Agradecemos o Dr. Yong-Ling Zhu por prevê construção Sinaptofisina-pHluorin2x e Dr. James E. Rothman fornecendo VAMP2-phluorin. Agradecemos o Dr. Susan Cheng e Virginia Crocker da facilidade de microscopia eletrônica de NINDS pelo apoio técnico e ajuda. Este trabalho foi financiado pelo Instituto Nacional de Disorders Neurological e curso Intramural Research Program nos EUA e uma bolsa da KRIBB iniciativa programa de pesquisa (Korean biomédica cientista Fellowship Program), Instituto de pesquisa da Coreia do Biociências e biotecnologia, República da Coreia.

Nota: O seguinte protocolo descreve os métodos de imagem pHluorin e métodos EM neurônios hippocampal culta. pHluorin monitores vesícula sináptica proteína absorção em células vivas e EM detecta a absorção da vesícula sináptica e as alterações ultra-estruturais.
 cuidados com animais e procedimento seguiu as diretrizes do NIH e foram aprovados pelo Comitê de uso e cuidados do Animal NIH.
 1. pHluorin Imaging <ol><li>cultura neurônio Hippocampal <ol><li>preparar hipocampo Buffer (HB) combinando NaHCO 3 de 4 mM e 5 mM HEPES e ajustar o pH 7.3 com 5 M de NaOH. Fazer o meio de cultura misturando neurobasal médio, 2% B27, 0.5 mM L-glutamina e 1% penicilina-estreptomicina. Além disso, prepare uma mistura do buffer de HB com 20% de soro Fetal bovino (HB/20% FBS). 
		Nota: Este protocolo cultivo baseia-se na Serra, et al 29 e Wu et al 24</li> <li>decapitar filhotes de rato entre pós-Natal dia 0 ao dia 2 em meio de cultura e extrair o cérebro em 4 ° C HB/20% FBS. Retire o tronco cerebral e tálamo para expor o hipocampo. Dissecar para fora o hipocampo, após expor-lo pela remoção do tronco cerebral e tálamo e transferir para a 4 ° C HB/20% FBS. 
		Nota: Normalmente, o rendimento é em torno de 4 x 10 5 células/mL para um filhote de cachorro (dois hipocampo). Limpar as membranas aderente, usando uma pinça ou tesoura, então a transferência para a 4 ° C HB/20% FBS. Desenrole o giro denteado e isolar o subiculum cortando com uma tesoura e, em seguida, transferir a 4 ° C HB/20% FBS.</li>
		<li>Dividir cada hipocampo, por um corte de ponta a ponta, em cerca de 10 fatias e transfira para um tubo cónico polipropileno de 15 mL.</li>
		<li>Depois permitindo que o tecido resolver, lavar com 10 mL de HB/20% FBS e depois lavar três vezes com 10ml de HB.</li>
		<li>Preparar a solução de digestão de 137 mM de NaCl, 5 mM KCl, 7mm Na2HPO 4 e 25mm HEPES e ajustar o pH 7.2 com 5 M de NaOH. Retire o sobrenadante do hipocampo. Adicionar 2 mL de solução de digestão 10 mg de tripsina e 1 mg de DNase e filtrar através de uma membrana esterilizada 0,22 µm diretamente sobre a pelota amostra.</li>
		<li>Incubar o hipocampo por 5 min a 37 ° C, em seguida, lave duas vezes com 10 mL de HB/20% FBS e então lave uma vez com 10 mL de HB.</li>
		<li>Dissociar as células com 6 mg de MgSO 4 &#8729; 7 H 2 O e 1 mg de DNase 2ml HB e filtro estéril sobre o hipocampo de pelotas através de um filtro de 0,22 µm estéril. Delicadamente, dissocia as células por moer cuidado, enquanto tendo o cuidado de evitar a introdução de bolhas de ar. Permitir que as partículas de tecido repousar por 2 min e em seguida lentamente transfira o sobrenadante para outro tubo.</li>
		<li>, Adicionar 3 mL HB/20% FBS para a suspensão de células e centrífuga ou 10 min a 4 ° C e 1.000 rpm. Desprezar o sobrenadante e ressuspender em meio de cultura.</li>
		<li>Placa de 60.000 células suspendidas em meio de cultura de 150 µ l sobre uma lamela de diâmetro de 25 mm de poli-D-lisina revestido, sem derramamento da lamela. Adicione 2 mL de meio de cultura previamente aquecido 2 h após chapeamento. As lamelas são mantidas em placas de cultivo de 6 boas ou pratos de Petri estéril.</li>
