Agradecemos sinceramente a la Fundación Whitehall por su apoyo financiero y fondos de inicio del Scripps Research Institute por llevar a cabo este trabajo.

1. Preparación De Ganglios Abdominales, Mediciones Electrofisiológicas, Y El Aislamiento De Las Neuronas Individuales Identificadas Del Ganglio Abdominal De Aplysia californica
<ol><li>Mantenga Aplysia en el acuario de laboratorio con agua de mar artificial circulante (ASW) a 16 ° C bajo condiciones de luz: oscuras de 12:12.</li>
	<li>Aislamiento del ganglio abdominal.<ol>
<li>Anestesiar a los animales inyectando 380 mM mgCl2 solución durante 5-10 min (equivalente al 30-35% del peso corporal del animal).</li>
		<li>Identificar el ganglio abdominal en función de su posición en el sistema nervioso central24,28.</li>
		<li>Retirar los ganglios mediante operación quirúrgica y almacenarlos inmediatamente en agua de mar artificial consistente en 450 mM de NaCl, 10 mM de KCl, 10 mM de CaCl2,55 mM de MgCl2,2,5 mM de NaHCO3,y 10 mM de HEPES (pH 7,4).</li>
	</ol>
</li>
	<li>Tratamiento con proteasas.<ol>
<li>Incubar ganglio abdominal durante 30 min a 34 °C±0,5 en una placa de Petri con proteasa (dispase) al 0,1% diluida en ASW descrita anteriormente. NOTA: La cantidad de la proteasa utilizada debe estandarizarse con la edad y el peso de los animales. En general, los animales más viejos y más grandes requerirían más proteasa.</li>
	</ol>
</li>
	<li>Retiro de la envaesda de los ganglios.<ol>
<li>Después del tratamiento enzimático, fije los ganglios a la base de silicona de Sylgard de una cámara celular (Ø = 1 cm; vol. = 0,3 ml) y perfunda con ASW (caudal: 150 μl/min) a temperatura ambiente (18 °C±2). NOTA: Con el fin de aumentar la visibilidad de las neuronas identificadas, coloque el ganglio en la parte superior de un cilindro de vidrio de policarbonato(Figura 1)(diámetro Ø = 2 mm de cámara celular modificada) que se puede iluminar fácilmente desde la parte inferior utilizando un estereomicroscopio binocular con aumento de 20X y 40X.</li>
		<li>Retire cuidadosamente la vainquia del ganglio mediante el uso de tórceps y microscisores.</li>
	</ol>
</li>
	<li>Grabación electrofisiológica simultánea de dos neuronas.<ol>
<li>Identificar la neurona de interés.</li>
		<li>Empalar la neurona con un solo microelectrodo agudo intracelular con resistencia 10-15 MΩ lleno de 3 M KCl solución.</li>
		<li>Registre la actividad neuronal (paso de banda de 10 kHz) utilizando un sistema de grabación intracelular.</li>
		<li>Procese los datos de registro utilizando software de electrofisiología como Axograph. NOTA: Uno puede llevar a cabo fácilmente la grabación de múltiples neuronas. Las neuronas L7, L11 o R15 y R2 se identifican fácilmente en función de su posición. Se requieren dos amplificadores y dos micromanipuladores para grabar simultáneamente desde dos neuronas L7 y R15 (Figura 2).</li>
	</ol>
</li>
	<li>Aplicación de fármacos.<ol>
<li>Aplicar el fármaco de elección en la cámara celular por pipeteo suave o inyectar directamente en las neuronas.</li>
		<li>Realizar mediciones electrofisiológicas. NOTA: Dependiendo del objetivo experimental, se podrían medir diferentes parámetros electrofisiológicos de las neuronas como los cambios en el potencial de membrana (MP) o el potencial de acción (AP) antes, durante y después de la aplicación del fármaco.</li>
	</ol>
</li>
	<li>Aislamiento de neuronas individuales.<ol>
<li>Al concluir los experimentos electrofisiológicos, detenga la perfusión de ASW y lave el ganglio con etanol al 100%.  La exposición al etanol petrificará las neuronas y, usando los pórceps finos, hará que sea fácil quitar las neuronas individuales sin dañar otras neuronas del ganglio.</li>
		<li>Retire las neuronas individuales debajo de un estereomicroscopio y transfiérelo a un pequeño tubo de Eppendorf que contenga un reactivo de extracción de ARN de 500 μl helado. Las neuronas individuales aisladas se pueden almacenar en reactivos de extracción de ARN a -80 °C para futuros análisis.</li>
	</ol>
</li>
</ol>2. Aislamiento de ARN de neuronas individuales y análisis de expresión génica por PCR cuantitativa en tiempo real (qPCR)
<ol><li>Precauciones para minimizar la degradación del ARN: Las ribonucleasas (RNasas) degradan el ARN y son enzimas muy estables y activas. Siga estos pasos durante el manejo de los ENR.<ol>
<li>Limpie el área de trabajo y los instrumentos con agentes desactivadores de RNase.</li>
