Agradecemos Dr. Yong Ling Zhu prevén construir pHluorin2x synaptophysin y el Dr. James E. Rothman proporcionando VAMP2-phluorin. Agradecemos a Dr. Susan Cheng y Virginia Crocker de NINDS microscopia electrónica por su soporte técnico y ayuda. Este trabajo fue financiado por el Instituto Nacional de desórdenes neurológicos y movimiento programa de investigación intramuros en Estados Unidos y una beca del programa de iniciativa de KRIBB en investigación (Coreano biomédico científico programa de becas), Instituto de investigación de Corea de Biociencia y biotecnología, República de Corea.

Nota: el siguiente protocolo describe los métodos de proyección de imagen de pHluorin y métodos EM en neuronas hippocampal cultivadas. pHluorin monitores vesícula sináptica proteína asimilación en las células vivas y EM detecta la absorción de la vesícula sináptica y cambios ultraestructurales.
 cuidado de los animales y procedimiento seguido las pautas de NIH y fueron aprobados por el Comité de uso y cuidado de animales de NIH.
 1. pHluorin imágenes <ol><li>cultura Hippocampal neuron <ol><li>preparar Buffer de hipocampo (HB) mediante la combinación de 4 mM NaHCO 3 y 5 mM HEPES y ajustar el pH a 7.3 con 5 M NaOH. Hacer el medio de cultivo mediante la mezcla de neurobasal medio, 2% B27, 0,5 mM L-glutamina y 1% de penicilina-estreptomicina. Además, preparar una mezcla de tampón de HB con el 20% de suero bovino Fetal (FBS HB/20%). 
		Nota: Este protocolo de cultivo se basa en Sankaranarayanan, et al. 29 y Wu et al. 24</li> <li>decapitar a crías de ratón entre postnatal día 0 a día 2 en medio de cultivo y extraer el cerebro en 4 ° C HB/20% FBS. Quitar el tronco encefálico y el tálamo para exponer a los hipocampos. Diseccionar a los hipocampos, después de exponer por el retiro del tallo cerebral y tálamo y transferir a fresca 4 ° C HB/20% FBS. 
		Nota: Normalmente, el rendimiento es alrededor de 4 x 10 5 células/mL para un cachorro (dos hipocampos). Limpie las membranas adherentes utilizando unas pinzas o tijeras, luego transferir a fresca 4 ° C HB/20% FBS. Desenrolle el giro dentado y aislar el subiculum cortando con unas tijeras, luego transferir a fresca 4 ° C HB/20% FBS.</li>
		<li>Dividir cada hipocampo, cortando de un extremo a extremo, en rodajas de unos 10 y transferirlos a un tubo cónico de polipropileno de 15 mL.</li>
		<li>Después de permitir que el tejido colocar, lavar con 10 mL de SBF HB/20% y luego lavar tres veces con 10 mL de HB.</li>
		<li>Preparar la solución de digestión de 137 mM NaCl, KCl de 5 mM, 7 mM Na2HPO 4 y 25 mM HEPES y ajustar pH a 7.2 con NaOH M 5. Retire el sobrenadante de los hipocampos. Añadir 10 mg de tripsina y 1 mg de la ADNsa a 2 mL de solución de digestión y filtrar a través de una membrana de 0,22 μm estéril directamente en el sedimento de la muestra.</li>
		<li>Incubar los hipocampos por 5 min a 37 ° C, luego lavar dos veces con 10 mL de SBF HB/20% y luego lavar una vez con 10 mL de HB.</li>
		<li>Disociar las células con 6 mg de MgSO 4 &#8729; 7 H 2 O y 1 mg de la ADNsa 2 mL HB y filtro estéril en los hipocampos de pellets a través de un filtro estéril de 0,22 μm. Suavemente disociar las células por trituración cuidadosa, mientras que teniendo cuidado de no introducir burbujas de aire. Permitir que las partículas de tejido a reposar 2 min y entonces lentamente transfiera el sobrenadante a otro tubo.</li>
		<li>Añadir 3 mL HB/20% FBS a la suspensión de la célula y centrífuga o 10 min a 4 ° C y 1.000 rpm. Deseche el sobrenadante y resuspender en medio de cultivo.</li>
		<li>Placa de 60.000 células suspendidas en 150 μL de medio de cultivo en un cubreobjetos de 25 mm de diámetro recubierto de Poly-D-lisina, sin derrame de cubreobjetos. Añadir 2 mL de medio de cultivo con 2 h después de la galjanoplastia. Cubreobjetos se mantienen en placas cultivo de 6 pocillos o platos de Petri estéril.</li>
		<li>Mantener las células a 37 ° C en un 5% CO 2 humidificado incubadora en medio de cultivo durante 14-21 días antes de la grabación. Durante el crecimiento de la cultura, cambiar la mitad superior del medio dos veces a la semana.</li>
	</ol></li> <li>Transfección <ol><li>6-7 días después de la galjanoplastia, transfectar synaptophysin-pHluorin 2 X (SpH) o VAMP2-pHluorin en neuronas hippocampal. Para SpH, utilizar un promotor de citomegalovirus (CMV), insertado en un vector pcDNA3 de 30. Para VAMP2-pHluorin, use un promotor CMV insertado en un vector de pCI 31.</li>
		<li>Transfectar cualquier vector por el portador del lípido en las células diana, con 1 μg de plásmido. Utilizar medio de cultivo de paso protocolo 1.1.1, que carece de suero, para la transfección. Cambiar el medio 2 h después de transfección para reducir toxicidad. 
