This work is supported by a National Science Foundation award (1145581) to LWG.

NOTA: Todo el procedimiento se realizó de conformidad con las directrices de los Institutos Nacionales de Salud, aprobadas por el Comité de Cuidado y Uso de Animales de la Universidad de Illinois en Chicago. 1. Soluciones y Medios de Cultivo Preparados <ol><li> Mantenga todas las soluciones a -20 ° C durante un máximo de seis meses. Mantenga los medios de cultivo a 4 ° C durante un máximo de 3 meses. </li><li> Preparar 100 ml de medio de cultivo mediante la mezcla de 1 ml de solución-pen estreptocócica y 1 ml de ITSX (insulina-transferrina-selenio-suplementación) a 100 ml con DMEM. </li><li> Preparar la solución de enzima mediante la mezcla de 250 ml de DMEM, 2,5 ml de 100 mM CaCl 2, y 2,5 ml de EDTA 50 mM. </li><li> Prepare la solución de inactivación mediante la mezcla de 225 ml de DMEM, 25 ml de FBS, 625 mg de albúmina, y 625 mg de inhibidor de tripsina. </li><li> Preparar la solución de NaCl 154 mM, KCl 5,6 mM, 5,0 mM HEPES, 3,6 mM NaHCO3, 5,6 mM de glucosa de un Locke. Ajustar el pH a 7,3 con NaOH. </li><li>Preparar una solución de poli-D-lisina (PDL) de 50 mg / ml. Utilice una concentración final de 1 mg / ml a cubreobjetos capa. </li><li> Preparar una solución extracelular de NaCl 140 mM, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM de HEPES-NaOH, y glucosa 10 mM. Ajustar el pH a 7.3 con NaOH con una osmolaridad de ~ 310 nmol / kg. </li><li> Preparar una solución de la pipeta de NaCl 50 mM, TEACl 100, 5 mM KCl, 2 mM CaCl2, 1 mM de MgCl 2, y 10 mM HEPES. Ajustar el pH a 7.3 con NaOH con una osmolaridad de ~ 290 nmol / kg. </li></ol> 2. glándula suprarrenal Aislamiento <ol><li> Autoclave un par de grandes y pequeñas tijeras de disección y dos pares de pinzas. Use guantes durante todo el proceso y coloque la bandeja de disección en un cubo de hielo. </li><li> Utilice los ratones tomadas en el día 0-3. La eutanasia a los animales con un tanque presurizado CO 2 seguido por decapitación. </li><li> Utilizar el más pequeño conjunto de tijeras para cortar por la parte posterior del cachorro después de la columna vertebral de Cervi<html> cal a caudal región. Asegúrese de cortar superficialmente bajo la piel y no penetrar en la cavidad del cuerpo. </li><li> A continuación, exfoliación de la piel lejos de la columna vertebral de modo que la musculatura está expuesto a tener una visión clara de la columna vertebral. A partir de aquí, hacer un corte transeccional en la médula espinal cervical y luego hacer dos cortes paralelos a lo largo de los lados de la columna vertebral. </li><li> Con un par de pinzas que sujetan el cuerpo principal del animal, utilice el otro par de pinzas para agarrar la columna vertebral y pelar la columna vertebral hasta los riñones están expuestos. </li><li> En este momento, visualizar las glándulas suprarrenales en la parte superior de los riñones. A continuación, coloque cuidadosamente las dos glándulas de cada animal en un tubo cónico lleno de solución de Locke. NOTA: A veces, las glándulas se apegará a los fórceps. Si este es el caso, utilice suavemente otro par de fórceps para ayudar en la eliminación de las glándulas de las pinzas y en la solución de Locke. Las glándulas se pueden mantener en esta condición para un máximo de 2 horas. </li></ol>e &quot;&gt; 3. Digestión de Tejidos <ol><li> Mientras tanto, burbuja de la solución de enzima con 5% de CO 2 + 95% de O 2 durante 15 min antes de su uso. Asegúrese de que la solución equilibrada pasa de un color rosa brillante en color rosado / naranja. </li><li> Añadir la papaína a la solución de enzima recién burbujeó a una concentración final de 20-25 unidades / ml. Incubar cada par de glándulas de cada animal para 40-60 min a 37 ° C en la solución de enzima con papaína. </li><li> Añadir 75% de solución de inactivación a cada tubo y se incuba a 37 ° C durante otros 10 min. Esta incubación inactiva la actividad de la enzima de la papaína. </li><li> Después de la incubación de 10 min con la solución de inactivación, transferir suavemente las glándulas en un tubo recién marcado y luego se lavan