Agradecemos al Dr. S. Stella y Helen Vuong de contribuciones para el protocolo de imágenes de calcio. Agradecemos Hojas Dr. K. para el rodaje de las escenas de la entrevista. Agradecemos al Dr. Arlene Hirano por sus comentarios sobre el manuscrito. Este proyecto de investigación y desarrollo se llevó a cabo por los autores en la Escuela David Geffen de Medicina en UCLA y es posible gracias a un acuerdo de contrato que se adjudicó y administrado por el Comando de Investigación y Material Médico del Ejército de EE.UU. (USAMRMC) y la tecnología de la telemedicina y Avanzado Centro de Investigación (TATRC), en Fort Detrick, MD con el Número de Contrato: W81XWH-10-2-0077. El apoyo a estos estudios también provino de NIH EY04067 y opiniones de Mérito VA (NB). BCN es un VA Carrera del Investigador Científico.

Todos los experimentos se llevaron a cabo de conformidad con las directrices para el bienestar de los animales de laboratorio emitidos por la Política de Servicio de Salud Pública en Cuidado Humano y Uso de Animales de Laboratorio y la Universidad de California-Los Ángeles (UCLA) Comité de Investigación Animal. Se utilizaron masculino y femenino adulto ratas Sprague-Dawley (Charles River Lab, Wilmington, MA) entre la edad de 3-5 semanas. 1. Animal y Tejidos Preparación para Wholemount Retina y Protocolo de inmunohistoquímica <ol><li> Prepare los siguientes materiales y herramientas: un microscopio de disección, 2 pinzas con puntas muy finas, tijeras, papel de filtro de celulosa, pipeta de plástico y un portaobjetos de microscopio. </li><li> La eutanasia a la rata en una cámara cerrada por profundamente anestesiar al animal con 1-3% de isoflurano seguido por decapitación con una guillotina. Quitar los ojos con un par de tijeras de iris y el lugar en una placa de Petri que contiene la solución extracelular. Hibernate A, Ames míSe recomiendan Dium o soluciones fisiológicas. </li><li> [Si se desea el relleno de la CGR con Fluo-4 Paso opcional]. Realizar el etiquetado Fluo-4 como se describe en el 2.3 a 2.4 pasos. </li><li> Usando el microscopio de disección (intensidad de la luz: 1,6 x 10 8 fotones / mm 2 ⋅sec), retire la córnea mediante la reducción de la parte frontal del globo ocular. Retire el objetivo y el vítreo de la superficie de la retina interna con fórceps. Para quitar el vítreo, estabilizar el globo ocular sosteniendo firmemente la esclerótica con una pinza. Tienes que despegar la base del vítreo hacia el centro de la retina con la otra pinza. La preparación en esta etapa se llama el ocular, que se utiliza para cryosections. Más detalles sobre cryosections se pueden encontrar en las referencias 14,15. </li><li> Retire la retina de la ojera. Luego, hacen cuatro cortes para permitir la retina para establecer planos. Montar suavemente la retina en un portaobjetos de microscopio con la capa de fotorreceptores arriba. Añadir una gota de solución a la retina y poner cepapel de filtro llulose en la retina. Una vez que la retina se adhiere al papel de filtro, colocar la retina en solución. Si es necesario, aplanar la retina con un cepillo o fórceps. </li><li> Coloque las retinas wholemounted en PFA al 4% durante 10-15 minutos para la fijación. </li><li> Lavar las retinas wholemounted tres veces durante 30 min en tampón fosfato 0,1 M (PB), pH 7,4. Bloquear las retinas con 5% de suero de cabra normal o suero de burro en PB 0,1 que contiene 0,3% de Triton X-100 y 0,1% NaN 3 a 4 ° C durante la noche. Se recomienda un volumen final de 500 l para todos los pasos para guardar las soluciones de bloqueo y anticuerpos. </li><li> Al día siguiente, se incuban los wholemounts retinae con los anticuerpos primarios 5-7 días a 4 ° C. Las diluciones óptimas deben ser determinadas por el usuario. </li><li> Lavar el retinas 3 x 30 min en PB 0,1 M y se incuba durante la noche a 4 ° C en el anticuerpo secundario conjugado con fluoróforo correspondiente que se dirige contra la especie de anticuerpo primario (concentración 1: 1000). </li><li> Después de tres lavados finales de 30 min cada uno con PB, montar las retinas en medio de montaje. Sellar el cubreobjetos con esmalte de uñas para el almacenamiento prolongado. Guardar las muestras a 4 ° C y proteger de la luz. </li></ol> 2. Etiquetado de las células ganglionares y axones en Rata Wholemount retinas con un calcio Indicador