		<li>Células mantêm a 37 ° C em um 5% CO 2 humedecidas incubadora em meio de cultura por 14-21 dias antes da gravação. Durante o crescimento da cultura, mudar a metade superior do meio duas vezes por semana.</li>
	</ol></li> <li>Transfection <ol><li>6-7 dias após o chapeamento, transfect Sinaptofisina-pHluorin 2 X (SpH) ou VAMP2-pHluorin em neurônios hippocampal. Para SpH, use um promotor de citomegalovírus (CMV), inserido em um vetor de pcDNA3 30. Para VAMP2-pHluorin, use um promotor CMV inserido em um vetor de pCI 31.</li>
		<li>Transfect qualquer vector por transportadora de lipídios em células-alvo, usando 1 µ g de plasmídeo. Use o meio de cultura de passo protocolo 1.1.1, que carece de soro, para o transfeccao. Alterar o meio 2 h após a transfeccao para reduzir a toxicidade. 
		Nota: Em caso de baixa expressão da SpH nos boutons, aumente a concentração de DNA de 2 µ g ou o tempo de incubação com a transportadora de lipídios, a menos que as células são insalubres. Normalmente, as células 4-10 (0,006-0,008%) de neurônios foram transfectadas.</li>
	</ol></li> <li>Microscopia de luz <ol><li>preparar solução salina normal é composto por 119 mM NaCl, 2 mM CaCl 2, 2,5 mM KCl, 25mm HEPES (pH 7,4), glicose de 30 mM, 2 mM MgCl 2, 6-cyano-7-nitroquinoxaline-2,3-dione (0.01 mM CNQX) e 0,05 mM ácido DL-2-amino-5-phosphonovaleric (AP5). Leve uma lamela da placa de meio de cultura e coloque em uma câmara de imagem que permite a estimulação do campo, usando lubrificante e selante para evitar vazamentos. Evite deixar o vidro seco durante a transferência de uma lamela da placa para a câmara, adicionando imediatamente a solução salina normal de 750 µ l. 
		Nota: CNQX AP5 utilizaram e bloquear a atividade pós-sináptica, que tem o potencial para atividade recorrente. Antes de colocar uma câmara em um microscópio de fluorescência invertido, use lenço de limpeza para confirmar que a câmara não vaza. Vazamento lento provoca alterações de foco durante a gravação, pela mistura de óleo normal saline e imersão, que resulta em mudanças no índice de refração.</li>
		<li>Estimulação e gravação de imersão de óleo <ol><li>imagem em um microscópio invertido widefield com um 60 X (1.4 abertura numérica) lente com uma lâmpada de iodetos metálicos. Visualizar pHluorin com um filtro definido para um pico de excitação de 480 nm, um espelho de passe longo 490 nm, um filtro de emissão 500-550 nm e um obturador manual de aleta. Capturar imagens cada 100 ms, com binning 2x2, usando uma câmera de elétron multiplicando carga acoplado dispositivo (EMCCD). 
			Nota: Vários recursos devem ser considerados para atingir as condições apropriadas para evitar fotobranqueamento, incluindo o intervalo de captura de imagem e filtrar e binning, que são dependentes de equipamentos. No caso da imagem latente confocal, tempo de alimentação e exposição do laser deve ser considerado para evitar fotobranqueamento. A instalação foi determinada após a gravação de pelo menos 3 min sem estímulo e a gravação foi realizada pelo menos 10 s sem estímulo para verificar se há fotobranqueamento.</li>
			<li>Escolher uma área com alta densidade de boutons para facilidade de análise em cada experimento. Identificar células transfectadas por seu sinal de fluorescência verde, com ênfase em padrões de expressão redondas ou ovais no bouton e expressão contínua entre boutons.</li>
			<li>Induzir endocitose por estimulação de campo usando um 1 pulso de ms, 20 mA ação potencial (AP) fornecida a partir de um estimulador de pulso e entregues através de um eletrodo de platina dentro da unidade de isolamento de estímulo. Atividade de fluorescência da imagem no decorrer da estimulação e durante a recuperação celular. 