las glándulas 3x con medios de cultivo. </li></ol> 4. Disociación Celular y Plating <ol><li> Después del lavado, colocar las dos glándulas en 200 l de los medios de cultivo y a continuación, valorar con cuidado las glándulas yabajo a través de la punta de pipeta con una pipeta de 200 l hasta que ya no hay un gran trozo de tejido en la solución. NOTA: Cuando se titulan, mantener glándulas en la parte inferior del tubo con una fuerza hacia abajo desde la punta de la pipeta 200 l y enganchar el tejido suave y lentamente. Asegúrese de utilizar, trazos continuos lentos, nunca pipeteo rápido o abrupto. </li><li> Añadir un medio de cultivo de 250 l adicional para llevar el volumen total de medios de cultivo de hasta 450 l. </li><li> Placa de 60 l de esta solución que contiene células en cada uno de siete 12 mm cubreobjetos PDL-pre-recubiertas, que se colocan en una placa de cultivo de 60 x 15 mm. </li><li> Una vez plateado, colocar la cápsula en la incubadora que se ajusta a 37 ° C y se mantuvo con un flujo / suministro constante de 5% de CO 2 durante 30 min para asegurar un entorno para la viabilidad celular. Después de la incubación, añadir con cuidado 5 ml de medios de cultivo pre-calentado en la placa de cultivo y devolver el plato de nuevo en la incubadora durante 24 horas antes de la célula-agregadograbaciones d. NOTA: Normalmente, las células se pueden utilizar para los registros electrofisiológicos para un máximo de 4 días. </li></ol> 5. Cell Identificación y gigaohmio Seal Formación <ol><li> Coloque el cubreobjetos de 12 mm en la solución extracelular. Desde el conjunto de glándulas suprarrenales se utilizan para la preparación de células, mezclar las células cromafines (típicamente muy brillantes con un marrón / coloración beige y una forma circular casi perfecta (Figura 1) en cultivo con células corticales (más pequeños y más débiles que las células cromafines). </li><li> Tire de las pipetas de parche en cuatro etapas, utilizando una programable P-97 extractor. Sumerja la punta de la pipeta en una cera derretida, lo que ayudará a reducir la capacitancia de la copa y luego disparar el pulimento de la punta a &quot;soplar-away&quot; esta cera desde el interior de la punta de la pipeta. NOTA: Cuando se llena con una solución de parche pipeta, pipetas de parche suelen tener una resistencia de ~ 2 mW. </li><li> Acérquese al parche pipeta cerca de la célula dentro de varios microns. Para lograr una configuración de célula-adjunto, buscar cualquier deformación celular ligero durante la aproximación de la pipeta de parche más cerca de la célula. Aplicar una suave succión hasta que se logre un sello GΩ. </li></ol> 6. Grabaciones capacitancia celular-Attached <ol><li> Una vez que un sello GΩ se revela, hacer grabaciones de células-unido a través de la utilización de un amplificador EPC-7 además de patch-clamp y un amplificador lock-in analógico de dos fases (ver tabla adjunta de equipos). Aplicar una onda sinusoidal con una amplitud de 50 mV (rms) y la frecuencia de 20 kHz desde el amplificador lock-in. </li><li> Ajuste el filtro de salida del amplificador lock-in 1 constante de tiempo ms y 24 db. Ajuste la fase del amplificador lock-in de tal manera que los cambios de capacitancia transitorios producidos por pulsos suave succión sólo aparecen en la traza capacitancia parche (Im) sin proyección en la traza de la conductancia parche (Re) (Figura 2). NOTA: Por favor, vea los detalles para la calibración del sistema de reConferencia 19. </li><li> Identificar los cambios de capacitancia típicos por pasos &quot;hacia abajo&quot; (Figura 3). NOTA: Una grabación sólida será visto como una &quot;escalera&quot; de tipo parecido con muchos pasos a la baja, lo que indica la internalización de las vesículas individuales. El proceso de actualización sirve como estimulación mecánica ya la acción actual puede ser típicamente registra en la mayoría de las células. </li><li> Nombre los archivos como carpetas individuales, incluyendo la fecha: mm / dd / año y cada grabación de células-que se adjunta como su propia numeración individuo (xx) dentro de ese archivo. </li></ol>