		<li>Use guantes en todo momento mientras manipula el ARN y cámbiese los guantes con frecuencia.</li>
		<li>Use solo plásticos libres de ARNsa para manejar los ARNs.</li>
	</ol></li>
	<li>Aislar el ARN total de las neuronas individuales:<ol>
<li>El protocolo trizol estándar para el aislamiento de ARN se puede utilizar para aislar LOS ARN de neuronas individuales. Añadir brevemente 20% v/v de cloroformo a Trizol, mezclar bien por breve vórtice.</li>
		<li>Coloque los tubos en el rotador durante 10 min a 4 °C. Haga girar la mezcla Trizol-Cloroformo a 12.000 x g durante 15 min a 4 °C.</li>
		<li>Recoger la fase acuosa y transferir a un tubo fresco.</li>
		<li>Añadir solución de acetato de sodio (pH 5,5) a una concentración final de 0,3 M, 100 ng/ml del coprecipitante GlycoBlue, y 2,5 volúmenes de etanol al 100% para precipitar el ARN.</li>
		<li>Mezcle bien las muestras e incube a -80 °C durante la noche.</li>
		<li>Gire las muestras a 12.000 x g durante 20 min a 4 °C y un pequeño pellet azul será visible en la parte inferior del tubo.</li>
		<li>Retire el sobrenadante y lave el pellet con 850 μl de etanol helado al 75% a 12.000 x g durante 10 min a 4 °C.</li>
		<li>Retire el sobrenadante con cuidado y seque al aire el pellet de ARN durante 7-10 min, máximo. NOTA: Asegúrese de que el ARN no se deja secar durante mucho tiempo como encima-secar el pellet de ARN hace que la solubilización sea muy difícil.
</li>
		<li>Una vez secado, resuspend el pellet de ARN en 10 μl de agua libre de RNAse y determinar la concentración de ARN. NOTA: Determine la concentración de ARN utilizando un espectrofotómetro. Cuando no se use de inmediato, guarde el ARN a -80 °C.</li>
	</ol></li>
	<li>Síntesis de aRNA a partir de neuronas individuales:<ol>
<li>Amplificar el ARN (una o dos rondas dependiendo del objetivo experimental) utilizando el sistema de amplificación de ARN lineal disponible en el comercio. NOTA: El ARN total de neuronas individuales varió de ~10-60 ng. El rendimiento esperado de la primera ronda de amplificación es de ~ 100-300 ng y la segunda ronda de amplificación es de ~ 0.8-1.5 μg de ARN.</li>
	</ol></li>
	<li>Transcripción inversa del ARN:<ol>
<li>Para generar ADNc a partir de aRNA, utilice 1,0 μg de ARN en 20 μl de una reacción de transcripción inversa. Varios kits están disponibles comercialmente para este propósito. NOTA: El ADNc se sintetizó de acuerdo con el protocolo del fabricante utilizando los siguientes ajustes en el ciclador térmico. 5 min a 25 °C 30 min a 42 °C 5 min a 85 °C Mantener a 4 °C</li>
		<li>Después del paso de transcripción inversa, almacene el ADNc a -20 °C para el almacenamiento a largo plazo.</li>
	</ol></li>
	<li>Diseño y estandarización de cebadores: Varios programas de software excelentes están disponibles, de forma gratuita, para el diseño de cebadores para amplificaciones en tiempo real.<ol>
<li>Seleccione 18-24 oligonucleótidos de mer que produzcan amplicones de 70-110 pb y sintetice los cebadores utilizando fuentes comerciales. NOTA: Se deben diseñar de dos a tres pares de cebadores para cada gen de interés. Cada conjunto de cebadores debe ser estandarizado por qPCR. Los pares de cebadores delanteros e inversos que se utilizaron para el gen CREB1(Figura 4)son los siguientes: Juego de cebadores 1 Adelante: 5'-TGATTCTGACGCAAAGAAAAGA-3' Reverso: 5'-ACCGTGAGCAGTCAGTTGTAGA-3' Juego de cebadores 2 Delantero: 5'-AGGGAATGTCGATGAAAGAAGA-3' Reverso: 5'-GACACACAGGAAGTATGCCAAA-3'</li>
		<li>Para seleccionar el mejor cebador que se utilizará para los análisis aguas abajo, monitoree el valor de Ct y la forma de la curva de amplificación en qPCR. NOTA: Los cebadores que dan valores de Ct (Umbral de ciclo) entre 10-35 en la amplificación de 40 rondas y curvas suaves durante la fase exponencial de la PCR seguida de una meseta suave son óptimos para los análisis de expresión génica.</li>
	</ol></li>
	<li>PCR cuantitativa en tiempo real (qPCR): Nota: Utilice tubos o placas apropiados que sean compatibles con la máquina qPCR.<ol>
<li>Diluya el ADNc a 5x con agua libre de nucleasa.</li>
		<li>A 2 μl de ADNc diluido, agregue 8 μl de una mezcla maestra qPCR que contenga 2 μl deH2O, 5 μl de 2x mezcla maestra verde SYBR y 1,0 μl de imprimación directa e inversa de 10 μM (cada una).</li>
		<li>Cierre los tubos y selle la placa después de pipetear el ADNc y la mezcla maestra. Mezcle el contenido tocando suavemente y girando hacia abajo a 1.500 rpm durante 30 segundos.</li>
		<li>Proceda a configurar la máquina qPCR.</li>
	</ol></li>
</ol>

