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

Todos los experimentos con animales se llevaron a cabo de acuerdo con los Institutos Nacionales de Salud de Guía para el Cuidado y Uso de Animales de Laboratorio (NIH Publicaciones Nº 80-23, revisado 2012) y los estudios fueron aprobados por Institucional Cuidado de Animales y el uso de los Comités de Investigación en Stony Brook University (# 1618 y # 1792). 1. vHipp-NACC sinápticas Co-culturas <ol><li> Ratones Sacrificio (día postnatal 0-3, de tipo salvaje (WT) o nAChR α7 línea de ratón transgénico) con CO 2. Decapitar a cada cachorro y guardar la cola en un tubo de 1,5 ml de centrífuga para el genotipado post facto. Realice los siguientes pasos en una campana esterilizada. </li><li> Retire la parte superior del cráneo con tijeras y pinzas para exponer todo el cerebro. A partir de la coyuntura en la que las cortezas cerebrales parte el uno del otro en sentido caudal, obtienen una sección coronal que contiene las cortezas posteriores incluyendo el vHipp 5,21 con un microscopio de disección. </li><li> Diseccionar vTiras de tejido Hipp contienen la región CA1-subiculum a cabo de acuerdo con un desarrollo de atlas de cerebro de ratón 22 (Figura 1A). Transferir a una placa de cultivo de 35 mm que contiene medio de cultivo frío (4 ° C), cortado en pequeñas rodajas finas (alrededor de 100 micras x 100 micras microslices). </li><li> Pre-incubar 12 mm poli-D-lisina / laminina cubreobjetos de vidrio recubiertos dentro de 24 placas de cultivo de tejido bien con medios de cultivo (~ 500 l) durante al menos 30 min en la incubadora a 37 ° C. Retire los medios de comunicación antes de chapado microslices. </li><li> Placa con una pipeta Pasteur pulida al fuego en el centro del cubreobjetos con cantidad mínima de medios de comunicación (~ 50 l, que contiene ~ 20 piezas de microslices) para facilitar la unión de los explantes. Placa microslices vHipp procedentes de un solo animal (ya sea el + / + o el - / - genotipo de la línea transgénica α7) en cada cubreobjetos. </li><li> Después de los explantes se asientan en el cubreobjetos, añadir con cuidado los medios de comunicación adicional (~ 100 l) por The pared de manera que el nivel de los medios de comunicación es lo suficientemente alto para cubrir completamente los explantes en la periferia, pero no los que están en el centro del cubreobjetos. </li><li> Después de un / N incubación O en 37 ° C, 5% de CO 2 incubadora, dispersar neuronas NACC de días embrionarios 18 de postnatal día 1 ratones WT y añadir a los explantes. <ol><li> Diseccionar tejidos NACC de acuerdo con un desarrollo de atlas de cerebro de ratón 20 (Figura 1B). </li><li> Cortar en trozos pequeños (alrededor de 500 m × 500 m microslices) y transferir a un tubo de 15 ml. </li><li> Tratar con tripsina al 0,25% (2 ml) durante 15 min a 37 ° C. Wash trozos de tejido tres veces con medios de lavado en frío (5 ml, 4 ° C) seguido de un lavado en medio de cultivo frío (5 ml, 4 ° C). Deje reposar la suspensión durante 5 minutos en cada etapa de lavado, y luego verter el sobrenadante con cuidado. </li><li> Disociar las células por trituración suave en 2 ml de medio de cultivo con una pipeta Pasteur pulida al fuego ligeramente. </li><li> Transfer suspensión celular a otro tubo de 15 ml y centrifugar a 2000 x rpm durante 5 min. Eliminar el sobrenadante y resuspender las células en medios de cultivo en 1 ml por 6 cachorros. Dispersar las células pipeteando suavemente en medios varias veces con una pipeta Pasteur pulida al fuego. </li><li> Añadir 0,25 ml de células NACC dispersas a cada cubreobjetos con explantes vHipp. </li><li> Mantener las culturas en un humidificado 37 ° C, 5% de CO 2 incubadora. Coloque las placas de cultivo en gasas estériles humedecidas para estabilizar mecánicamente y mantener la humedad, lo que facilita la formación de sinapsis. </li></ol></li></ol> 2. La inmunocitoquímica <ol><li> Fijar culturas (5-7 días in vitro) en paraformaldehído al 4% / 4% de sacarosa / PBS (~ 500 l por cubreobjetos, 20 min, RT). </li><li> Culturas permeabilizar con 0,25% de Triton X-100 / PBS (~ 500 l por cubreobjetos, 5 min, RT). </li><li> Culturas bloque con 10% de suero normal de burro en PBS (~ 500 l por cubreobjetos, 30 min, RT) antes de la antibodtinción y. Incubar con anticuerpos primarios O / N a 4 ° C. Usa los siguientes anticuerpos primarios: anti-nAChR (α 4 -ECD 1: 500; anti-α 5 subunidades, 1: 500), anti-transportador vesicular de glutamato 1 (1: 250), anti-GAD65 (1: 100). </li><li> Lavar anticuerpos