			Nota: No caso de células mortas, expressão foi mais forte do que viver boutons e mostrou um padrão desigual entre boutons.</li>
		</ol></li></ol></li> <li>Análise de imagem <ol><li>Para analisar a intensidade de fluorescência em um único bouton, definido por uma região de interesse (ROI) como uma de 1,5 x 1,5 µm quadrados; o tamanho de um bouton é dentro traços cerca 1,5 µm. uso antes da estimulação para verificar fotobranqueamento e subtrair como pano de fundo.</li>
		<li>Normalize fluorescência muda (ΔF) com a equação: <img alt="Equation" height="11" src="/files/ftp_upload/55862/55862eq1.jpg" width="172" /> onde F máx e F 0 referem-se a máximo aumento após estimulação e fluorescência de base, respectivamente. Medir as endocitose taxas como a taxa de decaimento durante os primeiros 4-10 s após o ponto máximo da fluorescência de pHluorin. Obter a constante de tempo (τ) de endocitose por encaixe o decaimento de fluorescência pHluorin do pico aumento da linha de base com uma mono-exponencial função.</li>
	</ol></li></ol> 2. Microscopia eletrônica de <ol><li>Prepare uma poli-D-lisina revestido 6 placa aplicando 1,5 mL de solução de poli-D-lisina 0,01% estéril filtrada a cada poço por 1h à temperatura ambiente e, em seguida, lavar três vezes com água esterilizada. Dissecar, cultura e manter os neurônios hippocampal como em etapas 1.1.1, 1.1.2 e 1.1.3, respectivamente.</li>
	<li>Prepare uma solução de estimulação K + alta com HRP como 31,5 mM NaCl, 2 mM CaCl 2, 90 mM KCl, 25mm HEPES (pH 7,4), glicose de 30 mM, 2 mM MgCl 2, 0,01 mM CNQX, 0,05 mM AP5 e 5 mg/mL HRP, então ajustam o pH 7,4 com 5 M NaOH.
	Solução de estimulação de K + <ol><li>estimular a cultura de neurônio hippocampal com 1,5 mL de alta à temperatura ambiente (referida como K +) por adição de 1,5 mL para cada poço para 90 s. Na condição de repouso (referida como R), aplicar a mesma concentração de 5mg/mL HRP para 90 s, mas com solução salina normal. Para o exemplo de recuperação, aplicar solução de estimulação alta K + como com a amostra de K +, em seguida, rapidamente lavar substituir com solução salina normal e incubar durante 10 min.</li>
	</ol></li> <li>Fixação e coloração <ol><li>preparar o tampão de cacodylate at 0,1 M usando at cacodylate de 21,4 g/L pH 7,4. Consertar as células com 4% de glutaraldeído no buffer de cacodylate at 0.1 M pelo menos 1 h à temperatura ambiente. Lavar três vezes com 0,1 M at cacodylate o buffer para 7 min cada.</li>
		<li>Solução de preparar diaminobenzidina (DAB), composto de 0,5 mg/mL de DAB com 0,3% H 2 O 2 em ddH 2 O e filtrar com um filtro de 0,22 µm. Aplicar 1,5 mL de solução DAB por 30 min a 37 ° C. lavar três vezes com 0,1 M at cacodylate o buffer para 7 min cada. 
		Cuidado: DAB é um carcinógeno tóxico e suspeito. Por favor, use luvas e jalecos. 
		Nota: Etiquetando com DAB ocorre devido à sua oxidação por H 2 O 2, como catalisada por HRP. Pequenos aumentos nos componentes resultam em um sinal de aumento na amostra. Aumentando a concentração de HRP acelera o efeito do catalisador. Suficientemente grandes concentrações de H 2 O 2 permitem debilitante reações colaterais com HRP, inibindo o efeito da rotulagem 32. Neste trabalho, as concentrações deste sistema de rotulagem foram escolhidas com base na pesquisa atualmente disponíveis 33 , 34.</li>
		<li>Incubar os neurônios com 1,5 mL de 1% OsO 4 no buffer de cacodylate at 0,1 M por 1 h a 4 ° C, como fixação de postar. Lavar três vezes com 1,5 mL de tampão de cacodylate de nd de 0,1 M por 7 min cada. 
		Atenção: Devido à toxicidade e reatividade de OsO 4, mantendo a amostra no gelo em uma capa de química é preferível em muitos casos usar uma geladeira para a incubação.</li>