		Nota: En caso de baja expresión de SpH en los botones, aumentar la concentración de ADN a 2 μg o el tiempo de incubación con el transportador lipídico, a menos que las células son poco saludables. Por lo general, de 4-10 células (0.006 0.008%) de las neuronas fueron transfectadas.</li>
	</ol></li> <li>Microscopía <ol><li>preparación de solución salina normal compuesta de 119 mM NaCl, CaCl 2, 2,5 mM de KCl, 25 mM HEPES (pH 7.4), glucosa de 30 mM, 2 mM de MgCl 2, (6-cyano-7-nitroquinoxaline-2,3-dione) 0,01 mM de 2 mM CNQX) y 0.05 m de ácido DL-2-amino-5-phosphonovaleric (AP5). Tomar un cubreobjetos de la placa de medio de cultivo y el lugar en una cámara de proyección de imagen que permite la estimulación de campo, usando lubricante y sellante para evitar fugas. Evitar que el vidrio seco durante la transferencia de un cubreobjetos de la placa de la cámara inmediatamente agregando solución salina normal de 750 μl. 
		Nota: CNQX y AP5 fueron utilizados para bloquear la actividad postsináptica, que tiene el potencial para la actividad recurrente. Antes de colocar una cámara en un microscopio de fluorescencia invertido, utilice limpieza tejido para confirmar que la cámara no gotear. Filtración lenta provoca cambios de enfoque durante la grabación por la mezcla de aceite salino e inmersión normal, que se traduce en cambios en el índice de refracción de.</li>
		<li>Estimulación y registro de inmersión de aceite (apertura numérica de 1.4) <ol><li>de imagen en un microscopio invertido de campo amplio con 60 X la lente con una lámpara de haluro de metal. Visualizar la pHluorin con un filtro para un pico de excitación de 480 nm, 490 nm pase largo espejo, un filtro de emisión de 500-550 nm y un obturador de tirón manual. Capturar imágenes cada 100 ms, con 2 x 2 binning, usando una cámara de electrones multiplicando carga acoplada dispositivo (EMCCD). 
			Nota: Varias características deben considerarse para lograr las condiciones adecuadas para evitar el fotoblanqueo, incluyendo el intervalo de captura de imagen y filtrar y binning, que dependen de equipos. En el caso de la proyección de imagen confocal, tiempo de exposición y potencia láser debe considerarse para evitar el fotoblanqueo. La configuración se determinó después de grabar al menos 3 minutos sin estímulo y la grabación fue realizada por al menos 10 s sin estímulo para comprobar fotoblanqueo.</li>
			<li>Elija un área con una alta densidad de botones para facilitar el análisis en cada experimento. Identificar células transfected por su señal de fluorescencia verde, con énfasis en los patrones de expresión redonda u ovalada en el bouton y expresión continua entre boutons.</li>
			<li>Endocitosis inducir por el estímulo de campo usando un 1 ms de pulso, 20 potencial de acción mA (AP) provee de un estimulador de impulsos y a través de un electrodo de platino dentro de la unidad de aislamiento del estímulo. Actividad de fluorescencia de imagen a lo largo de la estimulación y durante la recuperación celular. 
			Nota: En el caso de las células muertas, expresión era más fuerte que la vida botones y demostró un patrón desigual entre boutons.</li>
		</ol></li></ol></li> <li>Análisis de la imagen <ol><li>Para el análisis de la intensidad de fluorescencia en un sola bouton, establecer hasta una región de interés (ROI) como un 1.5 x 1.5 μm de cuadrados; el tamaño de un bouton está a unos 1,5 μm. uso los rastros antes de estimulación para comprobar fotoblanqueo y restar como fondo.</li>
		<li>Normalizar la fluorescencia cambia (ΔF) con la ecuación: <img alt="Equation" height="11" src="/files/ftp_upload/55862/55862eq1.jpg" width="172" /> donde F max y F 0 consulte aumento máximo después de la estimulación y de la fluorescencia basal, respectivamente. Medir tipos de endocitosis como la tasa de decaimiento durante los primeros 4 10 s después del punto máximo de fluorescencia pHluorin. Obtener la constante de tiempo (τ) de endocitosis encajando el decaimiento de la fluorescencia de pHluorin el aumento de pico a la línea de base con una función exponencial mono.</li>
	</ol></li></ol> 2. La microscopia electrónica <ol><li>Prepare una Poly-D-lisina cubierta placa de 6 pozos por aplicar 1.5 mL de solución estéril filtrada de Poly-D-lisina de 0.01% a cada pocillo por 1 h a temperatura ambiente, luego se lava tres veces con agua esterilizada. Disecar, la cultura y mantener las neuronas hippocampal en pasos 1.1.1, 1.1.2 y 1.1.3, respectivamente.</li>
	<li>Prepare solución de estimulación de alta K + con HRP como 31,5 mM NaCl, CaCl 2, de 2 mM 90 mM KCl, 25 mM HEPES (pH 7.4), glucosa de 30 mM, 2 mM de MgCl 2, 0,01 mM CNQX, 0,05 mM AP5 y 5 mg/mL HRP, luego ajustan a pH 7,4 con 5 M NaOH.
	Solución para estimulación K + <ol><li>estimular la cultura de neurona del hipocampo con 1,5 mL de alta temperatura (denominada K +) por la adición de 1,5 mL a cada pozo de 90 s. En la condición de reposo (denominada R), se aplica la misma concentración de 5 mg/mL HRP por 90 s, pero con solución salina normal. Para la muestra de recuperación, aplicar solución de estimulación alta K + como con la muestra de K +, entonces rápidamente lavado sustituir con solución salina normal e incubar durante 10 minutos</li>