Tinte <ol><li> Preparar 1000 ml de mamíferos de Ringer que contenía (en mM) 125 NaCl, KCl 3, 2 CaCl2, 1,25 NaH 2 PO 4, 1 MgCl 2, 25 NaHCO 3 y 10 de la glucosa burbujeado con 95% O 2/5% de CO &#173; 2. </li><li> Realizar la disección como se describe en el 1.2 hasta 1.4 pasos. </li><li> Para cargar las células ganglionares de la retina (CGR) y sus axones con un colorante indicador de calcio, inyectar 0,5 l de Fluo-4 sal pentapotásico (de stock 40 mM en H 2 O) en el muñón del nervio óptico de aproximadamente 1 mm posterior al globo ocular usando una jeringa . Para medir los aumentos dinámicos en células ganglionares de la [Ca 2 + </sup&gt;] i un indicador con alta afinidad, tales como Fluo-4 con su Kd de 345 nM, es óptima. Fluo-4 ofrece el beneficio adicional de tener una muy alta eficiencia cuántica que resulta en un incremento de las emisiones de&gt; 100 veces cuando se une a calcio. </li><li> Coloque el ocular en mamíferos de Ringer burbujeaba con 95% O 2/5% de CO &#173; 2 durante 1 hora a temperatura ambiente en la oscuridad. </li></ol> 3. Calcio Protocolo Imaging para células ganglionares de la retina y los axones. <ol><li> Retire el ojo, cristalino y humor vítreo como se describe en los pasos 1.1 hasta 1.4. Aislar la retina de la ojera y se dividen en cuadrantes. Mount lado de células ganglionares de un cuadrante sobre un portaobjetos de vidrio y utilizar una rejilla rebanada arpa para estabilizarlo. Mantenga las otras piezas oscuro-adaptado de mamíferos de Ringer burbujeaba con 95% O 2/5% de CO &#173; 2 en el hielo para un uso posterior. </li><li> Superfuse la cámara de grabación con solución de Ringer y mamíferosmantener la solución burbujeando continuamente con 95% O2 / 5% de CO 2. </li><li> Image el tejido bajo el microscopio. Realizar experimentos a temperatura ambiente (~ 23 ° C) o alcanzar temperaturas fisiológicas por el calentamiento de la etapa, calentar el baño de agua debajo de la muestra o la aplicación de aire caliente a la superficie de la muestra. Nota: Es importante tener en cuenta que muchas funciones celulares están regulados por la temperatura, que desempeña un papel clave en el mantenimiento de la homeostasis intracelular 16. Sin embargo, la introducción de medidas de calentamiento puede aumentar la tasa de photobleaching, la evaporación del medio y el enfoque deriva causada por la expansión térmica 17. El uso de la temperatura ambiente disminuye intracelular compartimentación de Fluo-4 18. </li><li> Realizar un control de dos de K + impulsos de alta pareadas en ausencia de fármacos. Despolarizar CGR axones de CGR y elevando la [K +] o de 3 mM a 60 mM durante 33 segundos durante cada pulso para activarVGCCs con un sistema de superfusión impulsado ocho gravedad canal. Reducir Na + por 57 mM de mantener isosmolarity en la solución elevada K +. </li><li> Evaluar la contribución de las diferentes VGCCs a la señal de calcio con el uso de bloqueadores de los canales generales o específicas Ca. Por ejemplo, la reducción de la señal de calcio tras la administración de un bloqueador no específica del canal de calcio, cobalto, proporcionó una medida semi-cuantitativa de la contribución de los VGCCs a la alta K + señal de calcio -evoked. </li><li> Adquirir valores de intensidad de fluorescencia mediante la colocación de una región de interés (ROI) en torno a la CGR de interés (Figura 2). </li><li> Normalizar el cambio en la intensidad de fluorescencia producida por el segundo K + pico alto con el cambio producido por la primera alta K + pico. Entonces, para las pruebas de los bloqueadores de canales de calcio específicos, aplicar medicamentos sólo durante el segundo pulso de alta K + de un par y normalizar este pico contrael primer valor pico. Para medir el efecto del fármaco sobre el alto K + pico, dividir la amplitud de la segunda K + pico por el de la primera. El análisis debe ser realizado en estos valores con respecto a sus controles coincidentes derivados de alta emparejado K + picos medida en ausencia de fármaco. </li><li> Adquirir imágenes a intervalos de 5 seg. Para evitar la foto-blanqueo, mantener la excitación láser tan bajo como sea posible. Proporcionar la excitación por la línea de 488 nm del láser de argón, mientras que el tubo fotomultiplicador recoge la emisión fluorescente a través de un filtro LP 505 nm. </li></ol>