Los autores no tienen ningún interés financiero en competencia.

La neurona R15 participa en la regulación de los sistemas cardiovascular, digestivo, respiratorio y reproductivo30. Una actividad que estalla regularmente rítmica del AP es una característica de R15. Como se muestra en la sección de resultados, el registro emparejado de R15 y L7 muestran que la preparación de los ganglios ha preservado la actividad de las neuronas R15. Las neuronas R15 y L7 respondieron apropiadamente a Ach. Esta preparación de los ganglios se podía mantener hasta 8-10 horas y la activi...

El cerebro humano es extraordinariamente complejo con casi 100 mil millones de neuronas y billones de conexiones sinápticas. Hay casi un número igual de células no neuronales que interactúan con las neuronas y regulan su función en el cerebro. Las neuronas se organizan en circuitos que regulan comportamientos específicos. A pesar de los avances en nuestra comprensión de las funciones cerebrales y los circuitos neuronales, poco se sabe sobre la identidad de los componentes de los circuitos que controlan un comportamiento específico. El conocimiento de las identidades de varios componentes de un circuito facilitará grandemente nuestra comprensión de la base celular y molecular del comportamiento y ayuda en el desarrollo de las estrategias terapéuticas nuevas para los desordenes neuropsiquiátricos.
El caracol marino Aplysia californica ha sido un caballo de batalla para determinar los circuitos neuronales subyacentes a comportamientos específicos1-14. El sistema nervioso de Aplysia contiene aproximadamente 20.000 neuronas que se organizan en 9 ganglios diferentes. Las neuronas de Aplysia son grandes y se pueden identificar fácilmente en función de su tamaño, propiedades eléctricas y posición en los ganglios. Aplysia tiene un rico repertorio de comportamientos que pueden ser estudiados. Uno de los comportamientos bien estudiados es el reflejo branquial-abstinencia (GWR). Los componentes centrales de este reflejo están situados en los ganglios abdominales. Se han mapeado componentes de los circuitos GWR y se han determinado las contribuciones de varios componentes. Es importante destacar que los circuitos GWR se someten a aprendizaje asociativo y no asociativo5,6,15-19. Décadas de estudio sobre este reflejo también han identificado varias vías de señalización que tienen un papel clave en el aprendizaje y la memoria20-24.
Varias preparaciones diferentes de Aplysia se utilizaron para estudiar las bases celulares y moleculares del almacenamiento de la memoria. Estos incluyen el animal intacto2,3,la preparación semi-intacta1,7,13,14,16 y la reconstitución de los principales componentes de los circuitos neuronales25-29. Una preparación reducida para explorar los ganglios de Aplysia para los análisis electrofisiológicos y moleculares de circuitos neuronales identificados se describe aquí. Las cuatro neuronas identificadas siguientes fueron estudiadas. R15, una neurona que estallaba, L7 y L11, dos diversas neuronas de motor y R2, una neurona colinérgica fueron estudiados. R2 es la neurona más grande descrita en el sistema nervioso de los invertebrados. Brevemente, esta metodología implica el tratamiento proteasa de ganglios, mediciones electrofisiológicas antes y después de los tratamientos farmacológicos, y el aislamiento de neuronas individuales para el análisis cuantitativo de la expresión génica. Esta metodología nos permite combinar análisis moleculares con el registro simultáneo de múltiples neuronas. Esta metodología se utilizó con éxito para estudiar las respuestas de las neuronas R15 y L7 a la acetilcolina (Ach) por grabaciones intracelulares emparejadas. Después de mediciones electrofisiológicas R15 y L7 y otras neuronas identificadas como L11 y R2 se aislaron para el análisis cuantitativo de reacción en cadena de la polimerasa (qPCR) de la expresión de CREB1, un factor de transcripción importante para el almacenamiento de memoria.