		<li>Preparar 0,1 M de tampão de acetato de sódio com 13,61 g/L de sódio acetato e 11,43 mL/L de ácido acético glacial em pH 5,0. Lavar três vezes com tampão de acetato 1,5 mL 0,1 M em pH 5.0 para 7 min cada e incubar com acetato de uranilo 1,5 mL 1% em tampão de acetato 0,1 M em pH 5.0 para 1 h a 4 ° C. Wash três vezes com tampão de acetato 1,5 mL 0,1 M por 7 min cada.</li>
	</ol></li> <li>Epóxi incorporação <ol><li>desidratar a cultura neurônio com lavagens único 1,5 mL de etanol a 50%, 70% e 90%, para 7 min em cada um e em seguida 3 lavagens de 1,5 mL de etanol a 100% por 7 min em uma coifa.</li>
		<li>Mix 485 mL/L bisphenol-A-(epichlorhydrin) epóxi resina, anidrido de 160 mL/L dodecenilo succínico (DDSA), 340 mL/L metil-5-Norborne-2,3-dicarboxílico fenol 2.4.6-tris (dimetilaminometil) anidrido (NMA) e 15 mL/L (DMP-30) para criar o epóxi resina. Mistura cuidadosamente, em seguida, armazenar sob vácuo para remover as bolhas de ar. 
		Nota: É fundamental para remover as bolhas de ar na resina, especialmente aqueles de menor do que é visível a olho nu, porque eles podem causar cáries na resina durante o corte.</li>
		<li>Infiltrar a amostra, substituindo o etanol com epóxi de 50% de resina em etanol por 30 min à temperatura ambiente em um shaker, então 70% epóxi resina em etanol por 30 min à temperatura ambiente num agitador.</li>
		<li>Interruptor o epóxi resina solução com resina epóxi de 100% e incubar durante 10 min a 50 bolsas de executar 2 ° C. de resina epóxi de 100% fresco com incubação por 1h em 50 ° C. Adicionar fresco 100% epóxi resina e permite a endurecer a 50 ° C durante a noite e depois no 60 & #17 6. o; C por mais de 36 h endurecer.</li>
	</ol></li> <li>Retire a placa de muti cada amostra com um joalheiro &#39; Serra de s. Selecione as regiões de interesse, densas concentrações de células, usando um microscópio invertido e depois cortar blocos de 70 a 80 nm para secionar pelo micrótomo. Montar a região de corte no mandril de micrótomo e carregar o micrótomo. Posicione o chuck em micrótomo e montar uma faca de diamante com a borda paralela à superfície do bloco. Recolher seções de espessura de 70 a 80 nm diretamente sobre grades individuais.</li>
	<li>Dissolver acetato de uranilo em água para uma solução de 1%, em peso e separadamente dissolver o citrato de chumbo na água para uma solução de 3% em peso. Counterstain as seções por submersão com acetato de uranilo aquosa de 1% por 15 min e, em seguida, citrato de chumbo aquoso 3% por 5 min aumentar o contraste das amostras.</li>
	<li>Imagem EM <ol><li>examinar as seções com um microscópio eletrônico de transmissão e gravar imagens com uma câmera digital CCD em uma ampliação primária de 10.000-20, 000 X 3.</li>
	</ol></li> <li>Estatísticas <ol><li>executar um t-teste para identificar diferenças significativas comparando-se a média e o desvio-padrão de medida (no MEV mostrou) entre o controle e amostras experimentais.</li>
	</ol>
	</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+recycling+of+synaptic+vesicle+membrane+during+transmitter+release+at+the+frog+neuromuscular+junction.">Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction.</a> J Cell Biol. 57 (2), 315-344 (1973).</li><li>Ceccarelli, B., Hurlbut, W. P., Mauro, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turnover+of+transmitter+and+synaptic+vesicles+at+the+frog+neuromuscular+junction.">Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction.</a> J Cell Biol. 57 (2), 499-524 (1973).</li><li>Watanabe, S., Rost, B. R., 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=Ultrafast+endocytosis+at+mouse+hippocampal+synapses.">Ultrafast endocytosis at mouse hippocampal synapses.</a> Nature. 504 (7479), 242-247 (2013).