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Los autores declaran que no tienen intereses financieros en competencia.

Mediciones de capacitancia celular conectado requieren varios pasos críticos a fin de recabar las grabaciones de alta calidad: 1) células viables y saludables preparados a partir de las glándulas suprarrenales; 2) recubrimiento PDL de los cubreobjetos; 3) la formación de sello gigaohmio; 4) nivel de ruido del sistema; y 5) de corrección de fase. Para la cirugía animal, modificaciones potencialmente uno puede hacer es ajustar el abordaje quirúrgico de la mejor destreza traje y para evi...

La transmisión sináptica es mediada por exocitosis de vesículas sinápticas que contienen neurotransmisores, y estas vesículas debe someterse a endocítica reciclado local dentro de la terminal nerviosa para mantener la comunicación neuronal en el largo plazo. Dado el papel esencial de la transmisión sináptica en el cerebro, la comprensión de la maquinaria molecular que constituye el ciclo de la vesícula sináptica es una base esencial para lograr una mejor comprensión en la comunicación celular en su conjunto. Entre los sistemas modelo de célula, la célula cromafines adrenales ha proporcionado algunas de la visión más definitivo en la maquinaria molecular subyacente de reciclado de vesículas sinápticas. La exocitosis, el paso final en la liberación de neurotransmisores, se ha estudiado y se examinaron mediante el uso de la célula cromafines adrenales 1,2 inmensamente. De hecho, la mayoría de los jugadores moleculares que orquestan la formación, la orientación, soporte, y la fusión de los gránulos secretores han sido identificados debido a la aplicación de divtécnicas ERSE en células cromafines 1. Además, al proporcionar una oportunidad para permitir la resolución de una sola vesícula de la maquinaria de proteína implicada en la exocitosis, la cromafín sigue siendo un modelo de gran alcance para hacer frente a las cuestiones de la fusión de vesículas 3. Mediciones de capacitancia de célula-adjunta se utilizaron primero en la solución de fusión de una sola vesícula durante la exocitosis 3. Exocitosis de vesículas tan pequeñas como ~ 60 nm de diámetro se ha demostrado que puede detectar mediante mediciones de admisión de la membrana celular con la técnica de patch clamp en la configuración de células vinculada 4-7. De admisión se define como una medida de la facilidad con que un circuito o dispositivo permitirá que fluya una corriente; es la inversa de la impedancia. Por lo tanto, las mediciones de admitancia proporcionan una comprensión de la capacitancia de la membrana. Esto se logra mediante la incorporación de la membrana vesicular en la membrana plasmática; esta incorporación revela cambios en la superficiezona 8. Cada vesícula fusión causa un aumento gradual en 9,10 capacitancia de la membrana. Además, esta medida admisión proporciona la conductancia de la membrana y la conductancia poro de fusión durante un evento de exocitosis 3. Como esta técnica ha proporcionado una herramienta única en la identificación de la cinética de una sola vesícula durante la exocitosis, nuestro laboratorio ha aplicado recientemente este concepto para detectar la endocitosis de vesículas individuales 11,12. Nuestro interés específico es mediada por clatrina endocitosis (CME), que ha sido considerada como un componente fundamental de limpieza en muchas células y 13 como una vía principal para la endocitosis de las vesículas sinápticas en los terminales neuronales 14,15. CME es conocido por ser biológicamente importante, sin embargo, su cinética no quedan bien entendidos debido a limitaciones técnicas en el seguimiento de eventos endocíticas singulares. Dadas las similitudes en los mecanismos exocytic entre las células cromafines y neuronas 1, es plausible que los mecanismos de fisión en células cromafines pueden aplicar probablemente a la endocitosis de vesículas sinápticas en las neuronas. Las mediciones de capacitancia de célula-adjunto se han utilizado para controlar los eventos de endocítica individuales y analizar la cinética de fisión, que la mayoría de los métodos son incapaces de resolver. En nuestras grabaciones celulares inscritos, una onda sinusoidal a 20 kHz se superpone sobre el potencial de mantenimiento, y la corriente de salida se separa en la conductancia de la membrana en un canal y la membrana capacitancia en el otro canal de dos fases amplificador lock-in de 16 18. A partir de los cambios en la conductancia de la membrana y la capacitancia, se puede calcular la cinética de la fisión de poros, que probablemente corresponde a la membrana tubular cuello que conecta la vesícula internalización a la membrana plasmática antes de la vesícula de pinzamiento. En conjunto, esta técnica nos da la oportunidad de examinar los mecanismos de regulación de la fisión de vesículas durante CME.

