Este trabajo fue financiado por los Institutos Nacionales de Salud (NIH) de subvención DA030396. Gracias a los miembros del laboratorio drenan útil para el debate y la crítica del manuscrito. Un agradecimiento especial a Mi Ran Kim para la asistencia técnica y Jonathan Thomas Ting para el asesoramiento con respecto a las secciones de cerebro de ratón adulto.

1. Preparación de las soluciones para la preparación de cortes de cerebro y electrofisiología <ol><li> Soluciones para la preparación de secciones de cerebro se han descrito anteriormente 3, 4. Preparar N-metil-D-glucamina (NMDG)-basado de corte y solución de recuperación de la siguiente composición (en mM): 93 N-metil-D-glucamina, 2,5 KCl, 1,2 NaH 2 PO 4, 30 NaHCO 3, 20 HEPES, 25 glucosa, 5 ascorbato de Na +, 2 tiourea, 3 Na + piruvato, 10 MgSO 4 • 7H 2 O, 0,5 CaCl 2 • 2H 2 O. Ajuste a 300-310 mOsm con NMDG. Ajustar el pH a 7.3-7.4 con 10 N HCl. <ol class="lalpha"><li> Disolver MgSO 4 • 7H 2 O y CaCl 2 • 2H 2 O en agua Millipore para hacer 2 M soluciones madre de cada uno. </li><li> Pesar otros componentes en el orden indicado y añade a un matraz volumétrico de llenado parcialmente con agua Millipore mientras se agita para disolver. Una vez que todo se añade, llenarmatraz aforado con agua Millipore. </li><li> Medir el pH y ajustar con 10 N HCl. </li><li> Medir la osmolaridad y ajustar con NMDG si es necesario. </li></ol></li><li> Preparar HEPES holding líquido cefalorraquídeo artificial (ACSF) de la siguiente composición (en mM): 92 NaCl, 2,5 KCl, 1,2 NaH 2 PO 4, 30 NaHCO 3, 20 HEPES, 25 glucosa, 5 ascorbato Na +, 2 tiourea, 3 Na piruvato +, 2 MgSO 4 • 7H 2 O, 2 CaCl 2 • 2H 2 O. Ajuste a 300-310 mOsm con sacarosa. Ajustar el pH a 7.3-7.4 con 1 N HCl o NaOH. <ol class="lalpha"><li> Pesar los componentes en el orden indicado y añade a un matraz volumétrico de llenado parcialmente con agua Millipore mientras se agita para disolver. Una vez que todo se añade, llenar matraz aforado con agua Millipore. </li><li> Medir el pH y ajustar con NaOH o HCl si es necesario. </li><li> Medir la osmolaridad y ajustar con sacarosa, si es necesario. </li></ol></li><li> Preparar solución estándar de grabación LCRa de la siguiente composición (en mM): 124 NaCl, 2,5 KCl, 1,2 NaH 2 PO 4, 24 NaHCO 3, 12,5 glucosa, 2 MgSO 4 • 7H 2 O, 2 CaCl 2 • 2H 2 O. Ajuste a 300-310 mOsm con sacarosa. Ajustar el pH a 7.3-7.4 con 1 N HCl o NaOH. </li></ol> 2. Preparación de las rebanadas cerebral agudo <ol><li> Corte de cortes de cerebro y recuperación se realizan como se ha descrito anteriormente 3, 4. Burbuja de dos platos de cristal lleno de NMDG solución de recuperación con 95% de oxígeno / 5% de gas CO 2 (carbógeno), un plato sobre el hielo, y la otra a 33 º C. </li><li> Burbuja de un plato de cristal lleno con HEPES que sostienen LCRa con carbógeno a temperatura ambiente. </li><li> Se anestesia el ratón adulto (de 2 a 12 meses) con Na + pentobarbital (200 mg / kg, ip). Continúe con el procedimiento cuando el animal no responde a un dedo del pie-pinch. </li><li> Si es necesario, corte una muestra de tejido de la cola para su posterior genotypING del animal. </li><li> Abrir la cavidad torácica, exponer el corazón, y la abrazadera de la aorta descendente. Lacerar la aurícula derecha. </li><li> Transcardially perfundir los animales con 5-10 ml de 0-4 ° C NMDG solución de recuperación. </li><li> Decapitar a los animales, retire el cerebro y lo coloca en 4 ° C NMDG solución de recuperación de 1 min. </li><li> En una placa metálica enfriada con hielo, recorte el cerebro en la orientación deseada (coronal, parasagital, horizontal, etc) para incluir el área de interés. </li><li> Coloque el bloque de cerebro con pegamento a la superficie etapa seca de una máquina de cortar vibrante (DSK-Zero 1; Dosaka), sumergir el bloque cerebro en 4 ° C NMDG solución de recuperación y continuamente