Financiado por el NIH P40 OD010952, la Fundación Korein, de la Universidad de Miami Fellowship de SLC y una beca de Maytag para ATK. Los autores agradecen al personal de la Nacional de Recursos para la Aplysia, así como Lauren Simonitis y Hannah Peck, quien proporcionó micrografías de una figura.

1. Preparación de células <ol><li> En el Día 1, pesar y anestesiar a los animales. <ol><li> Pesar unos 30 g, 1 kg animal. Se anestesia en animales 5-10 volúmenes de agua de mar 01:01: MgCl isotónica. 6H 2 0 durante 1 hora con aireación, tales como una bomba de aire de acuario eléctrico con piedra de aire adjunto. </li></ol></li><li> Prepare suministros de disección. <ol><li> Montar la bandeja de disección limpia con alfileres de acero inoxidable, tales como pasadores de tela. Montar varios platos de Petri que contenían agua de mar artificial (ASW; véanse las Tablas 1 y 3) + penicilina / estreptomicina (P / S). </li><li> Tenga listo un microscopio de baja potencia. Tener instrumentos de disección limpias listas, como la nariz crucería y pinzas quirúrgicas finas y finas y grandes tijeras quirúrgicas. Rocíe los instrumentos con un 70% de alcohol isopropílico antes de su uso y deje secar. </li></ol></li><li> Posición animal en la bandeja de disección. <ol><li> Posición animales cara ventral hacia abajo en la bandeja de disección. Pin animalesa través de los bordes de la pared del cuerpo y las fronteras parapodiales, pero evite la cola y la cabeza, para exponer la superficie dorsal y la ranura genital </li><li> Enjuague la superficie dorsal en el agua fría de la llave de ejecución lenta. Aplicar una corriente de luz de 70% de alcohol isopropílico a partir de una botella flexible a lo largo de la ranura genital y sobre la parte superior de la cabeza. </li></ol></li><li> Hacer la incisión inicial. <ol><li> El uso de un microscopio de baja potencia (véase la Tabla 2) y la luz reflejada para observar el punto en el que la ranura genital origina en el margen de cáscara anterior, mantener tensada con unas pinzas de punta acanalada un pliegue de tejido a la izquierda de este origen, mientras cortando un poco profunda el hoyo todo el camino a través del área lisa, plana de la pared del cuerpo justo a la derecha de la ranura genital con tijeras en ángulo finas. </li><li> Hemolinfa deberá observarse para verter en el agujero, a veces llevando consigo el ganglio abdominal y el estómago. </li></ol></li><li> Ampliar la incisión. <ol><li> Use tijeras grandes para expandir ªe incisión anterior a un punto entre los rinóforos, mientras tira hacia arriba con la cuchilla inferior para evitar cortes en el intestino. Se observaron los órganos internos que se derrame fuera de la incisión. </li></ol></li><li> Retire los ganglios. <ol><li> Vuelva a colocar la bandeja y reorientar el microscopio en la región de la cabeza. </li><li> Con unas pinzas limpias y tijeras finas, quitar la cabeza ganglios cortando en un momento los nervios que forman la cabeza de los ganglios en un anillo alrededor del esófago para eliminar pleural-pedal y cerebral ganglios como grupo. </li><li> Retire el ganglio vestibular que se adhiere a la parte ventral del esófago. </li><li> Retire el ganglio abdominal cortando los 4 grandes conectivas nerviosas que emiten desde este ganglio. </li></ol></li><li> Aislar los ganglios de interés. <ol><li> Recortar los ganglios de la cabeza aparte, dejando una longitud de conectores unidos a cada ganglio de interés que es igual a al menos el diámetro del ganglio; esto retardar enzima sobre-digestiónde las células de interés en el siguiente paso. </li><li> Ponga cada ganglio objetivo a través de 2 enjuagues de ASW + P / S, moviéndose entre enjuagues con pinzas limpias. </li><li> Si la orientación de las células de PVC, deje el hemiganglion pleural derecho accesorio a la hemiganglion pedal derecho, y del mismo modo dejar el hemiganglion pleural izquierdo conectado al hemiganglion pedal izquierdo. </li><li> Desechar los ganglios no deseado y el resto del animal de acuerdo con la práctica aceptada la investigación. </li></ol></li><li> Digerir enzimáticamente los ganglios. <ol><li> Para cada ganglio, preparar 1 ml de solución de enzima que consiste en 3,75 mg dispasa, 1 mg de hialuronidasa y 0,3 g de colagenasa tipo XI por ml de ASW + P / S en un tubo cónico de 15 ml de