Con el apoyo de premio a la Trayectoria para el Desarrollo de la Investigación y Prevención de la Ceguera, las subvenciones del NIH EY19312 y EY19543 (VJK), así como por la subvención sin restricciones de Investigación para Prevención de la Ceguera y la EY02687 (Departamento de Oftalmología y Ciencias Visuales de la Universidad de Washington).

1. Haciendo electrodos <ol><li> Preparar electrodos de vidrio. Pesar 120 mg de agar y se mezcla en 10 ml de agua destilada (concentración final de 1,2% de agar). Derretir la solución de agar en un baño de agua caliente. Llenar capilares de vidrio (que se usen instrumentos de Word Presision TW100-4 capilares con las siguientes dimensiones: longitud = 100 mm, OD / ID = 1/0.75 mm, y el volumen interno = 44 l) con la solución de agar con una jeringa de plástico. Solidificar el agar a temperatura ambiente durante 10 min. Así, hasta ~ 200 capilares puede ser llenado (el resto de la mezcla de agar puede ser almacenado a -4 ° C y reutilizados varias veces). </li><li> Cortar los capilares en mitades utilizando una cuchilla de diamante. </li><li> Remoje los electrodos de vidrio resultantes en solución ratón electrodo por lo menos 24 horas a 4 ° C. Almacenar los electrodos a 4 ° C y utilizarlos durante el período de 3-4 meses. </li></ol> 2. Configuración del experimento <ol><li> Dark-adaptación de un ratón durante la noche. El uso de tipo salvaje (por ejemplo, C57Bl / 6) para los ratones varilla impulsadas grabaciones transretinal y alfa T - / - ratones que carecen de barra de señalización de 8 para las grabaciones de cono transretinal. </li><li> Preparar una solución de perfusión L, 100 ml de solución electrodo, y 20 ml solución de incubación (véase más adelante) y filtrar la solución a través de perfusión un Millipore 0,45 micras o 0,22 micras filtro (opcional). </li><li> Calibrar la fuente de luz (505 nm LED o estimulador bombilla halógena óptico) utilizando un fotómetro colocado en el plano de la retina. En el caso de fuente de luz LED, utilizar un difusor óptico para conseguir una uniformidad de luz. </li><li> Prepare la cámara de perfusión para el experimento (construcciones de la cámara puede variar). Nuestra cámara está hecho de plexiglás 3 mm, con una parte inferior hecha de la tapa de un pequeño plato de Petri de plástico, y tiene un volumen de perfusión activa de ~ 400 l (l = 25 mm, W = 8 mm, H ≈ 2 mm). Llenar el espacio electrodo inferior de la cámara con una solución de electrodo que contiene 2 mM de L-glutamato y 10 mM de BaCl 2. Usando un tubo de plástico y de vidrio un capilar, realizar una conexión entre la solución en el espacio electrodo inferior (hecho de conector de tubo de plástico unido a la parte inferior de la cámara) y el soporte del electrodo. Asegúrese de que no hay burbujas en el electrodo, los tubos de conexión, o el titular de electrodo (purgar la solución con la jeringa de plástico si es necesario). </li><li> Montar la cámara de grabación en la platina del microscopio (opcional: el microscopio puede ser montado en una mesa anti-vibración y protegido dentro de una jaula de Faraday para minimizar el ruido mecánico y electromagnético). Conectar el soporte de electrodo inferior al polo positivo cabezal de la platina del amplificador diferencial (por ejemplo, Warner Instrumentos DP-311 amplificador). Centro y alinear el electrodo de la cámara baja la apertura de los puntos de luz de estímulo. Conecte la línea de perfusión a la cámara de grabación y gire a la perfusión en el. </li><li> Burbuja de la perfusión de solución con 95% de O 2/5% CO 2, mientras que la incubación de 40 ° C water baño. CO 2 ajusta el pH de la solución a 7,2. La solución fluye desde la botella a la cámara de grabación por la gravedad que pasa a través de una resistencia cerámica recalentar a 36-37 ° C. Para reducir la pérdida de calor, el calentador debe estar situada lo más cerca de la cámara de grabación como sea posible, idealmente en la platina del microscopio. </li><li> Con un regulador de flujo ajustar la velocidad de flujo a aproximadamente 1 ml / min. </li><li> Conectar el electrodo superior a la segunda (negativo) polo de la cabezal de la platina utilizando un soporte de electrodo segundos. Obligar a la sonda del termopar al electrodo superior mediante una junta tórica. Asegurarse de que las puntas de los electrodos y el termopar están muy juntas de modo que la lectura de la temperatura