burbujas de la solución con carbógeno. </li><li> Cortar las rebanadas de la zona del cerebro de interés. Espesor de la rebanada es de 200-400 micras, dependiendo del área del cerebro específica de interés, la edad del animal, y experimento a realizar. </li><li> Inmediatamente después del corte, incube las rebanadas deseadas en carbogenated, 33 ° C NMDG de recuperación solutidurante exactamente 12 minutos, luego se transfieren a HEPES carbogenated, sala de temperatura de mantenimiento solución por un período inicial de 60 min. Después de esta incubación de 60 min, las rodajas se puede utilizar para la grabación. Las láminas se mantuvieron en solución de HEPES que sostienen todo el día hasta que se utilizan para la grabación. </li></ol> 3. Patch Clamp grabación de neuronas en rodajas de cerebro <ol><li> Tire una micropipeta estándar con un parche programable Flaming-Brown extractor (Sutter P-97 o extractor verticales equivalente). Pipetas que son óptimas para la formación de sello giga-ohmios típicamente tienen una resistencia de 4-6 mW </li><li> Transferir una rebanada a la cámara de grabación (RC-27L; Instrumentos Warner) en la etapa de un microscopio vertical (Nikon FN-1). Continuamente superfuse (1,5 - 2,0 ml / min) en el sector con carbogenated, grabación estándar LCRa calentado y mantenido a 32 ° C. Alternativamente, las rebanadas se puede mantener a temperatura ambiente. </li><li> Y centre el área cerebral de interés utilizandouno de los objetivos del aire 10 veces (Nikon Plan Fluor 10X; NA 0,3; WD: 16 mm). </li><li> Visualizar las neuronas individuales utilizando un 40X en el infrarrojo cercano (IR) objetivo de inmersión en agua (Nikon APO 40XW; NA: 0,80; WD: 3,5 mm) conectado a contraste de interferencia IR-diferencial (DIC) óptica y una velocidad alta de infrarrojo cercano dispositivo acoplado cargado (CCD) video cámara (Hamamatsu C-7500). Bajo IR-DIC observación, las neuronas sanas presentan una superficie lisa, de color gris claro membrana plasmática. </li><li> Llenar una micropipeta con una solución de registro intracelular adecuado. Para la mayoría de aplicaciones, se utiliza la siguiente solución (en mM): 135 gluconato de potasio, 5 EGTA, 0,5 CaCl 2, 2 MgCl 2, 10 HEPES, 2 Mg-ATP, GTP y 0,1 (pH se ajustó a 7,25 con Tris base, osmolaridad ajustado a 290 mOsm con sacarosa). Establecer una grabación de célula entera de una neurona visualizado. </li></ol> 4. La aplicación local de medicamentos a las neuronas en rodajas <ol><li> Tire una micropipeta de patch clamp estándar como se describe anteriormente. Usoun extractor de pipeta como una Sutter P-97 que produce dos hermanas &quot;idénticas&quot; consejos de la misma pieza de vidrio es ventajoso cuando las soluciones de fármacos múltiples se van a utilizar en la misma célula. Tamaños de la punta que tienen una resistencia de ~ 5 mW si se llenaron con la solución intracelular se ha descrito anteriormente es la óptima. </li><li> Relleno de la micropipeta con una solución de fármaco se diluyó en aCSF carbogenated, grabación estándar. Dirija la punta hacia abajo, y la película de la micropipeta relleno de fármaco para eliminar las burbujas de aire atrapadas en la columna de solución. </li><li> Montar la micropipeta relleno de fármaco en un soporte de pipetas conectado con un tubo a un sistema de presión de expulsión adecuado, tal como un Picospritzer III (General Valve Co.). El titular de la pipeta debe ser montado en un micromanipulador con la misma resolución que manipuladores comúnmente utilizado para la grabación de patch clamp (por ejemplo, Sutter MP-285). Adicionalmente, el manipulador debe permitir el movimiento diagonal dentro y fuera de la rebanada. Hombreexpulsar manualmente presión 3 a 4 veces en la micropipeta con el fin de acumular la presión adecuada detrás de la columna de fluido. </li><li> Uso de los controles micromanipulador, baje