polipropileno. </li><li> Lugar enjuaga ganglios en la solución enzimática y coloque la tapa. Se coloca el tubo en posición horizontal sobre un agitador rotatorio (véase el cuadro 2) a baja velocidad durante 13-5 horas durante la noche a aproximadamente 23 ° C (temperatura ambiente). </li></ol></li><li> Prepare elplacas de cultivo. <ol><li> Antes de microdisección (Día 2), preparar poli-D-lisina (PDL) recubiertos con placas de cultivo en una campana de cultivo de tejido. Añadir ~ 0,5 ml PDL (0,2 mg / ml de agua estéril) para el centro de un plato de 35 mm para ~ 25 min. </li><li> Enjuague PDL 3 veces con agua estéril. Deje que se seque. UV-esterilizar las placas. </li></ol></li><li> Día 2 Aislar las neuronas. <ol><li> Llene el centro de 35 mm platos que eran PDL-revestido y UV esterilizada con aproximadamente 0,5 ml de la ASW + P / S para crear una isla de los medios de comunicación en el interior del plato de otro modo seco. Esto se puede hacer en el banco, fuera de la campana de cultivo de tejido. Uno de los platos debe estar preparado para cada grupo de células procedentes de una hemiganglion. </li><li> Tenga listo un plato de disección de 100 mm de diámetro hecha por Sylgard-recubrimiento y curado de un mm de la superficie de la disección profunda 5, equipado con pasadores de disección finas de varios tamaños para ser utilizados como alfileres y sondas (véase la Tabla 3). Enjuague este plato con un 70% de alcohol isopropílico y después con ASW + P / S, y finalmente fill con ASW + P / S. </li></ol></li><li> Prepare microdisección de los ganglios digerido. <ol><li> Verter la solución de enzima que contiene la pleural-pedal y bucal ganglios digerido en el plato de disección. </li><li> Coloque el plato en la platina del microscopio sobre una superficie negro con luz reflejada. </li><li> Utilizando el tejido conectivo adjunto, precisar cada pleural-pedal hemiganglion lado dorsal hacia arriba. Los tejidos deben ser suaves e intolerante de estiramiento. </li></ol></li><li> Microdissect neuronas PVC. <ol><li> Uso de 2 pares de pinzas finas limpias, y que sostiene un pasador de disección fina en un par de fórceps como una sonda, aislar las células de PVC a partir de una hemiganglion pleural. La digestión enzimática se han eliminado o roto aparte la vaina de tejido conectivo que cubre el clúster de PVC, sin embargo, las células se adhieren el uno al otro. </li><li> Utilice la sonda para separar estas células desde el pedal de conjuntivo-pleural 18, a continuación, dejar que la caída grupo de células a la parte inferior de la disecciónplato. </li></ol></li><li> Transferir las neuronas a placa de cultivo. <ol><li> Transferir el grupo de células a un preparado 35 mm de placa de cultivo por succión suavemente el clúster en la punta de un poco pulida al fuego desechable de pipeta Pasteur de vidrio, a continuación, dispensación lentamente en la isla los medios de comunicación en una placa de cultivo, teniendo cuidado de evitar la introducción de burbujas de aire en la pipeta. Repita el aislamiento del grupo PVC de la otra hemiganglion y colocar en una placa de cultivo independientes. </li></ol></li><li> Microdissect y transferir las neuronas BSC. <ol><li> De clavijas de salida del lado ventral ganglio vestibular arriba, con cuidado de sobra las células de interés. Repita el paso 1.12, y los 2 grupos de células BSC del ganglio vestibular 10. </li><li> Aislamiento de las neuronas de PVC y el BSC producirá 4 placas de cultivo que contienen grupos de neuronas: 2 platos que contienen grupos BSC y 2 platos que contienen grupos de PVC. Deseche el resto de los ganglios y dejar de lado el plato de 100 mm disección. </li></ol></li><li> DissocIATE los grupos de células. <ol><li> Coloque una placa de cultivo que contiene las células en la platina del microscopio de baja potencia y retire la cubierta. </li><li> Disociar el clúster de PVC con un movimiento rápido suavemente la parte inferior de la placa de cultivo adyacente a la agrupación de células con un pasador de disección fina celebrada en fórceps. Sacudiendo está cavando la punta del alfiler en el plástico de la parte inferior del plato como si tratara de cavar una mota de plástico. Cuando la fricción con el plástico libera el punto de pin, una ola de percusión se produce a través del medio que se rompe la mata cerca de las células. </li><li> Pueden