primarios a cabo con PBS (3 veces / ~ 500 l por cubreobjetos, 5 min) y luego incubar culturas en anticuerpos secundarios conjugados con Alexa 488 (~ 500 l por cubreobjetos, 1: 500) o Alexa 594 (~ 500 l por cubreobjetos , 1: 500) durante 1 hora a 37 ° C. </li><li> Incubar culturas en αBgTx conjugados con Alexa 594 (~ 500 l por cubreobjetos, 1: 1000 en medios de cultivo) durante 45 min a 37 ° C antes de la fijación para el etiquetado α7 superficie * nAChR. </li><li> Montaje y sello cubreobjetos. </li><li> Captura de imágenes con un microscopio de fluorescencia equipado con los objetivos del Plan-apocromáticos (20X con 0,8 NA o 63X aceite con 1,4 NA) y una cámara CCD. </li></ol> 3. FM1-43 Based vesicular Fusión <ol><li> Mantener culturas (5-7 días in vitro) en una cámara de imágenes, montar la cámara en el hilado disco microscopio confocal. Continuamente perfundir (1 ml / min) con cóctel de HBS a temperatura ambiente. </li><li> Detener culturas de perfusión y de carga con 10 M FM1-43 en 56 mM K + ACSF (~ 500 l) durante 90 segundos (tinción), lavar colorante externo de distancia durante 15 minutos con Ca 2+ HBS que contenía ADVASEP-7 (0,1 mM) para secuestrar FM1-43 unida a la membrana. </li><li> Estimular las culturas con 56 mM de K + ACSF (~ 500 l) sin FM1-43 durante 120 segundos (decoloración), vuelva a cargar las culturas con FM1-43 utilizando las mismas condiciones, y lavar de nuevo con Ca 2+ HBS que contenía ADVASEP-7 para 15 min. </li><li> Adquirir FM1-43 imágenes de fluorescencia con disco giratorio microscopio confocal equipado con un objetivo Plan-Apochromat (60X agua con 1,4 NA, de excitación 488 nm, emisión 530 nm) y capturar imágenes con una cámara CCD. </li><li> Recoge imágenes de fluorescencia FM1-43 basales (cada 2 s durante 1 min) Como control pre-nicotina; aplicar la nicotina (1 mM) por perfusión rápida (2 ml / min) durante 1 min, y luego lavar la nicotina a cabo con el coctel HBS. Mantenga imágenes con lapso de tiempo de captura antes, durante y después de la aplicación de nicotina cada 2 s durante 5 min. </li><li> Cuantificar la intensidad de fluorescencia antes de FM1-43 (tinción F) y después (F destaining) aplicación de nicotina en cada botón sináptico. </li><li> Calcular la cantidad total de liberables fluorescencia FM1-43 largo de los axones vHipp cuantificando la diferencia de intensidad de fluorescencia FM1-43 antes y después de la aplicación de la nicotina (Df = tinción F -F decoloración). Analizar fracción de disminución de intensidad de fluorescencia después de la nicotina con la siguiente ecuación: F =% de disminución Delta F / F de tinción. </li></ol> Imagen 4. Calcio <ol><li> Enjuague culturas (5-7 días in vitro) rápidamente con HBS normal, entonces cargar las culturas con 5 M Fluo-4 Ca 2+ indicador (AM éster) y 0,02% de Pluronic F-127 en HBS (2 ml) durante 30 min a 37 ° C y 5% de CO 2. </li><li> Lave Fluo-4 solución con HBS (3 veces / 5 min). Culturas Regreso a la incubadora (37 ° C / 5% de CO 2) por lo menos durante 30 minutos. </li><li> Mantener culturas en la misma cámara de imágenes y las mismas condiciones que se describen en la sección FM1-43 fusión vesicular en base (consulte el paso 3.1). </li><li> Recoge imágenes Fluo-4 de fluorescencia de proyecciones axonales de las vHipp micro-rebanadas con un objetivo Plan-Apochromat (60X agua con 1,4 NA, de excitación 488 nm, emisión 530 nm) y una cámara CCD. </li><li> Suma de línea de base Fluo-4 imágenes de fluorescencia (cada 10 seg durante 2 min) como control pre-nicotina, aplicar la nicotina (1 mM) por perfusión rápida (2 ml / min) durante 1 min, y luego lavar la nicotina a cabo con cóctel de HBS. Mantenga imágenes con lapso de tiempo de captura antes, durante y después de la aplicación de nicotina cada 10 segundos durante 30 minutos. </li><li> Guarde todos los marcos de las fluo-4 imágenes de fluorescencia primas, la exportación como una serie deImágenes en formato TIFF para su posterior análisis. </li><li> Recoge la intensidad integrada de fluo-4 de fluorescencia a lo largo de los axones vHipp antes y después de la aplicación de la nicotina. </li><li> Normalizar la intensidad de fluorescencia integrada con la siguiente ecuación: Delta F / F = 0 (0 FF) / F 0, donde F 0 es el pre-nicotina intensidad de fondo con corrección de fluorescencia integrado y F es la intensidad de fluorescencia integrada a lo largo de los axones vHipp en cada punto temporal después de la aplicación de nicotina. Analizar y trazar la intensidad de fluorescencia integrado normalizado. </li></ol>