	</ol></li> <li>Fijación y coloración <ol><li>preparar buffer de cacodilato de Na de 0.1 M con 21,4 g/L Na cacodilato a pH 7,4. Fijar las células con glutaraldehído al 4% en buffer de cacodilato de Na de 0.1 M por al menos 1 h a temperatura ambiente. Lavar tres veces con buffer de cacodilato de Na de 0.1 M por 7 min.</li>
		<li>Solución preparar diaminobenzidina (DAB), de 0,5 mg/mL de lenguado con 0.3% H 2 O 2 en ddH 2 O y el filtro con filtro de 0.22 μm. Aplique solución DAB 1.5 mL durante 30 min a 37 ° C. lavado tres veces con buffer de cacodilato de Na de 0.1 M por 7 min. 
		PRECAUCIÓN: DAB es un carcinógeno tóxico y sospecha. Utilice guantes y batas de laboratorio. 
		Nota: Etiquetado con el lenguado se produce debido a su oxidación por H 2 O 2, como catalizada por HRP. Pequeños incrementos en el resultado de los componentes de una señal creciente en la muestra. Aumento de la concentración de HRP acelera el efecto del catalizador. Suficientemente grandes concentraciones de H 2 O 2 permiten debilitante reacciones secundarias con HRP, inhibiendo el efecto de etiquetado 32. En este trabajo, las concentraciones de este sistema de etiquetado fueron seleccionadas en base a investigaciones actualmente disponibles 33 , 34.</li>
		<li>Incubar las neuronas con 1,5 mL de 1% OsO 4 en buffer de cacodilato de Na de 0.1 M por 1 h a 4 ° C como fijación. Lavar tres veces con 1,5 mL de buffer de cacodilato de Na de 0.1 M por 7 min. 
		PRECAUCIÓN: Debido a la toxicidad y reactividad de OsO 4, manteniendo la muestra en hielo en una campana química es preferible en muchos casos a utilizar una nevera para la incubación.</li>
		<li>Preparar 0.1 M tampón de acetato de sodio con 13,61 g/L sodio acetato y 11,43 mL/L de ácido acético glacial a pH 5,0. Lavar tres veces con tampón de acetato 1,5 mL de 0.1 M a pH 5.0 por 7 min e incubar con acetato de uranilo 1.5 mL de 1% en buffer de acetato de 0,1 M a pH 5,0 por 1 h a 4 ° C. Wash tres veces con tampón de acetato de 0,1 M 1,5 mL por 7 min.</li>
	</ol></li> <li>Epoxy incrustar <ol><li>deshidratar la cultura neurona con lavados de solo 1,5 mL de etanol 50%, 70% y 90%, durante 7 minutos en cada uno y luego 3 lavados de 1,5 mL de etanol de 100% durante 7 min en una campana de humos.</li>
		Bisphenol-A-(epichlorhydrin) de 485 mL/L de mezcla de <li>resina de epoxy, 160 mL/L dodecenilo succínico, anhídrido (DDSA), de 340 mL/L-metil-5-Norbornene-2, 3-Dicarboxylic fenol 2,4,6-tris (dimethylaminomethyl) anhídrido (NMA) y 15 mL/L (DMP-30) para crear el epoxi resina. Mezcla bien, luego almacenar bajo vacío para eliminar las burbujas de aire. 
		Nota: Es fundamental para eliminar las burbujas de aire en la resina, especialmente los inferiores visibles a simple vista, ya que pueden causar caries en la resina durante el seccionamiento.</li>
		<li>La muestra sustituyendo el etanol con 50% de epoxy de la resina en etanol por 30 min a temperatura ambiente en un agitador, entonces el 70% de epoxy resina en etanol durante 30 min a temperatura ambiente en un agitador infiltrado.</li>
		<li>Interruptor el epoxi resina solución con resina de epoxy del 100% e incubar 10 min a intercambios de realizar dos de 50 ° C. de resina epoxi 100% fresco con incubaciones por 1 h a 50 ° C. agregar dulce 100% de epoxy resina y deje que se endurezca a 50 ° C durante la noche y luego en el 60 y el #17 6; C de más de 36 h a.</li>
	</ol></li> <li>Con un joyero, retire cada muestra de la placa de la pozo muti &#39; Serrucho s. Seleccione las regiones de interés, las concentraciones densas de células, utilizando un microscopio de luz invertido y luego cortar bloques de 70 a 80 nm de seccionamiento por micrótomo. Monte de la región de corte en el mandril para Microtomo y carga el microtomo. Coloque el portabrocas en el microtomo y Monte un cuchillo del diamante con el borde paralelo a la superficie del bloque. Recoge secciones de espesor de 70 a 80 nm directamente sobre rejillas individuales.</li>
	<li>Acetato de uranilo se disuelven en agua para una solución al 1% en peso y por separado disolver citrato de plomo en el agua de una solución de 3% en peso. Contratinción las secciones por sumersión con acetato de uranilo acuosa 1% por 15 min y citrato de plomo acuosa 3% durante 5 minutos mejorar el contraste de las muestras.</li>
	<li>Proyección de imagen de EM <ol><li>examinar las secciones con un microscopio electrónico de transmisión y grabar imágenes con una cámara digital CCD a un aumento primario de 10.000-20, 000 X 3.</li>