ser necesarios 5-10 películas a casi disociar completamente las células, pero es mejor dejar pequeños grupos de células en lugar de a flick demasiado no sea que las células serán destruidas por pura mecánica. </li><li> Colocar la tapa y repita con otras placas de cultivo. </li></ol></li><li> Almacenar los cultivos celulares. <ol><li> Coloque las placas de cultivo que contienen islas de medios en sus centros con células disociadas enun gran contenedor que permite la circulación de aire, tal como una x 25 mm ronda placa Petri de plástico 150, y colocar este plato en una incubadora a 17 ° C. </li><li> Instalar en la cámara de un recipiente abierto de agua como una fuente de humedad. Dejar reposar durante la noche. Las células se adhieren al revestimiento de poli-D-lisina en el centro del plato. </li></ol></li><li> Placas de cultivo de inundación. <ol><li> El día 3, inundar suavemente los platos con 2,5 ml de ASW + P / S. Examinar y contar las células usando un microscopio invertido cultivo celular. Conservar en el 17 ° C incubadora. </li></ol></li></ol> 2. Electrofisiología El método es parche estándar de sujeción que se ha descrito en numerosos textos (por ejemplo, Sakmann y Neher, 1995) 16. Este protocolo funciona en células ≤ 100 pF de capacitancia o células &lt;60 m de diámetro durante los días 3 y 4 del protocolo. Las células sin procesos son óptimas para la grabación. La siguiente EXAMEN especialiones se aplican: <ol><li> Las células más grandes pueden ser acomodados con la opción de configuración del amplificador 200B Axopatch establecido en β = 0,1. La activación de corrientes muy grandes puede causar que las células se escapen de fijación de voltaje. Soluciones ASW sustituido para el contenido de sal (por ejemplo, una fracción de la de NaCl sustituido con impermeable al cloruro de N-metil-D-glucamina) se puede usar para reducir la amplitud de corriente de la célula entera. </li><li> Fibra llenos de borosilicato pipetas parche tirados en un extractor de pipeta y rellenada hasta la mitad con una solución intracelular (ICS) que consta de 450 mM KCl y otras sales (ver Tabla 1) son adecuados, y deben tener una resistencia de aproximadamente 0,5 a 1 MOhm. Si se desea el aislamiento de las corrientes iónicas específicas, por ejemplo, corrientes de Na + en la ausencia de corrientes de K, K + en esta solución puede ser sustituido por otros iones tales como Cs +. Cl - sustitución de ICS con un anión impermeant también se justifica si se desea un ICS más natural9. </li><li> Las células son robustos con&gt; 1 hr-largas grabaciones posibles a temperatura ambiente. La grabación es óptima en los días 3 y 4 del protocolo, correspondientes a 24-48 horas de cultivo. Después de 48 horas hay dificultades para formar sellos GigaOhm, quizás a causa de elaboración de un glicocálix sobre la superficie de la célula, y los problemas causados ​​por la abrazadera espacio excrecencia proceso. Las células son útiles, sin embargo, para los estudios de clamp no parches, tales como la toxicología, que se pueden completar en &lt;14 d. </li><li> ASW y otras soluciones para ser dispensado desde el dispositivo de solución por gravedad, así como la solución intracelular, deben ser filtrados a través de una jeringa montada en un filtro de 0,2 micras Acrodisk (Tabla 3) para eliminar las partículas finas que interfieren con la formación de sello GigaOhm. </li><li> La desensibilización se produce al utilizar agonistas tales como D-aspartato (D-Asp), por lo tanto, ~ 1-2 minutos entre las aplicaciones de los agonistas se recomienda. Por el contrario, la potenciación se observa a menudo con una rápida aplicación repetidacationes de agonistas tales como L-glutamato (L-Glu). </li><li> Agonista de corrientes activadas tales como L-Glu pueden ser estudiadas mediante la aplicación de agonistas con la Picospritzer pipeta. Esta pipeta se tira de la misma como la pipeta de parche y contiene agonista en ASW. Cuando la punta de la pipeta se coloca por debajo de la tubería de salida del dispositivo de solución por gravedad, y en un ángulo de 45 ° respecto a la tubería de salida (Figura 2F), agonista se lavó rápidamente fuera de la celda, y la desensibilización se reducirá al mínimo. El agonista de la pipeta debe crear un ángulo de 90 ° o más en relación con la pipeta de parche. </li></ol>