<ol>
	<li>Changeux, J. P., 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=Brain+nicotinic+receptors:+structure+and+regulation,+role+in+learning+and+reinforcement.">Brain nicotinic receptors: structure and regulation, role in learning and reinforcement.</a> Brain Res Brain Res Rev. 26 (2-3), 198-216 (1998).</li><li>Levin, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotinic+receptor+subtypes+and+cognitive+function.">Nicotinic receptor subtypes and cognitive function.</a> J Neurobiol. 53 (4), 633-640 (2002).</li><li>Dani, J. A., Bertrand, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotinic+acetylcholine+receptors+and+nicotinic+cholinergic+mechanisms+of+the+central+nervous+system.">Nicotinic acetylcholine receptors and nicotinic cholinergic mechanisms of the central nervous system.</a> Annu Rev Pharmacol Toxicol. 47, 699-729 (2007).</li><li>Mineur, Y. S., Picciotto, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotine+receptors+and+depression:+revisiting+and+revising+the+cholinergic+hypothesis.">Nicotine receptors and depression: revisiting and revising the cholinergic hypothesis.</a> Trends Pharmacol Sci. 31 (12), 580-586 (2010).</li><li>Zhong, C., Talmage, D. A., Role, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotine+elicits+prolonged+calcium+signaling+along+ventral+hippocampal+axons.">Nicotine elicits prolonged calcium signaling along ventral hippocampal axons.</a> PloS one. 8 (12), e82719 (2013).</li><li>McGehee, D. S., Heath, M. J., Gelber, S., Devay, P., Role, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotine+enhancement+of+fast+excitatory+synaptic+transmission+in+CNS+by+presynaptic+receptors.">Nicotine enhancement of fast excitatory synaptic transmission in CNS by presynaptic receptors.</a> Science. 269 (5231), 1692-1696 (1995).</li><li>Gray, R., Rajan, A. S., Radcliffe, K. A., Yakehiro, M., Dani, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+synaptic+transmission+enhanced+by+low+concentrations+of+nicotine.">Hippocampal synaptic transmission enhanced by low concentrations of nicotine.</a> Nature. 383 (6602), 713-716 (1996).</li><li>Dickinson, J. A., Kew, J. N., Wonnacott, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+alpha+7-+and+beta+2-containing+nicotinic+acetylcholine+receptors+modulate+excitatory+amino+acid+release+from+rat+prefrontal+cortex+nerve+terminals+via+distinct+cellular+mechanisms.">Presynaptic alpha 7- and beta 2-containing nicotinic acetylcholine receptors modulate excitatory amino acid release from rat prefrontal cortex nerve terminals via distinct cellular mechanisms.</a> Mol Pharmacol. 74 (2), 348-359 (2008).</li><li>Zappettini, S., Grilli, M., Salamone, A., Fedele, E., Marchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-synaptic+nicotinic+receptors+evoke+endogenous+glutamate+and+aspartate+release+from+hippocampal+synaptosomes+by+way+of+distinct+coupling+mechanisms.">Pre-synaptic nicotinic receptors evoke endogenous glutamate and aspartate release from hippocampal synaptosomes by way of distinct coupling mechanisms.</a> Br J Pharmacol. 161 (5), 1161-1171 (2010).</li><li>Zappettini, 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=Presynaptic+nicotinic+alpha7+and+non-alpha7+receptors+stimulate+endogenous+GABA+release+from+rat+hippocampal+synaptosomes+through+two+mechanisms+of+action.">Presynaptic nicotinic alpha7 and non-alpha7 receptors stimulate endogenous GABA release from rat hippocampal synaptosomes through two mechanisms of action.</a> PloS one. 6 (2), e16911 (2011).</li><li>Fabian-Fine, 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=Ultrastructural+distribution+of+the+alpha7+nicotinic+acetylcholine+receptor+subunit+in+rat+hippocampus.">Ultrastructural distribution of the alpha7 nicotinic acetylcholine receptor subunit in rat hippocampus.</a> J Neurosci. 21 (20), 7993-8003 (2001).</li><li>Jones, I. W., Barik, J., O'Neill, M. J., Wonnacott, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha+bungarotoxin-1.4+nm+gold:+a+novel+conjugate+for+visualising+the+precise+subcellular+distribution+of+alpha+7*+nicotinic+acetylcholine+receptors.">Alpha bungarotoxin-1.4 nm gold: a novel conjugate for visualising the precise subcellular distribution of alpha 7* nicotinic acetylcholine receptors.</a> J Neurosci Methods. 134 (1), 65-74 (2004).</li><li>Nashmi, 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=Assembly+of+alpha4beta2+nicotinic+acetylcholine+receptors+assessed+with+functional+fluorescently+labeled+subunits:+effects+of+localization,+trafficking,+and+nicotine-induced+upregulation+in+clonal+mammalian+cells+and+in+cultured+midbrain+neurons.">Assembly of alpha4beta2 nicotinic acetylcholine receptors assessed with functional fluorescently labeled subunits: effects of localization, trafficking, and nicotine-induced upregulation in clonal mammalian cells and in cultured midbrain neurons.</a> J Neurosci. 23 (37), 11554-11567 (2003).