	</ol></li> <li>Estadísticas <ol><li>realizar un t-test para identificar diferencias significativas al comparar la media y error estándar de medición (SEM) entre el control y las muestras experimentales.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Sankaranarayanan, S., 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=Real-time+measurements+of+vesicle-SNARE+recycling+in+synapses+of+the+central+nervous+system.">Real-time measurements of vesicle-SNARE recycling in synapses of the central nervous system.</a> Nature cell biol. 2 (4), 197-204 (2000).</li><li>Sun, T., Wu, X. 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=The+role+of+calcium/calmodulin-activated+calcineurin+in+rapid+and+slow+endocytosis+at+central+synapses.">The role of calcium/calmodulin-activated calcineurin in rapid and slow endocytosis at central synapses.</a> J Neurosci. 30 (35), 11838-11847 (2010).</li><li>Wu, X. -. S. S., Lee, S. H., 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=Actin+Is+Crucial+for+All+Kinetically+Distinguishable+Forms+of+Endocytosis+at+Synapses.">Actin Is Crucial for All Kinetically Distinguishable Forms of Endocytosis at Synapses.</a> Neuron. 92 (5), 1020-1035 (2016).</li><li>Granseth, B., Odermatt, B., Royle, S. J., Lagnado, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clathrin-mediated+endocytosis+is+the+dominant+mechanism+of+vesicle+retrieval+at+hippocampal+synapses.">Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses.</a> Neuron. 51 (6), 773-786 (2006).</li><li>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=The+kinetics+of+synaptic+vesicle+reacidification+at+hippocampal+nerve+terminals.">The kinetics of synaptic vesicle reacidification at hippocampal nerve terminals.</a> J Neurosci. 26 (8), 2313-2320 (2006).</li><li>Royle, S. J., Granseth, B., Odermatt, B., Derevier, A., Lagnado, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+phluorin-based+probes+at+hippocampal+synapses.">Imaging phluorin-based probes at hippocampal synapses.</a> Methods Mol Biol. 457, 293-303 (2008).</li><li>Moulder, K. L., Mennerick, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reluctant+vesicles+contribute+to+the+total+readily+releasable+pool+in+glutamatergic+hippocampal+neurons.">Reluctant vesicles contribute to the total readily releasable pool in glutamatergic hippocampal neurons.</a> J Neurosci. 25 (15), 3842-3850 (2005).</li><li>Li, Z., Burrone, J., Tyler, W. J., Hartman, K. N., Albeanu, D. F., Murthy, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+vesicle+recycling+studied+in+transgenic+mice+expressing+synaptopHluorin.">Synaptic vesicle recycling studied in transgenic mice expressing synaptopHluorin.</a> Proc Natl Acad Sci U S A. 102 (17), 6131-6136 (2005).</li><li>Morris, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elements+of+a+neurobiological+theory+of+hippocampal+function:+the+role+of+synaptic+plasticity,+synaptic+tagging+and+schemas.">Elements of a neurobiological theory of hippocampal function: the role of synaptic plasticity, synaptic tagging and schemas.</a> Eur J Neurosci. 23 (11), 2829-2846 (2006).</li><li>Sankaranarayanan, S., 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=Calcium+accelerates+endocytosis+of+vSNAREs+at+hippocampal+synapses.">Calcium accelerates endocytosis of vSNAREs at hippocampal synapses.</a> Nat Neurosci. 4 (2), 129-136 (2001).</li><li>Balaji, J., Armbruster, M., 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=Calcium+control+of+endocytic+capacity+at+a+CNS+synapse.">Calcium control of endocytic capacity at a CNS synapse.</a> J Neurosci. 28 (26), 6742-6749 (2008).</li><li>Ferguson, S. M., Brasnjo, G., 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=A+selective+activity-dependent+requirement+for+dynamin+1+in+synaptic+vesicle+endocytosis.">A selective activity-dependent requirement for dynamin 1 in synaptic vesicle endocytosis.</a> Science. 316 (5824), 570-574 (2007).</li><li>Baydyuk, M., Wu, X. S., He, L., Wu, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-derived+neurotrophic+factor+inhibits+calcium+channel+activation,+exocytosis,+and+endocytosis+at+a+central+nerve+terminal.">Brain-derived neurotrophic factor inhibits calcium channel activation, exocytosis, and endocytosis at a central nerve terminal.</a> J Neurosci. 35 (11), 4676-4682 (2015).</li><li>Wu, X. S., Zhang, Z., Zhao, W. D., Wang, D., Luo, F., Wu, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcineurin+is+universally+involved+in+vesicle+endocytosis+at+neuronal+and+nonneuronal+secretory+cells.">Calcineurin is universally involved in vesicle endocytosis at neuronal and nonneuronal secretory cells.</a> Cell Rep. 7 (4), 982-988 (2014).</li><li>Zhang, Z., Wang, D., 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=The+SNARE+proteins+SNAP25+and+synaptobrevin+are+involved+in+endocytosis+at+hippocampal+synapses.">The SNARE proteins SNAP25 and synaptobrevin are involved in endocytosis at hippocampal synapses.</a> J Neurosci. 