<ol>
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Las técnicas de disociación describen aquí dió cultivos de neuronas sensoriales que contienen 50-100 neuronas aisladas intercalados con pequeños números de células gliales y otras células no identificados. Los pasos más críticos en el protocolo son el tiempo de los ganglios permanecen en solución de enzima, y ​​agitando, la disociación de los grupos de células digeridas para romper el clúster en las células individuales. Digestión enzimática (paso 1.8) debe ser optimizado a la temperatura disponible...

El molusco opistobranch marino Aplysia, ha sido un modelo neurobiológico útil para muchas décadas. Es mejor conocido como modelo de habituación y condicionamiento clásico 7, 8. Los estudios sobre el aprendizaje y la memoria en este modelo ganó el Premio Nobel de Fisiología o Medicina en 2000 por Eric R. Kandel, en un premio que compartió con Arvid Carlsson y Paul Greengard 10. Los estudios que implican registros eléctricos a partir de preparaciones reducidas, en la que los elementos del sistema nervioso de este invertebrado se diseccionaron a partir del animal con los nervios y los músculos dejaron adjunta, han ayudado a aclarar los papeles de las neuronas individuales en Aplysia. Identificación de los mecanismos moleculares precisos que constituyen el aprendizaje en Aplysia sin embargo, a menudo empleado otra técnica, a largo plazo co-cultivos de una neurona sensorial y un motoneuronas, obtenido de uno en uno a partir de animales donantes individuales y se dejó formar una sinapsis en la placa de cultivo de 21 . Nosotros y otros 1, 3, 6, 14, 15, 16 hemos explotado la facilidad con la que identifican las neuronas se pueden orientar en este modelo, así como su resistencia en experimentos a largo plazo para hacer cultivos a corto plazo disociados de grupos de neuronas nominalmente homogéneos en el que se estudian las corrientes iónicas bajo fijación de voltaje en la configuración de patch clamp. Muchas neuronas Aplysia se levantan a las reiteradas rondas de parche de sujeción para dar tiempo a las manipulaciones experimentales de larga duración. La técnica es útil para las neuronas como las células neurosecretoras bolsa del ganglio abdominal, y las neuronas sensoriales de los ganglios pleural y bucal cuya disociación como se describe aquí, pero no para las neuronas muy grandes&gt; 60 m de diámetro, tales como L7 o R2 de el ganglio abdominal. No empleamos suero Aplysia en nuestras culturas, a diferencia de los senso-motoneuronas co-cultivos descritos en otro lugar. La mayoría de las neuronas obtenidas mediante este procedimiento no podrá contar con procesos para tél primero 48 h en cultivo, facilitando toda grabación de voltaje de la célula, pero entonces brotar y elaborar los axones y otros procesos durante aproximadamente 14 días antes de morir a causa de la falta de nutrientes y / o factores de crecimiento. Esta técnica produce cultivos primarios de neuronas 50-100 por plato de regiones fisiológicamente documentados de los ganglios bucal y pleural. Este protocolo es útil para los investigadores que estudian aspectos de la fisiología de células individuales en experimentos que requieren numerosos experimental repeticiones por animal. Se produce un par coincidente de los cultivos debido a la separación anatómica de las células diana en hemiganglia izquierda y derecha, permitiendo estudios que se benefician del tratamiento combinado y los cultivos de control. El protocolo se dirige bucales S racimo (BSC), las neuronas del ganglio vestibular, y ventrocaudal (PVC) pleurales neuronas del ganglio pleural. Estas células son de un tamaño apropiado para el conjunto de grabaciones de voltaje de la celda de visualización y Robust respuestas glutamatérgicas. La metodología discutida es apropiado para la mayoría de los ganglios en el sistema nervioso Aplysia.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Artificial seawater ASW</td>
 <td>Sigma-Aldrich</td>
 <td>assorted</td>
 <td>(mM): 417 NaCl, 10 KCl, 10 CaCl2 (2 H2O), 5MgCl2 (6H2O), 15 HEPES-NaOH, pH 7.6</td>