<ol>
	<li>Guenther, 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=Separation+of+calcium+currents+in+retinal+ganglion+cells+from+postnatal+rat.">Separation of calcium currents in retinal ganglion cells from postnatal rat.</a> Brain Res. 633, 223-235 (1994).</li><li>Farrell, S. 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=Modulation+of+voltage-gated+ion+channels+in+rat+retinal+ganglion+cells+mediated+by+somatostatin+receptor+subtype+4.">Modulation of voltage-gated ion channels in rat retinal ganglion cells mediated by somatostatin receptor subtype 4.</a> J Neurophysiol. 104, 1347-1354 (2010).</li><li>Caterall, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+regulation+of+voltage-gated+Ca2++channels.">Structure and regulation of voltage-gated Ca2+ channels.</a> Annu Rev Cell Dev Biol. 16, 521-555 (2000).</li><li>Berridge, M. 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=Calcium--a+life+and+death+signal.">Calcium--a life and death signal.</a> Nature. 395, 645-648 (1998).</li><li>Berridge, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+microdomains+Organization+and+function.">Calcium microdomains Organization and function.</a> Cell Calcium. 40, 405-412 (2006).</li><li>Dolphin, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+channel+diversity+multiple+roles+of+calcium+channel+subunits.">Calcium channel diversity multiple roles of calcium channel subunits.</a> Curr Opin Neurobiol. 19, 237-244 (2009).</li><li>Rottman, J. 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+ribonuclease+protection+assay+a+powerful+tool+for+the+veterinary+pathologist.">The ribonuclease protection assay a powerful tool for the veterinary pathologist.</a> Vet Pathol. 39, 2-9 (2002).</li><li>Briggman, K. L., Euler, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+electroporation+and+population+calcium+imaging+in+the+adult+mammalian+retina.">Bulk electroporation and population calcium imaging in the adult mammalian retina.</a> J Neurophysiol. 105, 2601-2609 (2011).</li><li>Segev, 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=Recording+spikes+from+a+large+fraction+of+the+ganglion+cells+in+a+retinal+patch.">Recording spikes from a large fraction of the ganglion cells in a retinal patch.</a> Nat Neurosci. 7, 1154-1161 (2004).</li><li>Jeon, C. 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=The+major+cell+populations+of+the+mouse+retina.">The major cell populations of the mouse retina.</a> J Neurosci. 18, 8936-8946 (1998).</li><li>Blankenship, A. 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=Synaptic+and+extrasynaptic+factors+governing+glutamatergic+retinal+waves.">Synaptic and extrasynaptic factors governing glutamatergic retinal waves.</a> Neuron. 62, 230-241 (2009).</li><li>Daniels, B. A., Baldridge, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=d-Serine+enhancement+of+NMDA+receptor-mediated+calcium+increases+in+rat+retinal+ganglion+cells.">d-Serine enhancement of NMDA receptor-mediated calcium increases in rat retinal ganglion cells.</a> J Neurochem. 112, 1180-1189 (2010).</li><li>Weitz, A. 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=Imaging+the+response+of+the+retina+to+electrical+stimulation+with+genetically+encoded+calcium+indicators.">Imaging the response of the retina to electrical stimulation with genetically encoded calcium indicators.</a> J Neurophysiol. 109, 1979-1988 (2013).</li><li>P&#233;rez de Sevilla M&#252;ller, L., 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=Expression+of+voltage-gated+calcium+channel+&#945;(2)&#948;(4)+subunits+in+the+mouse+and+rat+retina.">Expression of voltage-gated calcium channel &#945;(2)&#948;(4) subunits in the mouse and rat retina.