la pipeta fármaco en la porción mientras se visualiza la punta bajo IR-DIC óptica. Idealmente, se debe identificar visualmente y el centro de una neurona a grabarse, seguido por la colocación de la pipeta de drogas cerca de la célula antes de la reducción de la micropipeta grabación dentro de la rebanada. Colocación de la pipeta de drogas puede provocar el movimiento del tejido en la vecindad de la célula grabada que podría interrumpir la grabación si el fármaco pipeta se coloca en posición después de establecer un sello gigaohmio. </li><li> Utilización de vídeo de IR, la posición de la micropipeta fármaco en la superficie superior de la lámina de tejido. Ejecutar una presión de eyección y el monitor 1) la proximidad de la punta para el movimiento de los desechos en relación con la expulsión, y 2) la punta de la micropipeta para detectar cualquier signo de que la punta está tapada o bloqueada. Si la punta está obstruido / tapado, retraiga el pipeteee y reemplazarla con una nueva. </li><li> Enfoque de la célula grabada en diagonal desde la parte superior de la rebanada de tal manera que cuando está en posición final, la punta de la pipeta de relleno de fármaco es 1) en el mismo plano focal (o ligeramente por debajo) como la celda grabada y la punta del electrodo de registro , y 2) 10 a 40 micras de la célula registrada. Si la punta de pipeta relleno de fármaco está por encima de la celda grabada, la fuerza de la presión de eyección podría interrumpir el sello gigaohmio. </li><li> Registrar la respuesta celular deseada mientras mantiene la celda en modo de tensión de la abrazadera o el modo de abrazadera de corriente. En forma rutinaria grabar acetilcolina y / o la nicotina-suscitó corrientes celulares mientras mantiene las neuronas en el modo de fijación de voltaje. Aunque la presión de eyección puede ser activado manualmente cuando se utiliza un Picospritzer III, para obtener resultados más reproducibles se recomienda para activar la eyección de presión utilizando el sistema de adquisición (Axon Digidata 1440 y pClamp 10,3) con un pulso digital TTL. </li></ol> 5. ControladorLa micropipeta lleno de drogas con un traductor piezoeléctrico <ol><li> Si es posible, montar el soporte de pipetas relleno de fármaco a un traductor de una sola dimensión piezoeléctrico (por ejemplo Piezojena PA-100 o Burleigh PZM-150) que es capaz de aceptar una entrada de voltaje analógico (de nuevo, desde el sistema de adquisición), que es luego montado en el micromanipulador de alta precisión (Sutter MP-285). Un traductor piezoeléctrico es útil debido a que el movimiento de la pipeta relleno de fármaco dentro y fuera de la rodaja, incluyendo el tiempo y la velocidad de entrada / salida, puede ser más fácilmente controlados y reproducidos de experimento a experimento. Al estudiar los nAChR, los efectos desensibilizantes de fuga de la nicotina de la pipeta relleno de fármaco, si llegaran a ocurrir, se minimizan cuando la pipeta se mueve sólo cerca de la celda con un traductor piezoeléctrico para el momento de la presión de eyección. </li><li> A través del potenciómetro controlado manualmente en el amplificador de control de tensión piezoeléctrico, marque el voltajea su valor máximo. </li><li> Uso del micromanipulador, la posición de la pipeta relleno de fármaco en el segmento en el que la pipeta se expulsa su contenido sobre la celda grabada, y retraer manualmente la pipeta por la reducción de la tensión a cero del amplificador de tensión de control piezoeléctrica. </li><li> Uso de una señal de salida analógica desde el sistema de adquisición de grabación, mueva la pipeta relleno de fármaco en la rebanada durante un período de 250 mseg a partir de 300 mseg antes de la presión de eyección TTL pulso se produce. Retraer la pipeta durante un período de 250 ms, a partir de 50 mseg después del pulso TTL termina. </li></ol>