 </tr>
 <tr>
 <td>Intracellular solution</td>
 <td>Sigma</td>
 <td>assorted</td>
 <td>(mM): 450 KCl, 2.9 CaCl2 (2 H2O), 2.5 MgCl2 (6 H2O), 5 Na2ATP, 10 EGTA, and 40 HEPES-KOH, pH 7.4</td>
 </tr>
 <tr>
 <td>Poly-D-lysine</td>
 <td>Sigma</td>
 <td>P6407</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>penicillin/streptomycin added to ASW at 1:100</td>
 <td>Lonzo Walkersville, Inc.</td>
 <td>17-603E</td>
 <td>5,000 Units/ml penicillin plus 5,000 mg/ml streptomycin</td>
 </tr>
 <tr>
 <td>Neutral dispase II</td>
 <td>Roche Diagnostics</td>
 <td>10165859001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>hyaluronidase</td>
 <td>Sigma-Aldrich</td>
 <td>H4272</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>collagenase type XI</td>
 <td>Sigma-Aldrich</td>
 <td>C9407</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-Glutamate (L-Glu)</td>
 <td>Sigma-Aldrich</td>
 <td>49601-100G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>D-Aspartate (D-Asp)</td>
 <td>Sigma-Aldrich</td>
 <td>11200-10G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>N-methyl-D-aspartate (NMDA)</td>
 <td>Biomol</td>
 <td>100002-268</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-Asp</td>
 <td>Sigma</td>
 <td>A6683-25G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA)</td>
 <td>Sigma</td>
 <td>A6816-5MG</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-Glu R antagonists</td>
 <td>various</td>
 <td>various</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>agar</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>kynurenate</td>
 <td>Sigma-Aldrich</td>
 <td>61250</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>APV</td>
 <td>Sigma-Aldrich</td>
 <td>A5282</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DL-2-Amino-5-phosphonopentanoic acid (NMDAR antagonist)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>2-propanol</td>
 <td>VWRSP</td>
 <td>BDH1133</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Chloriding solution</td>
 <td>Sigma</td>
 <td>assorted</td>
 <td>25 g FeCl3 + 25 ml concentrated HCl + 50 ml H2O</td>
 </tr>
 <tr>
 <td>Sylgard silicone 2-part polymer</td>
 <td>World Precision Instruments (WPI)</td>
 <td>SYL184</td>
 <td>Provides pin-out surface for small dissection dishes</td>
 </tr>
 <tr>
 <td>0-40x zoom magnification microscope for dissections</td>
 <td>Wild</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Techniquip 150 Watts Fiber Optic Illuminator</td>
 <td>Microoptics of Florida</td>
 <td>TQ FOI-150</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>RotoMix 50800 orbital mixer</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nikon Diaphot inverted phase-contrast microscope with 4x, 20x (optional) &amp; 40x objectives</td>
 <td>SR Research Ltd.</td>
 <td>Eyelink II</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tektronix digital oscilloscope</td>
 <td>SR Research Ltd.</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>pClamp 10 data acquisition and analysis software</td>
 <td>Molecular Devices</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PC with Windows XP or higher operating system</td>
 <td>PC Solutions</td>
 <td>&nbsp;</td>
 <td>Thinkserver with solid state hard drives (80GB) and low noise monitors</td>
 </tr>
 <tr>
 <td>Flaming/Brown P87 micropipette puller</td>
 <td>Sutter Instruments, Novato, CA</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Axon Instruments Axopatch 200B clamp amplifier with a capacitance compensation range of 1-1000 pF; preamplifier</td>
 <td>Molecular Devices, Sunnyvale, CA</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Axon instruments electrode holder assembly for Axopatch 200B preamplifier</td>
 <td>Molecular Devices, Sunnyvale, CA</td>
 <td>CV203BU</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Digidata 1200 A/D converter</td>
 <td>Molecular Devices, Sunnyvale, CA</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Picospritzer, powered by N2 adjustable for pressure and duration</td>