33 (21), 9169-9175 (2013).</li><li>Wu, L. -. G. G., Hamid, E., Shin, W., Chiang, H. -. C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exocytosis+and+endocytosis:+modes,+functions,+and+coupling+mechanisms.">Exocytosis and endocytosis: modes, functions, and coupling mechanisms.</a> Annu Rev Physiol. 76 (1), 301-331 (2014).</li><li>Daniele, F., Di Cairano, E. S., Moretti, S., Piccoli, G., Perego, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TIRFM+and+pH-sensitive+GFP-probes+to+evaluate+neurotransmitter+vesicle+dynamics+in+SH-SY5Y+neuroblastoma+cells:+cell+imaging+and+data+analysis.">TIRFM and pH-sensitive GFP-probes to evaluate neurotransmitter vesicle dynamics in SH-SY5Y neuroblastoma cells: cell imaging and data analysis.</a> J Vis Exp. (95), e52267 (2015).</li><li>Shaner, N. C., Lin, M. Z., 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=Improving+the+photostability+of+bright+monomeric+orange+and+red+fluorescent+proteins.">Improving the photostability of bright monomeric orange and red fluorescent proteins.</a> Nat Methods. 5 (6), 545-551 (2008).</li><li>Li, Y., Tsien, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pHTomato,+a+red,+genetically+encoded+indicator+that+enables+multiplex+interrogation+of+synaptic+activity.">pHTomato, a red, genetically encoded indicator that enables multiplex interrogation of synaptic activity.</a> Nat Neurosci. 15 (7), 1047-1053 (2012).</li><li>Leitz, J., Kavalali, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+retrieval+and+autonomous+regulation+of+single+spontaneously+recycling+synaptic+vesicles.">Fast retrieval and autonomous regulation of single spontaneously recycling synaptic vesicles.</a> Elife. 3, e03658 (2014).</li><li>Bisht, K., El Hajj, H., Savage, J. C., S&#225;nchez, M. G., Tremblay, M. -. &. #. 2. 0. 0. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlative+Light+and+Electron+Microscopy+to+Study+Microglial+Interactions+with+&#946;-Amyloid+Plaques.">Correlative Light and Electron Microscopy to Study Microglial Interactions with &#946;-Amyloid Plaques.</a> J Vis Exp. (112), e54060 (2016).</li><li>Schikorski, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+Synaptic+Vesicle+Protein+Sorting+with+Enhanced+Horseradish+Peroxidase+in+the+Electron+Microscope.">Monitoring Synaptic Vesicle Protein Sorting with Enhanced Horseradish Peroxidase in the Electron Microscope.</a> High-Resolution Imaging of Cellular Proteins: Methods and Protocols. , 327-341 (2016).</li><li>Kononenko, N. L., Puchkov, D., 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/AP-2+mediate+synaptic+vesicle+reformation+from+endosome-like+vacuoles+but+are+not+essential+for+membrane+retrieval+at+central+synapses.">Clathrin/AP-2 mediate synaptic vesicle reformation from endosome-like vacuoles but are not essential for membrane retrieval at central synapses.</a> Neuron. 82 (5), 981-988 (2014).</li><li>Wu, Y., O'Toole, E. 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=A+dynamin+1-,+dynamin+3-+and+clathrin-independent+pathway+of+synaptic+vesicle+recycling+mediated+by+bulk+endocytosis.">A dynamin 1-, dynamin 3- and clathrin-independent pathway of synaptic vesicle recycling mediated by bulk endocytosis.</a> eLife. 2014 (3), e01621 (2014).</li><li>Heuser, J. E., Reese, T. 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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+activity-dependent+bulk+endocytosis+with+the+genetically-encoded+reporter+VAMP4-pHluorin.">Monitoring activity-dependent bulk endocytosis with the genetically-encoded reporter VAMP4-pHluorin.</a> J Neurosci Methods. 266, 1-10 (2016).</li><li>Burrone, J., Li, Z., Murthy, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+vesicle+cycling+in+presynaptic+terminals+using+the+genetically+encoded+probe+synaptopHluorin.">Studying vesicle cycling in presynaptic terminals using the genetically encoded probe synaptopHluorin.</a> Nat Protoc. 1 (6), 2970-2978 (2006).</li><li>Wisse, E., Braet, F., 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=Fixation+methods+for+electron+microscopy+of+human+and+other+liver.">Fixation methods for electron microscopy of human and other liver.</a> World journal of gastroenterology. 16 (23), 2851-2866 (2010).</li><li>Magidson, V., Khodjakov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circumventing+Photodamage+in+Live-Cell+Microscopy.">Circumventing Photodamage in Live-Cell Microscopy.</a> Methods in Cell Biology. 114, 545-560 (2013).</li><li>S&#248;ndergaard, C. R., Garrett, A. E., 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=Structural+artifacts+in+protein-ligand+X-ray+structures:+implications+for+the+development+of+docking+scoring+functions.">Structural artifacts in protein-ligand X-ray structures: implications for the development of docking scoring functions.</a> J Med Chem. 52 (18), 5673-5684 (2009).</li></ol>