<ol>
	<li>Tapper, A. 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+activation+of+?4*+receptors:+sufficient+for+reward,+tolerance,+and+sensitization.">Nicotine activation of ?4* receptors: sufficient for reward, tolerance, and sensitization.</a> Science. 306, 1029-1032 (2004).</li><li>Drenan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+activation+of+midbrain+dopamine+neurons+via+sensitized,+high-affinity+?6*+nicotinic+acetylcholine+receptors.">In vivo activation of midbrain dopamine neurons via sensitized, high-affinity ?6* nicotinic acetylcholine receptors.</a> Neuron. 60, 123-136 (2008).</li><li>Zhao, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+channelrhodopsin-2+transgenic+mice+for+optogenetic+dissection+of+neural+circuitry+function.">Cell type-specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function.</a> Nat. Methods. 8, 745-7452 (2011).</li><li>Peca, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shank3+mutant+mice+display+autistic-like+behaviours+and+striatal+dysfunction.">Shank3 mutant mice display autistic-like behaviours and striatal dysfunction.</a> Nature. 472, 437-442 (2011).</li><li>Zhou, F. M., Wilson, C. J., 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=Cholinergic+interneuron+characteristics+and+nicotinic+properties+in+the+striatum.">Cholinergic interneuron characteristics and nicotinic properties in the striatum.</a> J. Neurobiol. 53, 590-605 (2002).</li><li>Pidoplichko, V. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotine+activates+and+desensitizes+midbrain+dopamine+neurons.">Nicotine activates and desensitizes midbrain dopamine neurons.</a> Nature. 390, 401-404 (1997).</li><li>Nashmi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+nicotine+cell+specifically+upregulates+functional+?4*+nicotinic+receptors:+basis+for+both+tolerance+in+midbrain+and+enhanced+long-term+potentiation+in+perforant+path.">Chronic nicotine cell specifically upregulates functional ?4* nicotinic receptors: basis for both tolerance in midbrain and enhanced long-term potentiation in perforant path.</a> J. Neurosci. 27, 8202-8218 (2007).</li><li>Xiao, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+nicotine+selectively+enhances+?4?2*+nicotinic+acetylcholine+receptors+in+the+nigrostriatal+dopamine+pathway.">Chronic nicotine selectively enhances ?4?2* nicotinic acetylcholine receptors in the nigrostriatal dopamine pathway.</a> J. Neurosci. 29, 12428-12439 (2009).</li><li>Cohen, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotinic+cholinergic+mechanisms+causing+elevated+dopamine+release+and+abnormal+locomotor+behavior.">Nicotinic cholinergic mechanisms causing elevated dopamine release and abnormal locomotor behavior.</a> Neuroscience. 200, 31-41 (2012).</li><li>Drenan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+modulation+of+locomotion+and+striatal+dopamine+release+is+mediated+by+&#945;6&#946;4*+nicotinic+acetylcholine+receptors.">Cholinergic modulation of locomotion and striatal dopamine release is mediated by &#945;6&#946;4* nicotinic acetylcholine receptors.</a> J. Neurosci. 30, 9877-9889 (2010).</li><li>Grady, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+differences+determine+the+relative+selectivity+of+nicotinic+compounds+for+native+&#945;4&#946;2*-,+&#945;6&#946;2*-,+&#945;3&#946;4*-+and+&#945;7-nicotine+acetylcholine+receptors.">Structural differences determine the relative selectivity of nicotinic compounds for native &#945;4&#946;2*-, &#945;6&#946;2*-, &#945;3&#946;4*- and &#945;7-nicotine acetylcholine receptors.</a> Neuropharmacology. 58, 1054-1066 (2010).</li><li>Drenan, R. M. <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+&#945;6+and+&#946;3+subunits.">Subcellular trafficking, pentameric assembly, and subunit stoichiometry of neuronal nicotinic acetylcholine receptors containing fluorescently labeled &#945;6 and &#946;3 subunits.</a> Mol. Pharmacol. 73, 27-41 (2008).</li><li>Keath, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+modulation+by+nicotine+of+substantia+nigra+versus+ventral+tegmental+area+dopamine+neurons.">Differential modulation by nicotine of substantia nigra versus ventral tegmental area dopamine neurons.</a> J. Neurophysiol. 98, 3388-3396 (2007).</li><li>Mansvelder, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bupropion+inhibits+the+cellular+effects+of+nicotine+in+the+ventral+tegmental+area.">Bupropion inhibits the cellular effects of nicotine in the ventral tegmental area.</a> Biochem. Pharmacol. 74, 1283-1291 (2007).</li><li>Lee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+largest+group+of+superficial+neocortical+GABAergic+interneurons+expresses+ionotropic+serotonin+receptors.">The largest group of superficial neocortical GABAergic interneurons expresses ionotropic serotonin receptors.</a> J. Neurosci. 30, 16796-16808 (2010).</li><li>Margolis, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+in+the+identification+of+midbrain+dopamine+neurons.">Reliability in the identification of midbrain dopamine neurons.</a> PLoS One. 5, e15222 (2010).</li><li>Margolis, E. 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+ventral+tegmental+area+revisited:+is+there+an+electrophysiological+marker+for+dopaminergic+neurons.">The ventral tegmental area revisited: is there an electrophysiological marker for dopaminergic neurons.</a> J. Physiol. 577 (Pt 3), 907-924 (2006).</li><li>Couey, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distributed+network+actions+by+nicotine+increase+the+threshold+for+spike-timing-dependent+plasticity+in+prefrontal+cortex.">Distributed network actions by nicotine increase the threshold for spike-timing-dependent plasticity in prefrontal cortex.</a> Neuron. 54, 73-87 (2007).</li><li>Yang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinctive+nicotinic+acetylcholine+receptor+functional+phenotypes+of+rat+ventral+tegmental+area+dopaminergic+neurons.">Distinctive nicotinic acetylcholine receptor functional phenotypes of rat ventral tegmental area dopaminergic neurons.</a> J. Physiol. 587, 345-361 (2009).</li><li>Endo, T. <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+receptor+subtypes+involved+in+facilitation+of+GABAergic+inhibition+in+mouse+superficial+superior+colliculus.">Nicotinic acetylcholine receptor subtypes involved in facilitation of GABAergic inhibition in mouse superficial superior colliculus.</a> J. Neurophysiol. 94, 3893-3902 (2005).</li><li>Bouzat, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+structural+bases+of+activation+of+Cys-loop+receptors.">New insights into the structural bases of activation of Cys-loop receptors.</a> J. Physiol. Paris. , (2011).</li></ol>