</li><li>Drenan, R. M., 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=Subcellular+trafficking,+pentameric+assembly,+and+subunit+stoichiometry+of+neuronal+nicotinic+acetylcholine+receptors+containing+fluorescently+labeled+alpha6+and+beta3+subunits.">Subcellular trafficking, pentameric assembly, and subunit stoichiometry of neuronal nicotinic acetylcholine receptors containing fluorescently labeled alpha6 and beta3 subunits.</a> Mol Pharmacol. 73 (1), 27-41 (2008).</li><li>Wu, J., 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=Electrophysiological,+pharmacological,+and+molecular+evidence+for+alpha7-nicotinic+acetylcholine+receptors+in+rat+midbrain+dopamine+neurons.">Electrophysiological, pharmacological, and molecular evidence for alpha7-nicotinic acetylcholine receptors in rat midbrain dopamine neurons.</a> J Pharmacol Exp Ther. 311 (1), 80-91 (2004).</li><li>Parikh, V., Ji, J., Decker, M. W., Sarter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prefrontal+beta2+subunit-containing+and+alpha7+nicotinic+acetylcholine+receptors+differentially+control+glutamatergic+and+cholinergic+signaling.">Prefrontal beta2 subunit-containing and alpha7 nicotinic acetylcholine receptors differentially control glutamatergic and cholinergic signaling.</a> J Neurosci. 30 (9), 3518-3530 (2010).</li><li>Nayak, S. V., Dougherty, J. J., McIntosh, J. M., Nichols, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca(2+)+changes+induced+by+different+presynaptic+nicotinic+receptors+in+separate+populations+of+individual+striatal+nerve+terminals.">Ca(2+) changes induced by different presynaptic nicotinic receptors in separate populations of individual striatal nerve terminals.</a> J Neurochem. 76 (6), 1860-1870 (2001).</li><li>Richards, C. I., 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=Trafficking+of+alpha4*+nicotinic+receptors+revealed+by+superecliptic+phluorin:+effects+of+a+beta4+amyotrophic+lateral+sclerosis-associated+mutation+and+chronic+exposure+to+nicotine.">Trafficking of alpha4* nicotinic receptors revealed by superecliptic phluorin: effects of a beta4 amyotrophic lateral sclerosis-associated mutation and chronic exposure to nicotine.</a> J Biol Chem. 286 (36), 31241-31249 (2011).</li><li>Colombo, S. F., Mazzo, F., Pistillo, F., Gotti, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis,+trafficking+and+up-regulation+of+nicotinic+ACh+receptors.">Biogenesis, trafficking and up-regulation of nicotinic ACh receptors.</a> Biochem Pharmacol. 86 (8), 1063-1073 (2013).</li><li>St John, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+trafficking+of+nicotinic+acetylcholine+receptors.">Cellular trafficking of nicotinic acetylcholine receptors.</a> Acta Pharmacol Sin. 30 (6), 656-662 (2009).</li><li>Zhong, C., 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=Presynaptic+type+III+neuregulin+1+is+required+for+sustained+enhancement+of+hippocampal+transmission+by+nicotine+and+for+axonal+targeting+of+alpha7+nicotinic+acetylcholine+receptors.">Presynaptic type III neuregulin 1 is required for sustained enhancement of hippocampal transmission by nicotine and for axonal targeting of alpha7 nicotinic acetylcholine receptors.</a> J Neurosci. 28 (37), 9111-9116 (2008).</li><li>Jacobowitz, D. M., Abbott, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemoarchitectonic Atlas of the Developing Mouse Brain. , (1998).</li><li>Betz, W. J., Mao, F., Bewick, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+fluorescent+staining+and+destaining+of+living+vertebrate+motor+nerve+terminals.">Activity-dependent fluorescent staining and destaining of living vertebrate motor nerve terminals.</a> J Neurosci. 12 (2), 363-375 (1992).</li><li>Amaral, E., Guatimosim, S., Guatimosim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+fluorescent+styryl+dye+FM1-43+to+visualize+synaptic+vesicles+exocytosis+and+endocytosis+in+motor+nerve+terminals.">Using the fluorescent styryl dye FM1-43 to visualize synaptic vesicles exocytosis and endocytosis in motor nerve terminals.</a> Methods Mol Biol. 689, 137-148 (2011).</li><li>Garduno, J., 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=Presynaptic+alpha4beta2+nicotinic+acetylcholine+receptors+increase+glutamate+release+and+serotonin+neuron+excitability+in+the+dorsal+raphe+nucleus.">Presynaptic alpha4beta2 nicotinic acetylcholine receptors increase glutamate release and serotonin neuron excitability in the dorsal raphe nucleus.</a> J Neurosci. 32 (43), 15148-15157 (2012).</li><li>Guo, J. Z., Liu, Y., Sorenson, E. M., Chiappinelli, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptically+released+and+exogenous+ACh+activates+different+nicotinic+receptors+to+enhance+evoked+glutamatergic+transmission+in+the+lateral+geniculate+nucleus.">Synaptically released and exogenous ACh activates different nicotinic receptors to enhance evoked glutamatergic transmission in the lateral geniculate nucleus.</a> J Neurophysiol. 94 (4), 2549-2560 (2005).</li><li>Szabo, S. I., Zelles, T., Vizi, E. S., Lendvai, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+nicotine+on+spiking+activity+and+Ca2++dynamics+of+dendritic+spines+in+rat+CA1+pyramidal+neurons.">The effect of nicotine on spiking activity and Ca2+ dynamics of dendritic spines in rat CA1 pyramidal neurons.</a> Hippocampus. 18 (4), 376-385 (2008).</li></ol>