Los autores no tienen nada que revelar.

Aquí muestran dos métodos para el monitoreo de endocitosis vesícula sináptica. En el primer método, supervisamos pHluorin fusionado con una proteína de la vesícula sináptica en neuronas transfected y posteriormente eléctricamente estimulados. En segundo lugar, utilizamos proyección de imagen de EM de la absorción HRP como inducidas por KCl. Utilizamos diversos estímulos por dos razones. En primer lugar, aplicación de alto potasio induce la despolarización de todas las neuronas en la cultura. Esto facilita e...

Neurotransmisores se almacenan en vesículas sinápticas y liberados por exocitosis. La membrana de la vesícula sináptica y proteína son internalizados por endocitosis y reutilizadas en la próxima ronda de exocitosis. Endocitosis de vesículas sinápticas es importante para el mantenimiento de piscinas de la vesícula sináptica y quitan vesículas que sobresale de la membrana plasmática. La pHluorin pH-sensible a la proteína verde fluorescente, que se apagó en condiciones ácidas y dequenched en un pH neutro, se ha utilizado para medir el tiempo de endocitosis cursos en viven las células1,2,3. La proteína pHluorin se une típicamente al lado de las proteínas de la vesícula sináptica, como la sinaptofisina o VAMP2/synaptobrevin lumenal. En el resto, pHluorin se apaga en el lumen de pH 5.5 de vesículas sinápticas. Fusión de la vesícula a la membrana plasmática expone el lumen vesicular a la solución extracelular donde el pH es ~ 7.3, resultando en un incremento en la fluorescencia pHluorin. Después de la exocitosis, la fluorescencia creciente decae, debido a la endocitosis de las proteínas de la vesícula sináptica seguido de acidificación la vesícula dentro de las vesículas recuperadas. Aunque el decaimiento refleja endocitosis y acidificación vesicular de nuevo, en su mayoría refleja endocitosis, porque la acidificación es más rápida que la endocitosis en la mayoría condiciones1,4. La constante de tiempo de la acidificación es 3-4 s o menos5,6, que es generalmente más rápido que los 10 s o más necesarios para la endocitosis de vesículas4,5. Si los experimentos son necesarios para distinguir la endocitosis de la acidificación, experimentos de amortiguamiento ácidos con la solución de (MES) ácida 4-Morpholineethanesulfonic (25 mM) con un pH de 5.5 pueden utilizarse para determinar si las proteínas de la vesícula sináptica se recuperan de la membrana plasmática vía endocitosis1,3,4. Así, el aumento de intensidad de fluorescencia de pHluorin refleja un equilibrio de exo y endocitosis, y la disminución después de la estimulación nerviosa refleja específicamente endocitosis.
pHluorin la proyección de imagen puede utilizarse no sólo para medir el curso temporal de la endocitosis, pero también el tamaño de la vesícula sináptica piscinas7,8, y la probabilidad de liberación evocada y espontánea libertad9. Muchos factores y proteínas implicadas en la regulación de endocitosis, tales como calcio, proteínas solubles de receptores (trampa) de la proteína del NSF-accesorio, factor(BDNF) neurotrófico derivado del cerebro y calcineurina han sido identificadas usando proyección de imagen de pHluorin1 , 2 , 10 , 11 , 12 , 13 , 14 , 15 , 16. por otra parte, se podría detectar liberación de neurotransmisores en las neuronas no sólo primarias, sino en las células del neuroblastoma con TIRFM17. Recientemente, pHTomato, dsRed, mOrange y variantes de pHluorin fueron desarrollados para el monitoreo de grabaciones simultáneas de múltiples factores en una sola sinapsis18,19. Por ejemplo, pHTomato ha sido fusionado con synaptophysin y utilizado con un indicador de calcio genéticamente codificados (GCaMP5K) para controlar la fusión de la vesícula presináptica y Ca2 + afluencia en el compartimiento postsináptico20. Por lo tanto, pHluorin unida a las proteínas sinápticas proporciona un método útil para analizar la relación entre endocitosis y exocitosis.
EM es otro método comúnmente utilizado para el estudio de endocitosis, debido a la alta resolución espacial que muestra cambios ultraestructurales durante la endocitosis. Dos áreas generales son la capacidad de visualizar los cambios patológicos dentro de las células neuronales21 y seguir vesícula proteínas22. En particular, la observación de la absorción de la vesícula sináptica, curvatura de la membrana recubierta por clatrina de la zona de periactive y endosomal estructuras son posibles con EM3,23,24,25 ,26,27,28. Mientras que EM comprende artefactos potenciales, tales como malformaciones inducidas por el fijador, que puede afectar la endocitosis, y análisis de datos es mano de obra, que la resolución ofrece una atractiva oportunidad para visualizar la estructura celular. Posibles problemas de fijador y la limitación en la resolución temporal de EM pueden superarse por la alta presión de congelación, proporcionando un método rápido y no químicos de estabilización de las delicadas estructuras presentes durante la endocitosis27.