</li><li>Watanabe, S., Trimbuch, 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=Clathrin+regenerates+synaptic+vesicles+from+endosomes.">Clathrin regenerates synaptic vesicles from endosomes.</a> Nature. 515 (7526), 228-233 (2014).</li><li>Sankaranarayanan, S., Atluri, P. P., Ryan, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+has+a+molecular+scaffolding,+not+propulsive,+role+in+presynaptic+function.">Actin has a molecular scaffolding, not propulsive, role in presynaptic function.</a> Nat Neurosci. 6 (2), 127-135 (2003).</li><li>Zhu, Y., Xu, J., Heinemann, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+pathways+of+synaptic+vesicle+retrieval+revealed+by+single-vesicle+imaging.">Two pathways of synaptic vesicle retrieval revealed by single-vesicle imaging.</a> Neuron. 61 (3), 397-411 (2009).</li><li>Miesenbock, G., De Angelis, D. A., Rothman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+secretion+and+synaptic+transmission+with+pH-sensitive+green+fluorescent+proteins.">Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.</a> Nature. 394 (6689), 192-195 (1998).</li><li>Arnao, M. B. B., Acosta, M., del Rio, J. A. A., Garc&#237;a-C&#225;novas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+peroxidase+by+hydrogen+peroxide+and+its+protection+by+a+reductant+agent.">Inactivation of peroxidase by hydrogen peroxide and its protection by a reductant agent.</a> Biochim Biophys Acta. 1038 (1), 85-89 (1990).</li><li>De&#225;k, F., Schoch, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptobrevin+is+essential+for+fast+synaptic-vesicle+endocytosis.">Synaptobrevin is essential for fast synaptic-vesicle endocytosis.</a> Nat Cell Biol. 6 (11), 1102-1108 (2004).</li><li>Clayton, E. L., Evans, G. J. O., Cousin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+synaptic+vesicle+endocytosis+is+rapidly+triggered+during+strong+stimulation.">Bulk synaptic vesicle endocytosis is rapidly triggered during strong stimulation.</a> J Neurosci. 28 (26), 6627-6632 (2008).</li><li>Kavalali, E. T., Jorgensen, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+presynaptic+function.">Visualizing presynaptic function.</a> Nat Neurosci. 17 (1), 10-16 (2014).</li><li>Wienisch, M., Klingauf, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+proteins+exocytosed+and+subsequently+retrieved+by+compensatory+endocytosis+are+nonidentical.">Vesicular proteins exocytosed and subsequently retrieved by compensatory endocytosis are nonidentical.</a> Nat Neurosci. 9 (8), 1019-1027 (2006).</li><li>Fern&#225;ndez-Alfonso, T., Kwan, R., Ryan, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+vesicles+interchange+their+membrane+proteins+with+a+large+surface+reservoir+during+recycling.">Synaptic vesicles interchange their membrane proteins with a large surface reservoir during recycling.</a> Neuron. 51 (2), 179-186 (2006).</li><li>Gimber, N., Tadeus, G., Maritzen, T., Schmoranzer, J., Haucke, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusional+spread+and+confinement+of+newly+exocytosed+synaptic+vesicle+proteins.">Diffusional spread and confinement of newly exocytosed synaptic vesicle proteins.</a> Nat Commun. 6, 8392 (2015).</li><li>Nicholson-Fish, J. C., Smillie, K. J., Cousin, M. 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Aqui demonstramos dois métodos para monitorar a endocitose da vesícula sináptica. O primeiro método, temos monitorado pHluorin fundiu-se com uma proteína de vesícula sináptica nos neurônios transfectadas e posteriormente eletricamente estimulado. Em segundo lugar, usamos EM imagem latente de captação HRP como induzida por KCl. Usamos diferentes estímulos por duas razões. Primeiro, aplicação elevada de potássio induz a despolarização dos neurônios todos na cultura. Isso facilita o exame EM, dado que a no...