<table><tbody><tr><td>&lt;em&gt;Aplysia&lt;/em&gt;</td><td>National Aplysia Resource Facility, University of Miami</td><td></td><td></td></tr><tr><td>NaCl</td><td>SIGMA</td><td>S 3014-1KG</td><td></td></tr><tr><td>KCl</td><td>SIGMA</td><td>P 9333-500G</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;bull;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>SIGMA</td><td>C5080- 500G</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;bull;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher Scientific</td><td>BP 214-501</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;4&lt;/sub&gt;</td><td>SIGMA</td><td>S 6297-250G</td><td></td></tr><tr><td>HEPES</td><td>SIGMA</td><td>H 3375-500G</td><td></td></tr><tr><td>Protease</td><td>GIBCO</td><td>17105-042</td><td></td></tr><tr><td>Trizol</td><td>Ambion</td><td>15596-026</td><td></td></tr><tr><td>Chloroform</td><td>MP Biomedicals</td><td>2194002</td><td></td></tr><tr><td>100% Ethanol</td><td>ACROS</td><td>64-17-5</td><td></td></tr><tr><td>GlycoBlue</td><td>Ambion</td><td>AM9515</td><td></td></tr><tr><td>3 M NaOAc, pH 5.5</td><td>Ambion</td><td>AM9740</td><td></td></tr><tr><td>Nuclease free water</td><td>Ambion</td><td>AM9737</td><td></td></tr><tr><td>MessageAmp II aRNA Amplification Kit</td><td>Ambion</td><td>AM1751</td><td></td></tr><tr><td>qScript cDNA SuperMix</td><td>Quanta Biosciences</td><td>95048-100</td><td></td></tr><tr><td>Power SYBR Green PCR Master Mix</td><td>Applied Biosystems</td><td>4367659</td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11252-20</td><td></td></tr><tr><td>Scissors</td><td>Fine Science Tools</td><td>15000-08</td><td></td></tr><tr><td>Stainless Steel Minutien Pins&amp;nbsp;</td><td>Fine Science Tools</td><td>26002-10 or</td><td></td></tr><tr><td>26002-20</td><td></td><td></td><td></td></tr><tr><td>Veriti Thermal Cycler</td><td>Applied Biosystems</td><td>Veriti Thermal Cycler</td><td></td></tr><tr><td>5430R Centrifuge</td><td>Eppendorf</td><td>5430R Centrifuge</td><td></td></tr><tr><td>7900HT Fast Real-Time PCR</td><td>Applied Biosystems</td><td>7900HT Fast Real-Time PCR</td><td></td></tr><tr><td>Amplifier</td><td>BRAMP-01R</td><td>NPI Electronics</td><td></td></tr><tr><td>Digidata Converter</td><td>Instrutech ITC-18</td><td>HEKA ELEKTRONIK</td><td></td></tr><tr><td>Micro Manipulator</td><td>Patch Star</td><td>Scientifica</td><td></td></tr></tbody></table>

aplysia ganglia preparation for electrophysiological and molecular analyses of single neurons