<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 la endocitosis, vesículas sinápticas fundidas se recuperan en los terminales del nervio, permitiendo para el reciclaje de la vesícula y así el mantenimiento de la transmisión sináptica durante la cocción del repetidor del nervio. Endocitosis deteriorada en condiciones patológicas conduce a disminuciones en las funciones de cerebro y fuerza sinápticas. Aquí, describimos los métodos utilizados para medir la endocitosis de vesículas sinápticas en la sinapsis hippocampal mamífera en cultivo neuronal. Supervisamos la endocitosis de la proteína de la vesícula sináptica mediante la fusión de una proteína de la membrana vesicular sináptica, incluyendo synaptophysin y VAMP2/synaptobrevin, en el lado lumenal de vesicular, con pHluorin, una proteína fluorescente verde sensibles al pH que aumenta su intensidad de fluorescencia como el pH aumenta. Durante la exocitosis, pH del lumen vesicular aumenta, mientras que durante lumen vesicular endocitosis es acidificado a pH. Así, un aumento de la intensidad de la fluorescencia pHluorin indica fusión, mientras que indica que una disminución de la endocitosis de la proteína de la vesícula sináptica con. Además de utilizar el método de proyección de imagen de pHluorin para grabar endocitosis, supervisamos endocitosis la membrana vesicular por microscopia electrónica (EM) medidas de la absorción de peroxidasa (HRP) de rábano por vesículas. Por último, controla la formación de hoyos de membrana terminal del nervio en las varias horas después de la despolarización inducida por potasio alto. El curso del tiempo de formación de HRP absorción y membrana pit indica el curso del tiempo de endocitosis.