Os neurotransmissores são armazenados em vesículas sinápticas e liberados por exocitose. A membrana da vesícula sináptica e a proteína são então internalizadas por endocitose e reutilizadas na próxima rodada de exocitose. Endocitose das vesículas sinápticas é importante para a manutenção de piscinas de vesícula sináptica e remove salientes vesículas de membrana plasmática. O pHluorin sensíveis ao pH proteína verde fluorescente, que é saciada em circunstâncias ácidas e dequenched em pH neutro, tem sido usado para medir tempo de endocitose cursos em vivem células1,2,3. A proteína de pHluorin normalmente é unida ao lado lumenal das proteínas de vesículas sinápticas, tais como Sinaptofisina ou VAMP2/synaptobrevin da. No resto, pHluorin é extinguido no lúmen 5.5 pH das vesículas sinápticas. Fusão de vesículas para a membrana plasmática apresenta o lúmen vesicular à solução extracelular onde o pH é ~ 7.3, resultando em um aumento na fluorescência do pHluorin. Após a exocitose, decompõe-se o aumento da fluorescência, devido a endocitose das proteínas de vesículas sinápticas seguido por re-acidificação de vesículas dentro dessas vesículas recuperadas. Embora a decadência reflete tanto a endocitose e a re-acidificação vesicular, principalmente reflete endocitose, porque re-acidificação é mais rápido que endocitose na maioria das condições1,4. A constante de tempo de re-acidificação é 3-4 s ou menos5,6, que é geralmente mais rápido que o 10 s ou mais necessários para vesículas endocitose4,5. Se experimentos são necessários para distinguir a endocitose de re-acidificação, ácidos resfriamento experimentos usando a solução de (MES) ácido 4-Morpholineethanesulfonic (25 mM) com um pH de 5,5 podem ser usados para determinar se as proteínas de vesículas sinápticas são recuperadas da membrana plasmática através de endocitose1,3,4. Assim, o aumento de intensidade de fluorescência pHluorin reflete um equilíbrio de exo e endocitose, e a diminuição após a estimulação do nervo especificamente reflete endocitose.
pHluorin imagem pode ser usada não apenas para medir o tempo de curso de endocitose, mas também o tamanho da vesícula sináptica piscinas7,8, e9de lançamento a probabilidade de lançamento evocado e espontânea. Muitos fatores e proteínas envolvidas na regulação da endocitose, tais como cálcio, solúvel NSF-acessório da proteína do receptor (SNARE) proteínas, neurotrófico derivado do cérebro factor(BDNF) e calcineurina foram identificadas usando pHluorin de imagens1 , 2 , 10 , 11 , 12 , 13 , 14 , 15 , 16. Além disso, a liberação do neurotransmissor pode ser detectada nos neurônios não apenas primários, mas em células de neuroblastoma com TIRFM17. Recentemente, pHTomato, dsRed, mOrange e variantes de pHluorin foram desenvolvidos para o monitoramento de gravações simultâneas de vários fatores em uma única sinapse18,19. Por exemplo, pHTomato tem sido fundido com Sinaptofisina e usado com um indicador de cálcio geneticamente codificado (GCaMP5K) para monitorar fusão de vesícula pré-sináptica e Ca2 + influxo no compartimento pós-sináptica20. Portanto, pHluorin ligado a proteínas sinápticas fornece um método útil para analisar a relação entre endocitose e exocitose.
É outro método comumente usado para estudar a endocitose, devido a alta resolução espacial que mostra as alterações ultra-estruturais durante a endocitose. Duas áreas gerais são a capacidade de visualizar as alterações patológicas dentro de células neuronais21 e faixa de proteínas de vesícula22. Em particular, a observação da absorção da vesícula sináptica, curvatura de membrana revestida por Clatrina na zona de periactive, e CDDP estruturas são possíveis com EM3,23,24,25 ,26,,27,28. Enquanto EM envolve artefatos potenciais, tais como malformações induzidas pelo fixador, que pode afetar a endocitose e análise de dados é de trabalho intensivo, que a resolução prevê uma oportunidade atraente para visualizar a estrutura celular. Potenciais problemas de fixador e a limitação em resolução temporal EM podem ser superados por alta pressão, congelamento, fornecendo um método rápido e não-químicas de estabilizar as delicadas estruturas presentes durante a endocitose27.