El método presentado en este documento es en general útil para el estudio de ligando función del canal iónico en preparaciones de cortes de cerebro. Sin embargo, hay un número de factores que afecten significativamente a la calidad y reproducibilidad de los datos experimentales que resultan de la utilización de este método. Por ejemplo, las corrientes evocadas son muy sensibles al diámetro de la punta de la pipeta de relleno de fármaco. Pequeñas sugerencias causará dificultades con expulsar la solución de f?...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt; Nombre del reactivo</strong</td&gt;<td<strong&gt; Empresa</strong</td&gt;<td<strong&gt; Número de catálogo</strong</td&gt;</tr&gt;<tr&gt;<td&gt; N-metil-D-glucamina</td&gt;<td&gt; Sigma</td&gt;<td&gt; M2004</td&gt;</tr&gt;<tr&gt;<td&gt; KCl</td&gt;<td&gt; Sigma</td&gt;<td&gt; P3911</td&gt;</tr&gt;<tr&gt;<td&gt; NaH<sub&gt; 2</sub&gt; PO<sub&gt; 4</sub</td&gt;<td&gt; Sigma</td&gt;<td&gt; S9638</td&gt;</tr&gt;<tr&gt;<td&gt; NaHCO<sub&gt; 3</sub</td&gt;<td&gt; Sigma</td&gt;<td&gt; S6014</td&gt;</tr&gt;<tr&gt;<td&gt; HEPES</td&gt;<td&gt; Sigma</td&gt;<td&gt; H3375</td&gt;</tr&gt;<tr&gt;<td&gt; Glucosa</td&gt;<td&gt; Sigma</td&gt;<td&gt; G5767</td&gt;</tr&gt;<tr&gt;<td&gt; Na<sup&gt; +</sup&gt; Ascorbato</td&gt;<td&gt; Sigma</td&gt;<td&gt; A4034</td&gt;</tr&gt;<tr&gt;<td&gt; Tiourea</td&gt;<td&gt; Sigma</td&gt;<td&gt; T8656</td&gt;</tr&gt;<tr&gt;<td&gt; Na<sup&gt; +</sup&gt; Piruvato</td&gt;<td&gt; Sigma</td&gt;<td&gt; P2256</td&gt;</tr&gt;<tr&gt;<td&gt; MgSO<sub&gt; 4</sub&gt; • 7H<sub&gt; 2</sub&gt; O</td&gt;<td&gt; Sigma</td&gt;<td&gt; 230391</td&gt;</tr&gt;<tr&gt;<td&gt; De CaCl<sub&gt; 2</sub&gt; • 2H<sub&gt; 2</sub&gt; 0</td&gt;<td&gt; Sigma</td&gt;<td&gt; 223506</td&gt;</tr&gt;<tr&gt;<td&gt; NaCl</td&gt;<td&gt; Sigma</td&gt;<td&gt; S9625</td&gt;</tr&gt;<tr&gt;<td&gt; Na<sup&gt; +</sup&gt; Pentobarbital</td&gt;<td&gt; Vortech Farmacia</td&gt;<td&gt; 76351315</td&gt;</tr&gt;<tr&gt;<td&gt; Gluconato de potasio</td&gt;<td&gt; Sigma</td&gt;<td&gt; G4500</td&gt;</tr&gt;<tr&gt;<td&gt; EGTA</td&gt;<td&gt; Sigma</td&gt;<td&gt; E3889</td&gt;</tr&gt;<tr&gt;<td&gt; Mg-ATP</td&gt;<td&gt; Sigma</td&gt;<td&gt; A9187</td&gt;</tr&gt;<tr&gt;<td&gt; GTP</td&gt;<td&gt; Sigma</td&gt;<td&gt; G8877</td&gt;</tr&gt;<tr&gt;<td&gt; DSK-Zero 1 vibrante slicer</td&gt;<td&gt; Ted Pella, Inc.</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; P-97 Flaming / Brown micropipeta extractor</td&gt;<td&gt; Sutter</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Cámara RC-27 Grabación</td&gt;<td&gt; Warner</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; TC-344B perfusión controlador calentador</td&gt;<td&gt; Warner</td&gt;<td&gt; 640101</td&gt;</tr&gt;<tr&gt;<td&gt; SH-27B Solución calentador</td&gt;<td&gt; Warner</td&gt;<td&gt; 640102</td&gt;</tr&gt;<tr&gt;<td&gt; Nikon FN-1</td&gt;<td&gt; Nikon</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; C-7500 CCD cámara de vídeo</td&gt;<td&gt; Hamamatsu</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Picospritzer III</td&gt;<td&gt; General Valve Co.</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; MP-285 Micromanipulador</td&gt;<td&gt; Sutter</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; PA-100 traductor piezoeléctrico</td&gt;<td&gt; Piezosystem jena, Inc.</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; 12V40 piezo amplificador</td&gt;<td&gt; Piezosystem jena, Inc.</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Axopatch 200B</td&gt;<td&gt; Molecular Devices Corp.</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Digidata 1440A</td&gt;<td&gt; Molecular Devices Corp.</td&gt;<td</td&gt;</tr&gt;</tbody&gt;</table