Los autores no tienen nada que revelar.

La preparación de co-cultivo describe re-capitula circuitos del hipocampo ventral-accumbens in vitro. Este examen permite la preparación relativamente sencillo y fiable de los perfiles espaciales y temporales por el cual la activación de nAChR pre-sinápticos obtener mejor transmisión glutamatérgica 5, 21. Co-culturas se definen como el crecimiento de diferentes tipos de células específicas en un plato que puede proporcionar condiciones fisiológicas in vitro</...

Modulación de la excitabilidad colinérgica circuito contribuye a aspectos fundamentales de la cognición, y alterado modulación colinérgica es una característica de trastornos neurodegenerativos y neuropsiquiátricos incluyendo la enfermedad de Alzheimer, enfermedad de Parkinson, la esquizofrenia y la adicción 1-4. Un mecanismo establecido de la facilitación de la transmisión sináptica colinérgica en el sistema nervioso central es a través de la activación directa de nAChR localizadas en los sitios pre-sinápticas. La activación de estos receptores presinápticos conduce a un aumento de Ca2 + intracelular ([Ca 2 +] i) en los terminales presinápticos - tanto de forma directa, debido a la relativamente alta conductancia de calcio de ciertos subtipos de nAChR, e indirectamente, a través de cascadas de señalización intracelular 5, mejorando de este modo la liberación de neurotransmisores. De hecho, la activación de nAChR pre-sinápticos se ha relacionado con los cambios en la liberación de una amplia variedad de neurotransmisores incluyendo glutamato, GABA, ACh, unand dopamina 6-10. Aunque este proceso se ha estudiado indirectamente usando métodos electrofisiológicos en varias sinapsis, los reporteros ópticas de [Ca2 +] i y el reciclado de vesículas sinápticas permiten una medición más directa y temporalmente precisa de los fenómenos pre-sinápticos. Localización presináptica de nAChR se ha demostrado de forma convincente con el etiquetado inmuno-oro directa de nAChR en el microscopio electrónico (EM) nivel de 11,12. Varias otras técnicas también se han utilizado para tratar la localización nAChR indirectamente, incluyendo la detección de lugares de nAChRs subunit- quimeras de proteínas fluorescentes en neuronas cultivadas 13,14, registro electrofisiológico de las corrientes de nAChR en las terminales sinápticas 15,16, seguimiento de los cambios inducidos por la nicotina en [Ca 2 +] i en las terminales nerviosas sinápticas de imágenes en vivo de células 17, y la supervisión indirecta de la liberación de neurotransmisores en la terminal sináptica portécnicas de imagen de células vivas con indicadores fluorescentes, incluyendo exocitosis de vesículas sinápticas vistos por estirilo tintes anfipáticas FM (FM1-43 y FM4-64) y / o synapto-pHluorin y por los reporteros fluorescentes neurotransmisores específicos, como CNiFERs de ACh y iGluSnFr para el glutamato 18-20. En general, estos enfoques actuales para la identificación de la localización presináptica de nAChR son complicados y requieren sistemas y técnicas especiales para permitir la identificación fiable y monitorización fisiológica de la actividad presináptica. Aquí se describen los protocolos y equipos para un sistema de co-cultivo in vitro de un hipocampo ventral (vHipp) - núcleo accumbens (NACC) de circuito que proporciona acceso directo a identificar y analizar los dos componentes pre y post-sinápticos de la transmisión sináptica. Mostramos ejemplos de localización presináptica de nAChR y la imagen de células vivas de nAChR mediada por Ca 2 + de señalización y la liberación de neurotransmisores aloaxones ng vHipp. Una extensión natural (y sencillo) del protocolo que se presenta aquí es la preparación de los contactos pre y post-sináptica compuestos por neuronas de diferentes genotipos. De esta manera la contribución de un producto génico particular, a la pre y / o post-sinápticas mecanismos de modulación se puede evaluar directamente.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>1, Culture Media (50 ml)</td>