<table>
	<tbody>
		<tr>
			<td>Lipofectamine LTX with Plus</td>
			<td>Thermo Fisher</td>
			<td>15338-100</td>
			<td>Transfection of plasmid DNA including synaptophysin or VAMP2-pHluorin</td>
		</tr>
		<tr>
			<td>neurobasal medium</td>
			<td>Thermo Fisher</td>
			<td>21103-049</td>
			<td>Growth medium for neuron, Warm up to 37&#176;C before use</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Thermo Fisher</td>
			<td>17504-044</td>
			<td>Gradient for neuronal differentiation</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Thermo Fisher</td>
			<td>35050-061</td>
			<td>Gradient for neuronal culture</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine coated coverslip</td>
			<td>Neuvitro</td>
			<td>GG-25-pdl</td>
			<td>Substrate for neuronal growth and imaging of pHluorin</td>
		</tr>
		<tr>
			<td>Trypsin XI from bovine pancrease</td>
			<td>Sigma</td>
			<td>T1005</td>
			<td>Neuronal culture-digest hippocampal tissues</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I from bovine pancreas</td>
			<td>Sigma</td>
			<td>D5025</td>
			<td>Neuronal culture-inhibits viscous cell suspension</td>
		</tr>
		<tr>
			<td>pulse stimulator</td>
			<td>A-M systems</td>
			<td>model 2100</td>
			<td>Apply electrical stimulation</td>
		</tr>
		<tr>
			<td>Slotted bath with field stimulation</td>
			<td>Warner Instruments</td>
			<td>RC-21BRFS</td>
			<td>Apply electrical stimulation</td>
		</tr>
		<tr>
			<td>stimulus isolation unit</td>
			<td>Warner Instruments</td>
			<td>SIU102</td>
			<td>Apply electrical stimulation</td>
		</tr>
		<tr>
			<td>lubricant</td>
			<td>Dow corning</td>
			<td>111</td>
			<td>pHluorin imaging-seal with coverslip and imaging chamber, avoid leak from chamber</td>
		</tr>
		<tr>
			<td>AP5</td>
			<td>Tocris</td>
			<td>3693</td>
			<td>Gradient for normal saline, selective NMDA receptor antagonist, inhibit postsynaptic activity which have potential for recurrent activity</td>
		</tr>
		<tr>
			<td>CNQX</td>
			<td>Tocris</td>
			<td>190</td>
			<td>Gradient for normal saline, competitive AMPA/kainate receptor antagonist, inhibit postsynaptic activity which have potential for recurrent activity</td>
		</tr>
		<tr>
			<td>Illuminator</td>
			<td>Nikon</td>
			<td>C-HGFI</td>
			<td>Metal halide light source for pHluorin</td>
		</tr>
		<tr>
			<td>EMCCD camera</td>
			<td>Andor</td>
			<td>iXon3</td>
			<td>pHluorin imaging, detect pHluorin fluorescence intensity</td>
		</tr>
		<tr>
			<td>Inverted microscopy</td>
			<td>Nikon</td>
			<td>Ti-E</td>
			<td>Imaging for synaptophysin or VAMP2 pHluorin transfected cells</td>
		</tr>
		<tr>
			<td>NIS-Elements AR</td>
			<td>Nikon</td>
			<td>NIS-Elements Advanced Research</td>
			<td>Software for imaging acquisition and analysis</td>
		</tr>
		<tr>
			<td>Igor Pro</td>
			<td>WaveMetrics</td>
			<td>Igor pro</td>
			<td>Software for imaging analysis and data presentation</td>
		</tr>
		<tr>
			<td>imaging chamber</td>
			<td>Warner Instruments</td>
			<td>RC21B</td>
			<td>pHluorin imaging, apply field stimulation on living cells</td>
		</tr>
		<tr>
			<td>poly-l-lysine</td>
			<td>Sigma</td>
			<td>P4832</td>
			<td>Electron microscopy, substrate for neuronal growth, apply on multiwell plate for 1 h at room temperature then wash with sterilized water 3 times</td>
		</tr>
		<tr>
			<td>Horseradish peroxidase(HRP)</td>
			<td>Sigma</td>
			<td>P6782</td>
			<td>Electron microscopy, labeling of endocytosed synaptic vesicles by catalyzing DAB in presence hydrogen peroxide, final concentration is 5 mg/mL in normal saline, make fresh before use</td>
		</tr>
		<tr>
			<td>Na cacodylate</td>
			<td>Electron Microscopy Sciences</td>
			<td>12300</td>
			<td>Electron microscopy, buffer for fixatives and washing, final concentration is 0.1 N</td>
		</tr>
		<tr>
			<td>3,3&#8242;-Diaminobenzidine(DAB)</td>
			<td>Sigma</td>
			<td>D8001</td>
			<td>Electron microscopy, labeling of endocytosed synaptic vesicles, substrate for HRP, final concentration is 0.5 mg/mL in DDW and filtered, make fresh before use</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide solution</td>
			<td>Sigma</td>
			<td>H1009</td>
			<td>Electron microscopy, labeling of endocytosed synaptic vesicles by inducing HRP-DAB reaction, final concentration is 0.3% in DDW, make fresh before use</td>
		</tr>
		<tr>
			<td>glutaraldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>16365</td>
			<td>Electron microscopy, fixatives, final concentration is 4% in Na-cacodylate buffer, make fresh before use, shake well before to use</td>
		</tr>
		<tr>
			<td>TEM</td>
			<td>JEOL</td>
			<td>200CX</td>
			<td>Electron microscopy, imaging of endocytosed vesicles and ultrastructural changes</td>
		</tr>
		<tr>
			<td>CCD digital camera</td>
			<td>AMT</td>
			<td>XR-100</td>
			<td>Electron microscopy, capturing images</td>
		</tr>
		<tr>
			<td>Lead citrate</td>
			<td>Leica microsystems</td>
			<td>16707235</td>
			<td>Electron microscopy, grid staining</td>
		</tr>
	</tbody>
</table>