local application of drugs to study nicotinic acetylcholine receptor function in mouse brain slices

El consumo de tabaco provoca numerosos problemas de salud, incluyendo cáncer, enfermedades del corazón, enfisema y derrame cerebral. La adicción a fumar cigarrillos es un trastorno neuropsiquiátrico frecuente que se deriva de las acciones biofísicos y celulares de la nicotina sobre los receptores nicotínicos de acetilcolina (nAChR) en todo el sistema nervioso central. La comprensión de los diversos subtipos de nAChR que existen en las áreas cerebrales relacionadas con la adicción a la nicotina es una de las principales prioridades. Los experimentos que emplean técnicas tales como electrofisiología de pinzamiento zonal de célula completa o de dos electrodos grabaciones de tensión de la abrazadera son útiles para la caracterización farmacológica de nAChR de interés. NAChRs expresan las células, tales como células de cultivo de tejidos de mamífero u oocitos de Xenopus laevis, están físicamente aisladas y por lo tanto fácil de estudiar usando las herramientas de la farmacología moderna. Mucho se ha hecho uso de estas técnicas, en particular cuando el receptor diana ya se conocía unnd expresión ectópica se logró fácilmente. A menudo, sin embargo, es necesario estudiar nAChRs en su ambiente nativo: en neuronas dentro de las secciones de cerebro agudamente recogidas de ratones o ratas de laboratorio. Por ejemplo, ratones que expresan &quot;hipersensibles&quot; subunidades de nAChR tales como ratones α4 L9&#39;A 1 y ratones α6 L9 2, permiten la identificación inequívoca de las neuronas en base a su expresión funcional de una subunidad de nAChR específico. Aunque conjunto de células grabaciones patch clamp de neuronas en rodajas de cerebro se realiza rutinariamente por el electrofisiólogo experto, es difícil de aplicar localmente fármacos tales como la acetilcolina o la nicotina a la célula grabada dentro de un corte de cerebro. La dilución de la droga en el superfusato (baño de aplicación) no es rápidamente reversible, y tubo en U sistemas no se adaptan fácilmente para trabajar con secciones de cerebro. En este trabajo se describe un método para la rápida aplicación de activación de nAChR medicamentos a las neuronas registradas en adultos mOuse rodajas de cerebro. Estándar conjunto de células grabaciones están hechos de neuronas en rodajas, y una micropipeta segunda llena de un fármaco de interés es maniobrado en posición cerca de la célula registrada. Una inyección de aire a presión o nitrógeno inerte en la pipeta relleno de fármaco provoca una pequeña cantidad de solución de fármaco para ser expulsado de la pipeta sobre la celda de grabado. Usando este método, nAChR mediada por las corrientes son capaces de resolver con precisión de milisegundos. Tiempos de solicitud de medicamentos puede variarse fácilmente, y la pipeta de relleno de fármaco puede ser retraído y se reemplaza con una nueva pipeta, permitiendo curvas concentración-respuesta a crear para una sola neurona. Aunque se ha descrito en el contexto de nAChR neurobiología, esta técnica debe ser útil para el estudio de muchos tipos de canales iónicos activados por ligando o los receptores en las neuronas de rodajas de cerebro.