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			<td height="61" style="height:61px;width:88px;">Name of Material/ Equipment</td>
			<td style="width:88px;">Company</td>
			<td style="width:88px;">Catalog Number</td>
			<td style="width:88px;">Comments/Description</td>
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			<td height="43" rowspan="2" style="height:43px;width:88px;">Poly-D-Lysine</td>
			<td rowspan="2" style="width:88px;">Sigma</td>
			<td rowspan="2" style="width:88px;">P0899</td>
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		<tr height="22">
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			<td height="21" style="height:21px;width:88px;">DMEM&#160;&#160;</td>
			<td style="width:88px;"></td>
			<td rowspan="2" style="width:88px;">15066024</td>
			<td rowspan="2" style="width:88px;">Keep out of UV</td>
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			<td height="81" style="height:81px;width:88px;">Dulbecco&#8217;s Modified Eagle Medium</td>
			<td style="width:88px;">Life Technologies</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Cover Glass</td>
			<td style="width:88px;">Carolina Biological</td>
			<td style="width:88px;">633029</td>
			<td style="width:88px;">12mm</td>
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		<tr height="34">
			<td height="56" rowspan="2" style="height:57px;width:88px;">Penicillin Streptomycin</td>
			<td rowspan="2" style="width:88px;">Life Technologies</td>
			<td rowspan="2" style="width:88px;">15140122</td>
			<td rowspan="2" style="width:88px;">100mL</td>
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		<tr height="22">
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			<td height="61" style="height:61px;width:88px;">Insulin-Trans-Sel-X</td>
			<td style="width:88px;">Life Technologies</td>
			<td style="width:88px;">51500056</td>
			<td style="width:88px;">Only thaw on ICE!</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:88px;">Papain</td>
			<td style="width:88px;">Worthington</td>
			<td style="width:88px;">39S11614</td>
			<td style="width:88px;"></td>
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			<td height="56" rowspan="2" style="height:57px;width:88px;">EPC-7 plus patch amplifier</td>
			<td rowspan="2" style="width:88px;">HEKA</td>
			<td rowspan="2" style="width:88px;"></td>
			<td rowspan="2" style="width:88px;"></td>
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		<tr height="22">
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		<tr height="21">
			<td height="21" style="height:21px;">BNC-2090 data acquisition board</td>
			<td rowspan="2" style="width:88px;">National Instruments</td>
			<td rowspan="2" style="width:88px;"></td>
			<td rowspan="2" style="width:88px;"></td>
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			<td height="22" style="height:22px;"></td>
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		<tr height="61">
			<td height="61" style="height:61px;width:88px;">Igor data acquisition software</td>
			<td style="width:88px;">Wavemetrics</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">P-97 pipette puller</td>
			<td style="width:88px;">Sutter Instruments</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Microforge</td>
			<td style="width:88px;">Scientific Instruments</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="60">
			<td height="161" rowspan="3" style="height:161px;width:88px;">Borosilicate glass capillaries</td>
			<td rowspan="3" style="width:88px;">Sutter Instruments</td>
			<td rowspan="3" style="width:88px;">B150-110-10</td>
			<td style="width:88px;">Outer diameter-1.5 mm</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:88px;">Inner diameter 1.10 mm</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Length- 10 cm</td>
		</tr>
	</tbody>
</table>

methods for cell-attached capacitance measurements in mouse adrenal chromaffin cell

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion known as exocytosis that mediates synaptic transmission. Subsequent retrieval of synaptic vesicles is necessary to generate new neurotransmitter-filled vesicles in a process identified as endocytosis. During exocytosis, fusing vesicle membranes will result in an increase in surface area and subsequent endocytosis results in a decrease in the surface area. Here, our lab demonstrates a basic introduction to cell-attached capacitance recordings of single endocytic events in the mouse adrenal chromaffin cell. This type of electrical recording is useful for high-resolution recordings of exocytosis and endocytosis at the single vesicle level. While this technique can detect both vesicle exocytosis and endocytosis, the focus of our lab is vesicle endocytosis. Moreover, this technique allows us to analyze the kinetics of single endocytic events. Here the methods for mouse adrenal gland tissue dissection, chromaffin cell culture, basic cell-attached techniques, and subsequent examples of individual traces measuring singular endocytic event are described.

La viabilidad celular y la calidad del sello gigaohmio son críticos en la determinación de la calidad de las grabaciones capacitancia de célula-adjunto. Por lo tanto, es fundamental procurar un cultivo celular eficaz y eficiente antes de registros electrofisiológicos y células viables típicos se ilustran en la Figura 1. Práctica y la hora serán útiles en la consecución de un sello gigaohmio con alta calidad. Si uno puede ver claramente la deformación celular cuando la pipeta parche está acer...