measuring synaptic vesicle endocytosis in cultured hippocampal neurons

Durante a endocitose, fundidas vesículas sinápticas são recuperadas em terminais nervosos, permitindo a reciclagem de vesículas e, portanto, a manutenção da transmissão sináptica durante o disparo repetitivo do nervo. Endocitose prejudicada em condições patológicas leva a diminuições em funções sinápticas de força e cérebro. Aqui, descrevemos os métodos utilizados para medir a endocitose da vesícula sináptica na sinapse hippocampal mamífera na cultura neuronal. Temos monitorado endocitose de proteínas de vesícula sináptica pela fusão de uma proteína de membrana vesicular sináptica, incluindo Sinaptofisina e VAMP2/synaptobrevin da, o lado lumenal vesicular, com pHluorin, uma proteína fluorescente verde sensíveis ao pH que aumenta sua intensidade de fluorescência como o pH aumenta. Durante a exocitose, pH do lúmen vesicular aumenta, Considerando que durante o lúmen vesicular endocitose pH é re-acidificado. Assim, um aumento da intensidade de fluorescência pHluorin indica fusão, enquanto uma diminuição indica endocitose da proteína rotulados vesícula sináptica. Além de usar o pHluorin método de imagem para gravar endocitose, temos monitorado endocitose da membrana vesicular por microscopia de elétron (EM) medições de absorção de Horseradish peroxidase (HRP) por vesículas. Finalmente, temos monitorado a formação dos poços de membrana terminal do nervo em vários momentos após a despolarização induzida por potássio alto. O tempo de curso de formação de poço de captação e membrana HRP indica o tempo de curso de endocitose.

Usando o método de transportadora de lipídios, SpH foi expressa em neurônios hippocampal, permitindo a identificação de boutons (Figura 1a). Estimulação elétrica das células induzidas por exocitose e um correspondente aumento na intensidade de fluorescência. O aumento na fluorescência (ΔF) foi interrompido por encerrar o estímulo (Figura 1b). O aumento da fluorescência foi seguido por uma diminuição lenta, devido ...

Watch this Scientific Journal Video about Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons at JoVE.com

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

Endocitose da vesícula sináptica é detectada pela microscopia de luz de pHluorin fundido com proteínas de vesículas sinápticas e por microscopia eletrônica de absorção da vesícula.

Medindo a endocitose da vesícula sináptica nos neurônios Hippocampal culturas

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

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Neuroscience

9.8K visualizações.  National Institute of Neurological Disorders and Stroke. Endocitose da vesícula sináptica é detectada pela microscopia de luz de pHluorin fundido com proteínas de vesículas sinápticas e por microscopia eletrônica de absorção da vesícula.

Vídeo: Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye 1; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K+). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1Ai-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1Aiv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining).

Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy 2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.

FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.

Estudando Pools vesículas Synaptic usando fotoconversão de Corantes Styryl

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

Neurociência

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.

Recuperação de fluorescência Após Fotodegradação (FRAP) de fluorescência Proteínas Tagged em espinhas dendríticas dos neurônios do hipocampo Cultivadas

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

Análise de Morfologia Spine dendríticas em neurônios cultivados CNS

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

Análise quantitativa de Reposição da vesícula sináptica Piscina em Cultured neurônios granulares do cerebelo usando Dyes FM

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

Induzir o crescimento dendrítico no cultivadas neurônios simpáticos

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

Difusão lateral e Exocitose de proteínas de membrana em neurônios cultivados avaliada através da recuperação de fluorescência e fluorescência perda de fotobranqueamento

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

Detecção de palmitoylation proteína em neurônios cultivados por imunoprecipitação e Acil-Biotina Exchange (ABE)

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

Ensaio danos no DNA em neurônios do hipocampo Usando o Ensaio Cometa

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

Pós-incorporação de Etiquetagem immunogold de proteínas sinápticas em culturas de hipocampo

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using styryl FM dyes, which reveal membrane turnover. Conventional protocols for the use of FM dyes were designed for analyzing neurons following stimulated (evoked) synaptic activity. Recently, protocols have become available for analyzing the FM signals that accompany weaker synaptic activities, such as spontaneous or miniature synaptic events. Analysis of these small changes in FM signals requires that the imaging system is sufficiently sensitive to detect small changes in intensity, yet that artifactual changes of large amplitude are suppressed. Here we describe a protocol that can be applied to evoked, spontaneous, and miniature synaptic activities, and use cultured hippocampal neurons as an example. This protocol also incorporates a means of assessing the rate of photobleaching of FM dyes, as this is a significant source of artifacts when imaging small changes in intensity.

We describe the use of styryl FM dyes to image synaptic vesicle recycling in functional nerve terminals. This protocol can be applied not only to evoked, but also spontaneous and miniature synaptic activities. The protocol expands the variety of synaptic events that can be effectively evaluated.

Exame do Synaptic Vesicle Reciclagem Usando FM Corantes Durante Evocado, espontânea, e as atividades de Synaptic miniatura

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using ...