</a> J Comp Neurol. 521 (2), 2486-2501 (2013).</li><li>Hirano, A. A., 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=SNAP25+expression+in+mammalian+retinal+horizontal+cells.">SNAP25 expression in mammalian retinal horizontal cells.</a> J Comp Neurol. 519, 972-988 (2011).</li><li>Berridge, M. 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=Calcium+signalling+dynamics,+homeostasis+and+remodeling.">Calcium signalling dynamics, homeostasis and remodeling.</a> Nat Rev Mol Cell Biol. 4, 517-529 (2003).</li><li>Dailey, M. 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=Maintaining+live+cells+and+tissue+slices+in+the+imaging+setup.">Maintaining live cells and tissue slices in the imaging setup.</a> Cold Spring Harb ProtocI. , 373-379 (2011).</li><li>Spinelli, K. J., Gillespie, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+intracellular+calcium+ion+dynamics+in+hair+cell+populations+with+Fluo-4.">Monitoring intracellular calcium ion dynamics in hair cell populations with Fluo-4.</a> AM. PLoS One. 7, e51874 (2012).</li><li>Kwong, J. M. K., 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=RNA+binding+protein+with+multiple+splicing+A+new+marker+for+retinal+ganglion+cells.">RNA binding protein with multiple splicing A new marker for retinal ganglion cells.</a> Invest Ophthalmol Vis Sci. 51, 1052-1058 (2010).</li><li>P&#233;rez deSevilla M&#252;ller, L., 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=Displaced+amacrine+cells+of+the+mouse+retina.">Displaced amacrine cells of the mouse retina.</a> J Comp Neurol. 505, 177-189 (2007).</li><li>P&#233;rez de Sevilla M&#252;ller, L., 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=Tracer+coupling+of+intrinsically+photosensitive+retinal+ganglion+cells+to+amacrine+cells+in+the+mouse+retina.">Tracer coupling of intrinsically photosensitive retinal ganglion cells to amacrine cells in the mouse retina.</a> J Comp Neurol. 518, 4813-4824 (2010).</li><li>Raymond, I. 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=Cyan+fluorescent+protein+expression+in+ganglion+and+amacrine+cells+in+a+thy1-CFP+transgenic+mouse+retina.">Cyan fluorescent protein expression in ganglion and amacrine cells in a thy1-CFP transgenic mouse retina.</a> Mol Vis. 14, 1559-1574 (2008).</li><li>Raymond, I. 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=A+Thy1-CFP+DBA/2J+mouse+line+with+cyan+fluorescent+protein+expression+in+retinal+ganglion+cells.">A Thy1-CFP DBA/2J mouse line with cyan fluorescent protein expression in retinal ganglion cells.</a> Vis Neurosci. 26, 453-465 (2009).</li><li>Mayer, M. L., Westbrook, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeation+and+block+of+N-methyl-D-aspartic+acid+receptor+channels+by+divalent+cations+in+mouse+cultured+central+neurones.">Permeation and block of N-methyl-D-aspartic acid receptor channels by divalent cations in mouse cultured central neurones.</a> J Physiol. 394, 501-527 (1987).</li><li>Ascher, P., Nowak, L. <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+divalent+cations+in+the+N-methyl-D-aspartate+responses+of+mouse+central+neurons+in+culture.">The role of divalent cations in the N-methyl-D-aspartate responses of mouse central neurons in culture.</a> J Physiol. 399, 247-266 (1988).</li><li>Aizenman, 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=Responses+mediated+by+excitatory+amino+acid+receptors+in+solitary+retinal+ganglion+cells+from+rat.">Responses mediated by excitatory amino acid receptors in solitary retinal ganglion cells from rat.</a> J Physiol. 396, 75-91 (1988).