En nuestros experimentos, de manera rutinaria grabar de dopamina (DA)-producción de neuronas del área tegmental ventral (VTA) y la sustancia negra pars compacta (SNc). En el modo de fijación de voltaje, presión de aplicación de la acetilcolina o la nicotina a estas células normalmente se traducirá en una corriente rápida, catión hacia dentro que alcanza pico dentro de 100-200 ms (Figura 1A-B). Decaimiento de la corriente es dictada en gran medida por la difusión del fármaco desde el sitio de ...

Watch this Scientific Journal Video about Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices at JoVE.com

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

En este artículo, se describe un método útil para estudiar ligandos función del canal iónico en las neuronas de rodajas de cerebro agudamente aisladas. Este método implica el uso de una micropipeta de relleno de fármaco para aplicación local de medicamentos a las neuronas registradas utilizando técnicas estándar de patch clamp.

La aplicación local de medicamentos para estudiar la función del receptor nicotínico de acetilcolina en rodajas de cerebro de ratón

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent ...

local-application-drugs-to-study-nicotinic-acetylcholine-receptor

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Neuroscience

Unit 1

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

SCIENTISTS IN ACTION

18.9K vistas.  Purdue University. En este artículo, se describe un método útil para estudiar ligandos función del canal iónico en las neuronas de rodajas de cerebro agudamente aisladas. Este método implica el uso de una micropipeta de relleno de fármaco para aplicación local de medicamentos a las neuronas registradas utilizando técnicas estándar de patch clamp.