Un desafío importante en neurobiología es entender los fundamentos moleculares de los circuitos neuronales que gobiernan un comportamiento específico. Una vez identificados los mecanismos moleculares específicos, se pueden desarrollar nuevas estrategias terapéuticas para tratar anomalías en comportamientos específicos causadas por enfermedades degenerativas o envejecimiento del sistema nervioso. El caracol marino Aplysia californica es muy adecuado para las investigaciones de las bases celulares y moleculares del comportamiento porque los circuitos neuronales subyacentes a un comportamiento específico podrían determinarse fácilmente y los componentes individuales de los circuitos podrían ser fácilmente manipulados. Estas ventajas de Aplysia han llevado a varios descubrimientos fundamentales de la neurobiología del aprendizaje y de la memoria. Aquí se describe una preparación del sistema nervioso Aplysia para los análisis electrofisiológicos y moleculares de las neuronas individuales. Brevemente, el ganglio diseccionado del sistema nervioso se expone a la proteasa para eliminar la vaina ganglionar de tal manera que las neuronas están expuestas pero conservan la actividad neuronal como en el animal intacto. Esta preparación se utiliza para llevar a cabo mediciones electrofisiológicas de neuronas únicas o múltiples. Es importante destacar que, después de la grabación utilizando una metodología simple, las neuronas podrían aislarse directamente de los ganglios para el análisis de expresión génica. Estos protocolos se utilizaron para llevar a cabo grabaciones electrofisiológicas simultáneas de las neuronas L7 y R15, estudiar su respuesta a la acetilcolina y la cuantificación de la expresión del gen CREB1 en aislados de las neuronas L7, L11, R15 y R2 de Aplysia.

Los pesos de los animales que fueron utilizados en este estudio se extendieron a partir del 100-200 g. Siguiendo los protocolos descritos, se realizaron mediciones electrofisiológicas y análisis moleculares de neuronas de ganglios abdominales aislados de animales que oscilaban entre 2-5 g y 200-300 g.
La estandarización del tratamiento con proteasa es importante para el éxito de las mediciones electrofisiológicas de las neuronas en los ganglios. Inicialmente, se utilizaron concentraciones...

Watch this Scientific Journal Video about Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons at JoVE.com

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

El caracol marino Aplysia californica ha sido ampliamente utilizado como modelo neurobiológico para los estudios sobre bases celulares y moleculares del comportamiento. Aquí se describe una metodología para explorar el sistema nervioso de Aplysia para los análisis electrofisiológicos y moleculares de neuronas individuales de circuitos neuronales identificados.

Aplysia Preparación De Ganglios Para Análisis Electrofisiológicos Y Moleculares De Neuronas Individuales

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific ...

aplysia-ganglia-preparation-for-electrophysiological-molecular

Research

JoVE Journal

Bioengineering

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

9.0K vistas.  The Scripps Research Institute, Florida. El caracol marino Aplysia californica ha sido ampliamente utilizado como modelo neurobiológico para los estudios sobre bases celulares y moleculares del comportamiento. Aquí se describe una metodología para explorar el sistema nervioso de Aplysia para los análisis electrofisiológicos y moleculares de neuronas individuales de circuitos neuronales identificados.

Video: Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

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

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

Las culturas de las células nerviosas de Aplysia para imágenes de alta resolución de los Conos de Crecimiento