</li><li>Taschenberger, 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=Synaptic+current+kinetics+in+a+solely+AMPA-receptor-operated+glutamatergic+synapse+formed+by+rat+retinal+ganglion+neurons.">Synaptic current kinetics in a solely AMPA-receptor-operated glutamatergic synapse formed by rat retinal ganglion neurons.</a> J Neurophysiol. 74, 1123-1136 (1995).</li><li>Chen, S., Diamond, J. S. <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+glutamate+activates+extrasynaptic+NMDA+receptors+on+cells+in+the+ganglion+cell+layer+of+rat+retina.">Synaptically released glutamate activates extrasynaptic NMDA receptors on cells in the ganglion cell layer of rat retina.</a> J Neurosci. 22, 2165-2173 (2002).</li><li>Jakobs, T. 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=Expression+of+mRNA+for+glutamate+receptor+subunits+distinguishes+the+major+classes+of+retinal+neurons,+but+is+less+specific+for+individual+cell+types.">Expression of mRNA for glutamate receptor subunits distinguishes the major classes of retinal neurons, but is less specific for individual cell types.</a> Mol Vis. 13, 933-948 (2007).</li><li>Margolis, D. J., Detwiler, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+mechanisms+generate+maintained+activity+in+ON+and+OFF+retinal+ganglion+cells.">Different mechanisms generate maintained activity in ON and OFF retinal ganglion cells.</a> J NeurosciI. 27, 5994-6005 (2007).</li><li>Margolis, D. 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=Dendritic+calcium+signaling+in+ON+and+OFF+mouse+retinal+ganglion+cells.">Dendritic calcium signaling in ON and OFF mouse retinal ganglion cells.</a> J Neurosci. 30, 7127-7138 (2010).</li><li>Baldridge, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+recordings+of+the+effects+of+cholinergic+ligands+on+neurons+in+the+ganglion+cell+layer+of+mammalian+retina.">Optical recordings of the effects of cholinergic ligands on neurons in the ganglion cell layer of mammalian retina.</a> J Neurosci. 16, 5060-5072 (1996).</li><li>Hartwick, A. 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=Functional+assessment+of+glutamate+clearance+mechanisms+in+a+chronic+rat+glaucoma+model+using+retinal+ganglion+cell+calcium+imaging.">Functional assessment of glutamate clearance mechanisms in a chronic rat glaucoma model using retinal ganglion cell calcium imaging.</a> J Neurochem. 94, 794-807 (2005).</li><li>Badea, T. C., Nathans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+neuronal+morphologies+in+the+mouse+retina+visualized+by+using+a+genetically+directed+reporter.">Quantitative analysis of neuronal morphologies in the mouse retina visualized by using a genetically directed reporter.</a> J Comp Neurol. 480, 331-351 (2004).</li><li>Huberman, A. 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=Architecture+and+activity-mediated+refinement+of+axonal+projections+from+a+mosaic+of+genetically+identified+retinal+ganglion+cells.">Architecture and activity-mediated refinement of axonal projections from a mosaic of genetically identified retinal ganglion cells.</a> Neuron. 59, 425-438 (2008).</li><li>Kim, I. 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=Molecular+identification+of+a+retinal+cell+type+that+responds+to+upward+motion.">Molecular identification of a retinal cell type that responds to upward motion.</a> Nature. 452, 478-482 (2008).</li><li>Munch, T. A., 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=Approach+sensitivity+in+the+retina+processed+by+a+multifunctional+neural+circuit.">Approach sensitivity in the retina processed by a multifunctional neural circuit.</a> Nat Neurosci. 12, 1308-1316 (2009).</li><li>Feng, 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=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28, 41-51 (2000).</li></ol>