		</tr>
		<tr>
			<td>Neurobasal&#160;</td>
			<td>GIBCO</td>
			<td>10888022</td>
			<td>48 ml</td>
		</tr>
		<tr>
			<td>B-27 Supplements</td>
			<td>GIBCO</td>
			<td>0080085-SA</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>GIBCO</td>
			<td>10908-010</td>
			<td>0.5 ml</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>GIBCO</td>
			<td>35050-061</td>
			<td>0.5 ml</td>
		</tr>
		<tr>
			<td>Brain-derived neurotrophic factor (BDNF)</td>
			<td>GIBCO</td>
			<td>15140-122</td>
			<td>20 ng/ml</td>
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		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>2, washing media (HBSS, 100 ml)</td>
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		<tr>
			<td>HBSS, no calcium, no magnesium, no phenol red&#160;</td>
			<td>GIBCO</td>
			<td>14175-095</td>
			<td>99 ml</td>
		</tr>
		<tr>
			<td>HEPES ( 1M)</td>
			<td>GIBCO</td>
			<td>15630-130</td>
			<td>1 ml</td>
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		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>3, HEPES buffered saline&#160; (HBS)&#160;&#160; pH=7.3</td>
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		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888&#160;</td>
			<td>135 mM</td>
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		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333&#160;</td>
			<td>5 mM</td>
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		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266&#160;</td>
			<td>1 mM</td>
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		<tr>
			<td>CaCl2,</td>
			<td>Sigma</td>
			<td>C1016&#160;</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
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		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>4, HBS Cocktail for live imaging pH=7.3</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888&#160;</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333&#160;</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266&#160;</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>CaCl2,</td>
			<td>Sigma</td>
			<td>C1016&#160;</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>tetrodotoxin&#160;</td>
			<td>Tocris</td>
			<td>1078</td>
			<td>2 &#181;M</td>
		</tr>
		<tr>
			<td>bicuculline</td>
			<td>Tocris</td>
			<td>131</td>
			<td>10 &#181;M</td>
		</tr>
		<tr>
			<td>D-AP-5</td>
			<td>Tocris</td>
			<td>105</td>
			<td>50 &#181;M</td>
		</tr>
		<tr>
			<td>CNQX</td>
			<td>Tocris</td>
			<td>1045</td>
			<td>20 &#181;M</td>
		</tr>
		<tr>
			<td>LY341495</td>
			<td>Tocris</td>
			<td>1209</td>
			<td>10 &#181;M</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>5, Calcium-free&#160; HBS&#160;&#160; pH=7.3</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888&#160;</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333&#160;</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266&#160;</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>6, 56 mM Potassium ACSF pH=7.4</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td>119 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td>56 mM</td>
		</tr>
		<tr>
			<td>MgSO4.7H</td>
			<td>Sigma</td>
			<td>M1880</td>
			<td>1.3 mM</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td>2.5 mM</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma</td>
			<td>S8282</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td>26.2 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
		</tr>
	</tbody>
</table>