Watch this Scientific Journal Video about Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell at JoVE.com

Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

Los métodos para mediciones de capacitancia celulares inscritos en ratón suprarrenal Cromafín Cell

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion ...

methods-for-cell-attached-capacitance-measurements-mouse-adrenal

Research

JoVE Journal

Neuroscience

8.7K vistas.  University of Illinois at Chicago. After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

Video: Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

Guiada por ultrasonido microinyección en el cerebro anterior Ratón In Utero En E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

Investigación

Neurociencias

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

Múltiples imágenes del ratón neuroanatómicos de Resonancia Magnética

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

En electroporación in vivo de la retina del ratón en desarrollo

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

Patch-clamp mediciones de capacitancia y Ca 2 + Imágenes en las terminaciones nerviosas individuales en rebanadas de retina

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

Methods for Intravenous Self Administration in a Mouse Model

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages of using an IVSA paradigm to study the reinforcing properties of drugs of abuse such as cocaine include the fact that the drug is self-administered instead of experimenter-administered, the schedule of reinforcement can be altered, and accurate measurement of the quantities of drug consumed as well as the timing and pattern of IV injections can be obtained. Furthermore, the intravenous route of administration avoids potential confounds related to first pass metabolism or taste, and produces rapid increases in blood and brain drug levels. As outlined in this video, intravenous self-administration can be obtained without prior food restriction or prior drug training following careful catheter placement during surgery and meticulous daily catheter flushing and maintenance. Experimental procedures outlined in this paper include a description of animal housing and acclimation methods, operant training using sweetened milk solutions, and catheter implantation surgery.

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

Los métodos para Autoadministración intravenosa en un modelo de ratón

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the Modulation and Analysis of NF-&amp;#954;B-dependent Adult Neurogenesis

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

Los métodos para la modulación y Análisis de NF-KB dependiente de la neurogénesis adulta

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

En Vivo electrofisiológicos mediciones sobre los nervios ciáticos de ratones

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Imágenes en vivo de la mitosis en el desarrollo de la corteza de embriones de ratón

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

A Behavioral Screen for Heat-Induced Seizures in Mouse Models of Epilepsy

Transgenic mouse models have proved to be powerful tools in studying various aspects of human neurological disorders, including epilepsy. The SCN1A-associated genetic epilepsies comprise a wide spectrum of seizure disorders with incomplete penetrance and clinical variability. SCN1A mutations can result in a large variety of seizure phenotype ranging from simple, self-limited fever-associated febrile seizures (FS), moderate-level genetic epilepsy with febrile seizures plus (GEFS+) to more severe Dravet Syndrome (DS). Although FS are commonly seen in children below 6-7 years of age who do not have genetic epilepsy, FS in GEFS+ patients continue to occur into adulthood. Traditionally, experimental FS have been induced in mice by exposing the animal to a stream of dry air or heating lamps, and the rate of change in body temperature is often not well controlled. Here, we describe a custom-built heating chamber, with a plexiglass front, that is fitted with a digital temperature controller and a heater-equipped electric fan, which can send heated forced air into the test arena in a temperature-controlled manner. The body temperature of a mouse placed in the chamber, monitored through a rectal probe, can be increased to 40-42 &#176;C in a reproducible manner by increasing the temperature inside the chamber. Continual visual monitoring of the animals during the heating period demonstrates induction of heat-induced seizures in mice carrying an FS mutation at a body temperature that does not elicit behavioral seizures in wild-type litter mates. Animals can be easily removed from the chamber and placed on a cooling pad to rapidly return body temperature to normal. This method provides for a simple, rapid, and reproducible screening protocol for the occurrence of heat-induced seizures in epilepsy mouse models.

The goal of the method is to screen for hyperthermia or heat-induced seizures in mouse models. The protocol describes the use of a custom-built chamber with continuous monitoring of the body temperature to determine whether elevated body temperature leads to seizures.

Una prueba conductual para las convulsiones inducidas por el calor en modelos de epilepsia en ratones

Transgenic mouse models have proved to be powerful tools in studying various aspects of human neurological disorders, including epilepsy. The ...

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are laterally packed in the outermost layer of the retina, the photoreceptor layer, resulting in alignment of analogous subcellular compartments. Traditionally, tangential sectioning of the frozen flat-mounted retina has been used to study the movement and localization of proteins within different rod compartments. However, the high curvature of the rod-dominant mouse retina makes tangential sectioning challenging. Motivated by the study of protein transport between compartments, we developed two peeling methods that reliably isolate the rod outer segment (ROS) and other subcellular compartments for western blots. Our relatively quick and simple techniques deliver enriched and subcellular-specific fractions to quantitatively measure the distribution and redistribution of important photoreceptor proteins in normal rods. Moreover, these isolation techniques can also be easily adapted to isolate and quantitatively investigate the protein composition of other cellular layers within both healthy and degenerating retinae.

This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

Dos métodos de peeling para el aislamiento de compartimentos de células fotorreceptoras en la retina del ratón para el análisis de proteínas

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...