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

Biología

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard of electrophysiology applied to disease models and pharmaceutical screens alike 1. The method traditionally involves gently contacting a cell with a glass pipette filled by a physiological solution in order to isolate a patch of the membrane under its apex 2. An electrode inserted in the pipette captures ion-channel activity within the membrane patch or, when ruptured, for the whole cell. In the last decade, patch-clamp chips have been proposed as an alternative 3, 4: a suspended film separates the physiological medium from the culture medium, and an aperture microfabricated in the film replaces the apex of the pipette. Patch-clamp chips have been integrated in automated systems and commercialized for high-throughput screening 5. To increase throughput, they include the fluidic delivery of cells from suspension, their positioning on the aperture by suction, and automated routines to detect cell-to-probe seals and enter into whole cell mode. We have reported on the fabrication of a silicon patch-clamp chip with optimized impedance and orifice shape that permits the high-quality recording of action potentials in cultured snail neurons 6; recently, we have also reported progress towards interrogating mammalian neurons 7. Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre 8, a commercial foundry, and are available in large series. We are eager to engage in collaborations with electrophysiologists to validate the use of the NRCC technology in different models. The chips are used according to the general scheme represented in Figure 1: the silicon chip is at the bottom of a Plexiglas culture vial and the back of the aperture is connected to a subterranean channel fitted with tubes at either end of the package. Cells are cultured in the vial and the cell on top of the probe is monitored by a measuring electrode inserted in the channel.The two outside fluidic ports facilitate solution exchange with minimal disturbance to the cell; this is an advantage compared to glass pipettes for intracellular perfusion.
<img alt="Figure 1" src="/files/ftp_upload/3288/3288fig1.jpg" />
Figure 1. Principle of measurement using the NRCC patch-clamp chip
We detail here the protocols to sterilize and prime the chips, load them with medium, plate them with cells, and finally use them for electrophysiological recordings.

We show how planar patch-clamp chips fabricated at the National Research Council of Canada are sterilized, primed, loaded with medium, plated with cells, and used for electrophysiological recordings.

El cultivo y la electrofisiología de las células en NRCC de patch-clamp chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard ...

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation) 1. These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 &deg;C) that are natural for Aplysia, preparations are stable for long periods of time. 
To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange 2 which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties 3 and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3) 2,4. Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. 
An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record synaptic transmission in identified pre- and postsynaptic neurons. At the conclusion of each trial, a custom script combines electrophysiology and imaging data. To ensure proper synchronization we use a light pulse from a LED mounted in the camera port of the microscope. Manipulation of presynaptic calcium levels (e.g. via intracellular EGTA injection) allows us to test specific hypotheses, concerning the role of intracellular calcium in mediating various forms of plasticity.

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

We demonstrate how changes in the intracellular free calcium concentration and synaptic efficacy can be simultaneously monitored in a ganglion preparation of Aplysia. We image intracellular calcium using a fluorescent dye, Calcium Orange, and induce and monitor synaptic transmission with sharp (intracellular) electrodes.

Seguimiento de los cambios en la concentración de calcio intracelular y la eficacia sináptica en el molusco<em&gt; Aplysia</em

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., ...

An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica

Multifunctionality, the ability of one peripheral structure to generate multiple, distinct behaviors1, allows animals to rapidly adapt their behaviors to changing environments. The marine mollusk Aplysia californica provides a tractable system for the study of multifunctionality. During feeding, Aplysia generates several distinct types of behaviors using the same feeding apparatus, the buccal mass. The ganglia that control these behaviors contain a number of large, identified neurons that are accessible to electrophysiological study. The activity of these neurons has been described in motor programs that can be divided into two types, ingestive and egestive programs, based on the timing of neural activity that closes the food grasper relative to the neural activity that protracts or retracts the grasper2. However, in isolated ganglia, the muscle movements that would produce these behaviors are absent, making it harder to be certain whether the motor programs observed are correlates of real behaviors. In vivo, nerve and muscle recordings have been obtained corresponding to feeding programs2,3,4, but it is very difficult to directly record from individual neurons5. Additionally, in vivo, ingestive programs can be further divided into bites and swallows1,2, a distinction that is difficult to make in most previously described in vitro preparations.
The suspended buccal mass preparation (Figure 1) bridges the gap between isolated ganglia and intact animals. In this preparation, ingestive behaviors - including both biting and swallowing - and egestive behaviors (rejection) can be elicited, at the same time as individual neurons can be recorded from and stimulated using extracellular electrodes6. The feeding movements associated with these different behaviors can be recorded, quantified, and related directly to the motor programs. The motor programs in the suspended buccal mass preparation appear to be more similar to those observed in vivo than are motor programs elicited in isolated ganglia. Thus, the motor programs in this preparation can be more directly related to in vivo behavior; at the same time, individual neurons are more accessible to recording and stimulation than in intact animals. Additionally, as an intermediate step between isolated ganglia and intact animals, findings from the suspended buccal mass can aid in interpretation of data obtained in both more reduced and more intact settings. The suspended buccal mass preparation is a useful tool for characterizing the neural control of multifunctionality in Aplysia.