live imaging of nicotine induced calcium signaling and neurotransmitter release along ventral hippocampal axons

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

La preparación empleada consiste en genes quiméricos co-cultivos de circuitos vHipp-NACC in vitro. Las proyecciones que emanan de microslices vHipp, como entrada axonal presináptica, pueden hacer contactos sinápticos con las metas post-sinápticas, las neuronas dispersas de NACC. Indujo un (≥ 30 min) la facilitación sostenida de la transmisión glutamatérgica de las neuronas NACC inervados por vHipp nicotina axones 21 y prolongada de calcio de señalización a lo largo de los axones vHipp <su...

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

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

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

Imágenes en vivo de nicotina señalización de calcio inducido y neurotransmisor lanzamiento Junto ventrales hipocampo axones

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

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

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Drugs Acting on Autonomic Nervous System: Adrenergic Agonists and Antagonists Agents

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

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

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

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

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

La neuromodulación y mitocondrial de transporte: Imágenes en vivo en las neuronas del hipocampo durante largos períodos de tiempo

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

Investigación

Neurociencias

Live Imaging of Dorsal Root Axons after Rhizotomy

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

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

Imágenes en vivo de los axones de la raíz dorsal después de rizotomía

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

Analyzing Murine Schwann Cell Development Along Growing Axons

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

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

Analizar el desarrollo de células murinas Schwann largo de los axones en crecimiento

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

Calcium Phosphate Transfection of Primary Hippocampal Neurons

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

Fosfato de Calcio La transfección de Primaria neuronas del hipocampo

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

Live Imaging of Drosophila Larval Neuroblasts

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

Live Imaging of Drosophila Larval Neuroblasts

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

Imágenes en vivo de<em&gt; Drosophila</em&gt; larvales neuroblastos

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

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

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

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

Aislamiento de los ganglios de raíz dorsal y en cultivo primario para el estudio de liberación de neurotransmisor

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

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

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

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

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

Ex Vivo Proyección de imagen de calcio para la visualización de las respuestas cerebrales a la señalización endocrina en Drosophila

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

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

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

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

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

Microscopía espectral reflectométrico en axones mielinizados en Situ

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

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

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

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

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

Ex Vivo Proyección de imagen del calcio celular específicos de señalización en la sinapsis tripartita del diafragma del ratón

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

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

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

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

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