 <td>Parker Hannifin, Cleveland, OH</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>TMC Micro-G Vibration isolation table</td>
 <td>Ametek</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Faraday cage</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>custom manufacture</td>
 </tr>
 <tr>
 <td>Burleigh Piezoelectric Clamshell Micromanipulators</td>
 <td>Burleigh Instruments; Thorlabs</td>
 <td>&nbsp;</td>
 <td>presently PCS-5000; -6000 series + mounts</td>
 </tr>
 <tr>
 <td>Narishige M-152 manual manipulators (for perfusion system and picospritzer)</td>
 <td>Narishige USA</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Filament pipette glass,1.5 mm OD, 0.84 mm ID -</td>
 <td>WPI</td>
 <td>1B150-3</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>3 inch length</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ag/AgCl half cell</td>
 <td>WPI</td>
 <td>EP4</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>15 ml centrifuge tubes, 35-2097 BD Falcon* Centrifuge Tubes</td>
 <td>VWRSP</td>
 <td>21008-918</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Angled Scissors</td>
 <td>Fine Science Tools</td>
 <td>15006-09</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dumostar Fine forceps</td>
 <td>Fine Science Tools</td>
 <td>11295-00</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>35 mm falcon tissue culture dishes</td>
 <td>VWRSP</td>
 <td>25382-064</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>falcon 150 x 25 mm tissue culture dishes; 1013</td>
 <td>VWRSP</td>
 <td>1013</td>
 <td>also can be made into small dissection dishes with sylgard</td>
 </tr>
 <tr>
 <td>sylgard</td>
 <td>WPI</td>
 <td>SYL184</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>animal dissection tray</td>
 <td>various</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>15 ml centrifuge tubes, 35-2097 BD Falcon</td>
 <td>VWRSP</td>
 <td>21008-918</td>
 <td>For 6-bore gravity-fed perfusion system</td>
 </tr>
 <tr>
 <td>Aluminum clips with screw hole ends</td>
 <td>hardware store</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>23 gauge needles (manually file off points)</td>
 <td>VWRSP</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>Polyethylene tubing 0.022"ID x 0.042"OD; 427411</td>
 <td>Becton-Dickinson</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>H-7 pipette stand/holder for microcap perfusion array</td>
 <td>Narishige USA</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>one-way valves</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>Drummond Microcaps 1 &mu;l</td>
 <td>VWRSP</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>18 gauge needles for suction (filed off points)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Polyethylene tubing</td>
 <td>Cole Parmer</td>
 <td>4.27436E+11</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>fine dissection pins</td>
 <td>Fine Science Tools</td>
 <td>26002-20</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>capillary tubes</td>
 <td>Kimble 71900-100</td>
 <td>&nbsp;</td>
 <td>fire-polished and U-shaped in a Bunsen burner flame and filled with 3% agar in ECS</td>
 </tr>
 <tr>
 <td>modeling clay</td>
 <td>craft store</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>dish holder for microscope stage with isolated ground bath</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Custom manufacture</td>
 </tr>
 <tr>
 <td>pasteur pipettes</td>
 <td>VWRSP</td>
 <td>14672-412</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>pipette bulbs</td>
 <td>VWRSP</td>
 <td>53283-911</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>acrodisk syringe filters</td>
 <td>VWRSP</td>
 <td>28144-040</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>thick-walled 1.5 mm diameter borosilicate filament glass</td>
 <td>WPI</td>
 <td>1B150F-3</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>High purity nitrogen cylinder and bifurcating regulator</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>
Tables 1-3. Lists of Reagents, Materials, and Equipment.