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En este artículo, hemos descrito dos técnicas diferentes: 1) La inmunohistoquímica para mostrar Fluo-4 a la localización de las células ganglionares de la retina en wholemounts, y 2) El calcio de imágenes para analizar la dinámica de calcio en las células ganglionares de la retina y sus axones. La inmunohistoquímica utilizando wholemounts se ha usado para revelar la localización de las proteínas en el roedor retina 20-23. Sin embargo, hay algunas limitaciones. La calida...

Las células ganglionares de la retina (CGR) expresan L, N, P / Q y de tipo T VGCC como se determina a través del bloqueo farmacológico de estos componentes de la célula entera Ca canal actual 1,2. VGCC son proteínas multiméricas transmembrana que están implicados en la liberación del transmisor, la transcripción de genes, la regulación celular y 3,4,5 plasticidad sináptica. VGCCs funcionales se componen de al menos tres clases distintas de subunidades: grandes, transmembrana α 1 formador de poros subunidades que establecen propiedades biofísicas y farmacológicas del canal, principalmente extracelular auxiliar α 2 δ subunidades y las subunidades β intracelulares. Los dos últimos complejos heteroméricos forma con diferentes subunidades α 1 y alterar la cinética de activación periódica y el tráfico de los canales a la membrana plasmática 6. En las últimas décadas, muchas técnicas se han empleado para estudiar la proteína expresión, como la inmunohistoquímica, ensayo inmunoenzimático, el análisis occidental y citometría de flujo. Estas técnicas requieren el uso de anticuerpos específicos para la detección de una proteína dada de interés y proporcionan potentes herramientas para la localización y distribución de proteínas específicas en diferentes tejidos. Las técnicas utilizadas para detectar y cuantificar los niveles de expresión de ARNm de una proteína en particular, como el norte de blot, RT-PCR en tiempo real cuantitativa RT-PCR, hibridación in situ, microarrays de ADNc y ensayo de protección de ribonucleasa proporcionan un enfoque alternativo cuando los anticuerpos no son fácilmente los niveles de expresión disponible o si de una proteína particular son bajos 7. Sin embargo, una limitación al uso de tales técnicas moleculares es la identificación requerido de la secuencia del gen. Para localizar proteínas en la retina, inmunohistoquímica se puede realizar en retinas wholemount. Debido a la accesibilidad de la CGR, la wholemountpreparación proporciona una excelente plataforma para el estudio de la localización de las proteínas específicas del somata RGC y sus axones. Además de su localización, algunas propiedades funcionales de los VGCCs en CGR se pueden demostrar mediante el uso de técnicas de imagen de calcio. Se describe un protocolo de imágenes de calcio para etiquetar selectivamente CGR con un colorante indicador de calcio para medir la dinámica del calcio intracelular. La contribución de las diferentes VGCCs a la señal de calcio en diferentes compartimentos celulares se puede aislar con el uso de bloqueadores de los canales de Ca subtipo específico. Quizás uno de los aspectos más beneficiosos de la técnica de imagen de calcio descrito aquí es la capacidad de grabar simultáneamente y de forma independiente a partir de múltiples CGR y sus axones. Aunque muchas técnicas fisiológicas, como la grabación de patch clamp de célula entera, proporcionan altos grabaciones resolución temporal de las corrientes de membrana, la somática o fuente axonal de thescorrientes e grabados no pueden ser discriminados y grabaciones sólo se pueden hacer de una sola neurona a la vez. Arrays multielectrodo (AAM) son capaces de registrar simultáneamente picos de muchas células, pero tampoco pueden detectar ni discriminar la activación de, por ejemplo, los diferentes subtipos de canales de calcio. Meas preferentemente grabar a partir de células que se encuentran en las proximidades de un electrodo 8 y registro de las células que generan grandes picos 9. Métodos de imagen ópticos proporcionan una estrategia alternativa para que las grabaciones simultáneas e independientes de toda la población de células que se pueden integrar con la información obtenida a partir de una sola célula de microelectrodos y grabación de patch clamp y grabación MEA. Aunque se emplearon las técnicas de imagen de calcio descritos aquí para estudiar la dinámica de calcio de CGR, patch clamp y AAM también se pueden utilizar en paralelo a aclarar aún más las corrientes iónicas y propiedades enriquecidas de CGR. <pclass = &quot;jove_content&quot;&gt; Dado que las células amacrinas desplazadas constituyen aproximadamente el 60% de la población neuronal en la capa de células ganglionares de la retina en el ratón 10, nuestro objetivo era utilizar una técnica de carga que etiqueta selectivamente CGR con un colorante indicador de calcio sintético en una preparación wholemount. Aunque colorantes indicadores de calcio sintéticos proporcionan una excelente plataforma para el estudio de la dinámica de calcio intracelular, su uso generalizado se ha visto obstaculizado por la incapacidad para cargar eficazmente poblaciones específicas de neuronas dentro de una red dada. Se han realizado muchas técnicas, tales como carga a granel 11 y 8,12 electroporación para cargar poblaciones enteras de células, sin embargo, estas técnicas no discriminan entre tipos de células específicos. Genéticamente codificados indicadores de calcio proporcionan la capacidad de etiquetar selectivamente poblaciones específicas de células, sin embargo, tales métodos requieren la generación de animales transgénicos 13. Nuestra técnica describe un method etiquetar selectivamente CGR en la preparación wholemount vía óptica inyección muñón del nervio de un colorante indicador de calcio. En conjunto, las técnicas estructurales y fisiológicos descritos en este artículo proporcionan una plataforma para estudiar la localización y la contribución de los VGCCs a la señal de calcio en la CGR y sus axones.