Video: Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation de las neuronas del cerebro de roedores Rodajas para el Estudio de la transmisión sináptica y la imagen terminales presinápticos

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Investigación

Neurociencias

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

Respuestas imágenes de calcio en las neuronas GFP-etiquetados de hipotalámicas rodajas de cerebro de ratón

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Las fibras serotoninérgicos proyección de imagen en la médula espinal de ratón El uso de la claridad / Técnica CÚBICO

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2. Consequently, investigation of structural details of the interaction between 5-HT2A and mGlu2 affecting schizophrenia-related behaviors represents a powerful translational tool. As previously shown, the head-twitch response (HTR) in mice is elicited by hallucinogenic drugs and this behavioral response is absent in 5-HT2A knockout (KO) mice3,4. Additionally, by conditionally expressing the 5-HT2A receptor only in cortex, it was demonstrated that 5-HT2A receptor-dependent signaling pathways on cortical pyramidal neurons are sufficient to elicit head-twitch behavior in response to hallucinogenic drugs3. Finally, it has been shown that the head-twitch behavioral response induced by the hallucinogens DOI and lysergic acid diethylamide (LSD) is significantly decreased in mGlu2-KO mice5. These findings suggest that mGlu2 is at least in part necessary for the 5-HT2A receptor-dependent psychosis-like behavioral effects induced by LSD-like drugs. However, this does not provide evidence as to whether the 5-HT2A-mGlu2 receptor complex is necessary for this behavioral phenotype. To address this question, herpes simplex virus (HSV) constructs to express either mGlu2 or mGlu2&#916;TM4N (mGlu2/mGlu3 chimeric construct that does not form the 5-HT2A-mGlu2 receptor complex) in the frontal cortex of mGlu2-KO mice were used to examine whether this GPCR heteromeric complex is needed for the behavioral effects induced by LSD-like drugs6.

This article describes how to inject viral vectors into the mouse frontal cortex to test behavioral assays that require GPCR heteromeric formation.

HSV mediada por la expresión transgénica de constructos quiméricos para estudiar la función del comportamiento de los GPCR heterómeros en ratones

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2.  ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

Aplicación de la abrazadera de parche guiada por imágenes automatizada para el estudio de neuronas en cortes cerebrales

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug application during electrophysiological recording is the puff technique, which can be used to study the effect of pharmacological reagents on neuronal activities in brain slices. The greatest advantage of puff application is that the drug concentration around the recording site increases rapidly, thus preventing desensitization of membrane receptors. Successful use of puff application involves careful attention to the following elements: the concentration of the drug, the parameters of the puff micropipette, the distance between the tip of a puff micropipette and the neuron recorded, and the duration and pressure driving the puff (pounds per square inch, psi). This article describes a step-by-step procedure for recording whole-cell currents induced by puffing gamma-aminobutyric acid (GABA) onto a neuron of a prefrontal cortical slice. Notably, the same procedure can be applied with minor modifications to other brain areas such as the hippocampus and the striatum, and to different preparations, such as cell cultures.

We describe the puff technique, by which pharmacological reagents can be administered during whole-cell patch-clamp recording, and highlight various aspects of the features that are crucial for its success.

Celulares corrientes inducidas por Puff aplicación de GABA en rebanadas de cerebro

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Un enfoque alternativo para estudiar acontecimientos primarios en neurodegeneración con rodajas de cerebro de rata Ex Vivo

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Probing Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices via Laser Flash Photolysis of Photoactivatable Nicotine

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with subcellular location within cells where this function is carried out. To study the subcellular location of nicotinic ACh receptors (nAChRs) in native brain tissue, an optical method was developed for precise release of nicotine at discrete locations near neuronal membranes during electrophysiological recordings. Patch-clamped neurons in brain slices are filled with dye to visualize their morphology during 2-photon laser scanning microscopy, and nicotine uncaging is executed with a light flash by focusing a 405 nm laser beam near one or more cellular membranes. Cellular current deflections are measured, and a high-resolution three-dimensional (3D) image of the recorded neuron is made to allow reconciliation of nAChR responses with cellular morphology. This method allows for detailed analysis of nAChR functional distribution in complex tissue preparations, promising to enhance the understanding of cholinergic neurotransmission.

This article presents a method for studying nicotinic acetylcholine receptors (nAChRs) in mouse brain slices by nicotine uncaging. When coupled with simultaneous patch clamp recording and 2-photon laser scanning microscopy, nicotine uncaging connects nicotinic receptor function with cellular morphology, providing a deeper understanding of cholinergic neurobiology.

Sondeo de la función de Receptor nicotínico de la acetilcolina en rebanadas de cerebro de ratón mediante fotólisis de destello láser de la nicotina Photoactivatable

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Implantación crónica de todo cortical matriz electrocorticográfica en el tití común

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Evaluación de la inducción a largo plazo de la depresión en rodajas de cerebelosores adultos

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...