBS Cocktail for live imaging pH=7.3</td>
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		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888&#160;</td>
			<td>135 mM</td>
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		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333&#160;</td>
			<td>5 mM</td>
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		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266&#160;</td>
			<td>1 mM</td>
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		<tr>
			<td>CaCl2,</td>
			<td>Sigma</td>
			<td>C1016&#160;</td>
			<td>2 mM</td>
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		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375&#160;</td>
			<td>10 mM</td>
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		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
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		<tr>
			<td>tetrodotoxin&#160;</td>
			<td>Tocris</td>
			<td>1078</td>
			<td>2 &#181;M</td>
		</tr>
		<tr>
			<td>bicuculline</td>
			<td>Tocris</td>
			<td>131</td>
			<td>10 &#181;M</td>
		</tr>
		<tr>
			<td>D-AP-5</td>
			<td>Tocris</td>
			<td>105</td>
			<td>50 &#181;M</td>
		</tr>
		<tr>
			<td>CNQX</td>
			<td>Tocris</td>
			<td>1045</td>
			<td>20 &#181;M</td>
		</tr>
		<tr>
			<td>LY341495</td>
			<td>Tocris</td>
			<td>1209</td>
			<td>10 &#181;M</td>
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		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>5, Calcium-free&#160; HBS&#160;&#160; pH=7.3</td>
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		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888&#160;</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333&#160;</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266&#160;</td>
			<td>1 mM</td>
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		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375&#160;</td>
			<td>10 mM</td>
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		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
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		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>6, 56 mM Potassium ACSF pH=7.4</td>
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		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td>119 mM</td>
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			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td>56 mM</td>
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		<tr>
			<td>MgSO4.7H</td>
			<td>Sigma</td>
			<td>M1880</td>
			<td>1.3 mM</td>
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		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td>2.5 mM</td>
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		<tr>
			<td>NaH2PO4</td>
			<td>Sigma</td>
			<td>S8282</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td>26.2 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
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live imaging of nicotine induced calcium signaling and neurotransmitter release along ventral hippocampal axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

A preparação utilizada consiste de gene quimérico co-culturas de circuitos vHipp-NACC in vitro. Projeções que emanam microslices vHipp, como entrada axonal pré-sináptico, pode fazer contatos sinápticos com metas pós-sinápticos, os neurônios dispersos de NACC. A nicotina induziu um (≥ 30 min) facilitação sustentado de transmissão glutamatérgica de neurônios NACC inervados por vHipp axônios sinalização ao longo vHipp axônios 5 via α7 pré-sináptica * nAChRs 21 e prolo...

Watch this Scientific Journal Video about Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons at JoVE.com

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Imagens ao vivo de nicotina Induzida Sinalização de Cálcio e Neurotransmitter lançamento Junto ventrais do hipocampo Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

live-imaging-nicotine-induced-calcium-signaling-neurotransmitter

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8.9K visualizações.  Stony Brook University. We developed a gene-chimeric preparation of ventral hippocampal – accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Vídeo: Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

Neuromodulação e Transportes mitocondrial: Imagens ao vivo em neurônios do hipocampo durante muito tempo

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Neurociência

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

Imagens ao vivo de axônios da raiz dorsal após rizotomia

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Analyzing Murine Schwann Cell Development Along Growing Axons

The development of peripheral nerves is an intriguing process. Neurons send out axons to innervate specific targets, which in humans are often more than 100 cm away from the soma of the neuron. Neuronal survival during development depends on target-derived growth factors but also on the support of Schwann cells (SCs). To this end SC ensheath axons from the region of the neuronal soma (or the transition from central to peripheral nervous system) to the synapse or neuromuscular junction. Schwann cells are derivatives of the neural crest and migrate as precursors along emerging axons until the entire axon is covered with SCs. This shows the importance of SC migration for the development of the peripheral nervous system and underlines the necessity to investigate this process. In order to analyze SC development, a setup is needed which next to the SCs also includes their physiological substrate for migration, the axon. Due to intrauterine development in vivo time-lapse imaging, however, is not feasible in placental vertebrates like mouse (mus musculus). To circumvent this, we adapted the superior cervical ganglion (SCG) explant technique. Upon treatment with nerve growth factor (NGF) SCG explants extend axons, followed by SC precursors migrating along the axons from the ganglion to the periphery. The beauty of this system is that the SC are derived from a pool of endogenous SC and that they migrate along their own physiological axons which are growing at the same time. This system is especially intriguing, because the SC development along axons can be analyzed by time-lapse imaging, opening further possibilities to gain insights into SC migration.

Here we describe a Schwann cell (SC) migration assay in which SCs are able to develop along extending axons.

Analisando o desenvolvimento de células de Schwann Murino Junto axônios em crescimento

The  development of peripheral nerves is an intriguing process. Neurons send out  axons to innervate specific targets, which in humans are often more than ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

Fosfato de cálcio transfecção de primário neurônios do hipocampo

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

Fluorescência e imagem de bioluminescência subcelular Ca<sup&gt; 2+</sup&gt; Aged em neurônios do hipocampo

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

Isolamento de gânglios das raízes dorsais e cultura primária para estudar a liberação de neurotransmissores

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Ex Vivo Imagem de cálcio para visualizar as respostas do cérebro a sinalização endócrina em Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its highly-organized subcellular architecture, we recently developed a new imaging modality, named spectral reflectometry (SpeRe), which enables unprecedented label-free nanoscale imaging of the live myelinated axons in situ. The underlying principle is to obtain nanostructural information by analyzing the reflectance spectrum of the multilayered subcellular structure. In this article, we describe a detailed step-by-step protocol for performing a basic SpeRe imaging of the nervous tissues using a commercial confocal microscopic system, equipped with a white-light laser and a tunable filter. We cover the procedures of sample preparation, acquisition of spectral data, and image processing for obtaining nanostructural information.

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Here, we present a step-by-step protocol for imaging myelinated axons in a fixed brain slice using a label-free nanoscale imaging technique based on spectral reflectometry.

Microscopia de reflectométrica espectral em dendritos In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

Ex Vivo Imagem de célula específica cálcio sinalização em sinapse tripartida do diafragma de rato

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.