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			<td>A rabbit polyclonal antibody&#160;was generated against the N-terminus of the RBPMS&#160;polypeptide (RBPMS4-24), GGKAEKENTPSEANLQEEEVR, by a commercial vendor (ProSci, Poway, CA).</td>
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immunohistochemical and calcium imaging methods in wholemount rat retina

En este trabajo se describen las herramientas, los reactivos y las medidas prácticas que se necesitan para: 1) preparación exitosa de retinas wholemount para inmunohistoquímica y, 2) imágenes de calcio para el estudio de los canales de calcio dependientes de voltaje (VGCC) mediada por la señalización de calcio en la retina células ganglionares. El método de imagen de calcio describimos elude cuestiones relativas a la carga no específico de células amacrinas desplazadas en la capa de células ganglionares.

Inmunomarcaje con anticuerpos específicos proporciona una plataforma para estudiar la localización de proteínas particulares de interés en la retina y la técnica de imagen de calcio permite el estudio de la contribución de los VGCCs a la dinámica de calcio en las células ganglionares de la retina y sus axones. Mediante el uso de un anticuerpo contra las células ganglionares RBPMS proteína (proteína de unión de ARN con múltiples empalme) que etiqueta selectivamente CGR 19,</...

Watch this Scientific Journal Video about Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina at JoVE.com

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

Protocolos de inmunohistoquímica se utilizan para estudiar la localización de una proteína específica en la retina. Se emplean técnicas de imagen de calcio para estudiar la dinámica del calcio en las células ganglionares de la retina y sus axones.

Inmunohistoquímicos y métodos de imagen de calcio en Wholemount Rata Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

immunohistochemical-calcium-imaging-methods-wholemount-rat

Research

JoVE Journal

Neuroscience

14.4K vistas.  University of California, Los Angeles.  Protocolos de inmunohistoquímica se utilizan para estudiar la localización de una proteína específica en la retina. Se emplean técnicas de imagen de calcio para estudiar la dinámica del calcio en las células ganglionares de la retina y sus axones. 

Video: Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

Imagen de calcio funcionales en el desarrollo de redes corticales

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

Investigación

Neurociencias

Wholemount Immunohistochemistry for Revealing Complex Brain Topography

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its relatively uniform cytoarchitecture is a complex array of parasagittal domains of gene and protein expression. The molecular compartmentalization of the cerebellum is mirrored by the anatomical and functional organization of afferent fibers. To fully appreciate the complexity of cerebellar organization we previously refined a wholemount staining approach for high throughput analysis of patterning defects in the mouse cerebellum. This protocol describes in detail the reagents, tools, and practical steps that are useful to successfully reveal protein expression patterns in the adult mouse cerebellum by using wholemount immunostaining. The steps highlighted here demonstrate the utility of this method using the expression of zebrinII/aldolaseC as an example of how the fine topography of the brain can be revealed in its native three-dimensional conformation. Also described are adaptations to the protocol that allow for the visualization of protein expression in afferent projections and large cerebella for comparative studies of molecular topography. To illustrate these applications, data from afferent staining of the rat cerebellum are included.

Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum.

La inmunohistoquímica wholemount de Topografía Revelando Complex Brain

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

Formación de imágenes in vivo de calcio neuronal en C. elegans

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

El análisis inmunohistoquímico en el sistema nervioso central y periférico de la rata de nodo linfático secciones de tejido

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

Parénquima no invasiva, vascular y metabólico de alta frecuencia de ultrasonido y fotoacústica Rata cerebral profunda Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Proyección de imagen de calcio de dos fotones en las dendritas neuronales en rebanadas de cerebro

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

Imágenes transpupilares de dos fotones in vivo de la retina del ratón

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

In vivo Imágenes de calcio en la aceituna inferior de ratón

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the &#945;-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

Calcio de pericitos cerebrales e imágenes hemodinámicas en ratones transgénicos in vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

Imágenes subcelulares del manejo neuronal del calcio in vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...