Usando el método del portador lípido, SpH se expresó en neuronas hipocampales, lo que permite la identificación de los botones (Figura 1a). Estimulación eléctrica de las células inducido por exocitosis y un correspondiente aumento en la intensidad de la fluorescencia. El aumento de fluorescencia (ΔF) fue detenido por fin el estímulo (Figura 1b). El aumento de la fluorescencia fue seguida por un lento descenso, debido a l...

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

Endocitosis de vesículas sinápticas se detectan por microscopia ligera de pHluorin fusionado con la proteína de la vesícula sináptica y por microscopia electrónica de la absorción de la vesícula.

Medición de endocitosis vesícula sináptica en neuronas Hippocampal cultivadas

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

measuring-synaptic-vesicle-endocytosis-in-cultured-hippocampal-neurons

Research

JoVE Journal

Neuroscience

9.8K vistas.  National Institute of Neurological Disorders and Stroke. Endocitosis de vesículas sinápticas se detectan por microscopia ligera de pHluorin fusionado con la proteína de la vesícula sináptica y por microscopia electrónica de la absorción de la vesícula.

Video: 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.

El estudio de Piscinas vesícula sináptica con fotoconversión de tintes estirilo

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

Investigación

Neurociencias

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.

La recuperación de fluorescencia después de photobleaching (FRAP) de fluorescencia de proteínas marcadas en las espinas dendríticas de las neuronas del 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.

Análisis de la morfología espina dendrítica en las neuronas del SNC cultivadas

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.

Análisis cuantitativo de Reposición Synaptic piscina vesículas en cultivos de neuronas granulares del cerebelo con tintes 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.

Inducir el crecimiento dendríticas en las neuronas simpáticas cultivadas

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.

Difusión lateral y exocitosis de proteínas de membrana en neuronas cultivadas evaluó a través de la recuperación de fluorescencia y la pérdida de fluorescencia photobleaching

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.

La detección de palmitoilación proteínas en neuronas del hipocampo cultivadas por inmunoprecipitación e Intercambio Acyl-Biotina (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.

Ensayo de daño del ADN en las neuronas del hipocampo utilizando el ensayo 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.

Post-Etiquetado Inmunoelectrónica incorporación de proteínas sinápticas en cultivos de cortes 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.

El examen de la vesícula sináptica Reciclaje utilizando tintes FM Durante Evocados, espontánea, y actividades sinápticas miniatura

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