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.

<table border="0" cellpadding="0" cellspacing="0">
	<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>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>GIBCO</td>
			<td>10908-010</td>
			<td>0.5 ml</td>
		</tr>
		<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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		<tr>
			<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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		<tr>
			<td>HEPES ( 1M)</td>
			<td>GIBCO</td>
			<td>15630-130</td>
			<td>1 ml</td>
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		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>3, HEPES buffered saline&#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>
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		<tr>
			<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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		<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>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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
		</tr>
		<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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		<tr>
			<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>
		</tr>
		<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>
		</tr>
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</table>

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. ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

Preparação de fatias agudas zona subventricular for Imaging Cálcio

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

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 ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

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 ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

Imagens ao vivo de<em&gt; Drosophila</em&gt; larval Neuroblastos

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

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.