isolation of sensory neurons of aplysia californica for patch clamp recordings of glutamatergic currents

El molusco gasterópodo marino Aplysia californica tiene una historia venerable como un modelo de funcionamiento del sistema nervioso, con especial importancia en los estudios sobre el aprendizaje y la memoria. Las preparaciones típicas para este tipo de estudios son aquellos en los que la sensorial y motoneuronas se dejan intactos en un animal disecado mínimamente, o neuronal de co-cultivo técnicamente elaborada de sensorial y motoneuronas individual. Menos común es la preparación neuronal aislado en el que pequeños grupos de neuronas nominalmente homogéneos se disocian en células individuales en cultivo a corto plazo. Tales células aisladas son útiles para la caracterización biofísica de las corrientes de iones utilizando técnicas de patch clamp, y la modulación selectiva de estos conductancias. Se describe un protocolo para la preparación de tales cultivos. El protocolo se aprovecha de los fácilmente identificables las neuronas sensoriales de los ganglios de la glutamatérgicas pleural y bucal, y describe su disociación y un mantenimiento mínimo in la cultura durante varios días sin suero.

La ubicación de las neuronas sensoriales en los ganglios que están dirigidos en este protocolo, las neuronas BSC y PVC se muestran en la Figura 1. Las neuronas BSC están situados en 2 grupos ovales simétricas en el lado ventral del ganglio vestibular, la superficie que se enfrenta lejos de la masa en el ganglio vestibular intacta (Figura 1A). Las neuronas de PVC forman grupos bilaterales, en forma de V que se envuelven alrededor de la superficie dorsal del ganglio pleural hacia el e...

Watch this Scientific Journal Video about Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents at JoVE.com

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

Se describe la disección del sistema nervioso de la liebre de mar marino<em&gt; Aplysia</em&gt; Después de la anestesia, el aislamiento de las neuronas para el cultivo a corto plazo de los tejidos, y las grabaciones de las corrientes de iones de células individuales a través de la técnica de patch clamp.

El aislamiento de las neuronas sensoriales de<em&gt; Aplysia californica</em&gt; Se Patch clamp grabaciones de Corrientes glutamatérgicos

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

isolation-sensory-neurons-aplysia-californica-for-patch-clamp

Research

JoVE Journal

Neuroscience

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

12.2K vistas.  University of Miami. Se describe la disección del sistema nervioso de la liebre de mar marino Aplysia Después de la anestesia, el aislamiento de las neuronas para el cultivo a corto plazo de los tejidos, y las grabaciones de las corrientes de iones de células individuales a través de la técnica de patch clamp.

Video: Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal. Simultaneously recording from multiple nerves and muscles gives us precise information about the timing of neural activity. Previous recording methods have worked for two electrodes, but the study of additional nerves or muscles required combining and averaging the recordings of multiple animals, which made it difficult to determine fine details of timing and phasing, because of variability from response to response, and from animal to animal. Implanting four individual electrodes has a very low success rate due to the formation of adhesions that prevent animals from performing normal feeding movements. We developed a new method of electrode fabrication that reduces the bulk of the electrodes inside the animal allowing for normal feeding movements. Using a combination of glues to attach the electrodes results in a more reliable insulation of the electrode which lasts longer, making it possible to record for periods as long as a week. The fabrication technique that we describe could be extended to incorporate several additional electrodes, and would be applicable to vertebrate animals.

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

A technique is described for implanting four in vivo electrodes to monitor the neuromuscular control of feeding behavior in Aplysia californica.

Electrodo de fabricación e implantación de Aplysia californica Para grabaciones multi-canal neural y muscular en los animales intactos, libremente Comportarse

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal.  ...

Investigación

Neurociencias

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

Grabaciones de patch clamp de neuronas de ratón de retina en una rebanada preparación adaptada a la oscuridad

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (&alpha;, &beta;I, &beta;II and &gamma;), novel type I PKCs (&#949; and &eta;), novel type II PKCs (&delta; and &theta;), and atypical PKCs (&zeta; and &iota;). The classical PKCs are activated by Ca2+ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca2+-independent. The atypical PKCs are activated by neither Ca2+ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation 1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail 3. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article 4, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

En vivo de imágenes de la translocación de PKC en células Sf9 y en las neuronas sensoriales Aplysia

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

Grabaciones de patch clamp en Inner células del oído pelo Aislado de pez cebra

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation8 or in vivo9. This process can also be applied in other motor pools10,11,12 in Aplysia or in other animal systems2,3,13,14.

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.

Extracelularmente Identificar las neuronas motoras de un músculo en la piscina Motor<em&gt; Aplysia californica</em

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

Patch Clamp Las grabaciones de las células embrionarias de pez cebra Mauthner

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Perforado grabación de patch-clamp de ratón neuronas sensoriales olfativas en neuroepitelio Intacto: Análisis funcional de las neuronas que expresan un odorante del receptor Identificado

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

Las grabaciones de células enteras Patch-clamp en rodajas de cerebro

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

Subcelulares patch-clamp grabaciones desde el dominio somatodendrítica de la sustancia nigra dopamina Neuronas

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.