An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica

We describe a technique to extracellularly record and stimulate from nerves, muscles, and individual identified neurons in vitro while eliciting and observing different types of feeding behaviors in the feeding apparatus of Aplysia.

Un<em&gt; In Vitro</em&gt; Preparación para Obtener y grabar programas de alimentación del motor con los movimientos fisiológicos en<em&gt; Aplysia californica</em

Multifunctionality, the ability of one peripheral  structure to generate multiple, distinct behaviors1, allows animals  to rapidly adapt their behaviors ...

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

Preparación de Aplysia Sensorio-motor culturas de células neuronales

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

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 ...

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

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

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

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

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 ...

Characterizing RNA Modifications in Single Neurons Using Mass Spectrometry

Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous system (CNS). Recent evidence has linked specific neuronal RNA modifications to learning and memory paradigms. Unfortunately, conventional methods for the detection of these epitranscriptomic features are only capable of characterizing highly abundant RNA modifications in bulk tissues, precluding the assessment of unique PTM profiles that may exist for individual neurons within the activated behavioral circuits. In this protocol, an approach is described&#8212;single-neuron RNA modification analysis by mass spectrometry (SNRMA-MS)&#8212;to simultaneously detect and quantify numerous modified ribonucleosides in single neurons. The approach is validated using individual neurons of the marine mollusk, Aplysia californica, beginning with surgical isolation and enzymatic treatment of major CNS ganglia to expose neuron cell bodies, followed by manual single-neuron isolation using sharp needles and a micropipette. Next, mechanical and thermal treatment of the sample in a small volume of buffer is done to liberate RNA from an individual cell for subsequent RNA digestion. Modified nucleosides are then identified and quantified using an optimized liquid chromatography-mass spectrometry method. SNRMA-MS is employed to establish RNA modification patterns for single, identified neurons from A. californica that have known morphologies and functions. Examples of qualitative and quantitative SNRMA-MS are presented that highlight the heterogeneous distribution of RNA modifications across individual neurons in neuronal networks.

Post-transcriptional modifications of RNA represent an understudied layer of translation regulation that has recently been linked to central nervous system plasticity. Here, sample preparation and liquid chromatography-tandem mass spectrometry approach is described for simultaneous characterization of numerous RNA modifications in single neurons.

Caracterización de modificaciones de ARN en neuronas individuales mediante espectrometría de masas

Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous ...

Química

Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in the brain stem. Loss of hair cells is a major cause of sensory hearing loss. Prosthetic devices such as cochlear implants function by bypassing lost hair cells and directly stimulating SGNs electrically, allowing for restoration of hearing in deaf patients. The performance of these devices depends on the functionality of SGNs, the implantation procedure and on the distance between the electrodes and the auditory neurons. 
We hypothesized, that reducing the distance between the SGNs and the electrode array of the implant would allow for improved stimulation and frequency resolution, with the best results in a gapless position. Currently we lack in vitro culture systems to study, modify and optimize the interaction between auditory neurons and electrode arrays and characterize their electrophysiological response. To address these issues, we developed an in vitro bioassay using SGN cultures on a planar multi electrode array (MEA). With this method we were able to perform extracellular recording of the basal and electrically induced activity of a population of spiral ganglion neurons. We were also able to optimize stimulation protocols and analyze the response to electrical stimuli as a function of the electrode distance. This platform could also be used to optimize electrode features such as surface coatings.

We present a protocol to culture primary murine spiral ganglion neuron explants on multi electrode arrays to study neuronal response profiles and optimize stimulation parameters. Such studies aim to improve the neuron-electrode interface of cochlear implants to benefit hearing in patients as well as the energy consumption of the device.

Ganglio espiral neurona explante cultura y Electrofisiología de multi electrodos matrices

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in ...

Aplysia Preparación De Ganglios Para Análisis Electrofisiológicos Y Moleculares De Neuronas Individuales

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