se toma como cerca del centro de la retina como sea posible. Sumergir la punta del electrodo superior en la solución de perfusión y lo coloca en el centro de la cámara. Permitir 10-15 minutos para la estabilización de referencia. </li><li> Ajustar la tensión de CC para el calentador de manera que la temperatura de la solución es 36 -37 ° C. </li></ol> 3. Aislamiento de la retina del ratón <ol><li> Bajo una luz roja tenue, la eutanasia el ratón adaptado a la oscuridad con CO 2 y la dislocación cervical. Opcional: hacer una marca de referencia en cada ojo del ratón mediante la cauterización, el punto más dorsal de la esclerótica con una pluma de cauterio o pines climatizada de disección. Todos los procedimientos posteriores se realizan bajo la luz infrarroja usando convertidores de imagen infrarroja de ajuste a un microscopio de disección. </li><li> Diseccionar los ojos con tijeras curvas, aplicando una presión suave y estiramiento de la piel a los lados de cada ojo. Hemisect ambos ojos bajo iluminación infrarroja usando microtijeras o una hoja de afeitar. </li><li> Retire la córnea y el cristalino. Eliminar la mayor cantidad de vítreo como sea posible. </li><li> Pelar la retina primero desde la capa de epitelio de pigmento y, si es necesario, es importante limpiarlo con pinzas para eliminar gránulos restantes de epitelio de pigmento. Estamos típicamente usando retina completa tanto para la varilla y transret conograbaciones Inal. Sin embargo, si desea grabar en concreto de la parte dorsal de la retina del ratón que tiene más de M / L-9 conos, antes de pelar la retina quitar la parte ventral de la ojera con una hoja de afeitar o microtijeras uso de la marca cauterio como una referencia. </li><li> Coloque el globo ocular hemiseccionaron segundos en una pequeña placa de Petri con solución de incubación y se incuba la preparación en una caja hermética a la luz saturada con oxígeno puro hasta su uso. Normalmente, en estas condiciones la copa ocular segundo ratón se pueden almacenar durante varias horas a temperatura ambiente y se utiliza para las grabaciones. </li><li> Utilizando una pipeta de plástico o vidrio transferir la retina aislada a una gota de solución de perfusión (ca. 300 l) en la tapa de una placa de Petri. Añadir 5-6 gotas de solución de electrodos que contienen BaCl 2 al pre-incubar la retina con BaCl 2. Coloque un pedazo cuadrado (5x5 mm CA) de la parte de atrás de esquina engrasado (se utiliza el lubricante Dow Corning 111 de la válvula y sellador) Millipore filtro de papel (0,45 micras HARG tipo) con pre-hecho agujero en su centro (2-2,5 mm de diámetro) a la solución gota mismo. Utilizando posición fórceps la retina en la parte superior del papel de filtro (lado fotorreceptor arriba) y presione sus bordes en la periferia a plana montarla. La retina debe estar centrada sobre la abertura en el papel de filtro. </li><li> Con unas pinzas transferir el papel de filtro con la retina de la cámara de perfusión y colocarlo por encima del punto de estímulo-alineados espacio inferior del electrodo. Presione ligeramente los bordes engrasadas de papel de filtro para unir con la cámara. </li><li> Coloque el electrodo superior sobre el centro de la retina (ligeramente tocar la capa de fotorreceptores). Centrado el electrodo superior en la retina aumenta la amplitud de la señal (hasta ~ 20-40% en comparación con colocarlo más cerca del borde retina). Aunque la localización del electrodo distorsiona ligeramente la intensidad de la luz estímulo eficaz de llegar a un pequeño sector de la retina (~ 12-15% de su superficie total), sigue siendo la mejor forma de colocar el electrodoatropelló. La preparación está ahora listo para las grabaciones transretinal. </li></ol> 4. Grabaciones Transretinal <ol><li> Dependiendo de su línea de ratones, registro de varilla o M / L de cono-impulsadas por las respuestas de destello de prueba desde tenue hasta la intensidad luminosa de la luz calibrada 505 nm siempre de la fuente de luz LED o estimulador óptico. En el caso de nuestra fuente de luz LED, la intensidad del flash se controla mediante una combinación de control computarizado de tensión de entrada de LED, resistencias conmutables, y un conjunto de filtros de densidad neutra (usamos E-Colour # 211 0,9 filtros ND de cine de Rosco Laboratories ) colocado entre el LED y la retina. La duración de la prueba de flash LED (20 ms) es controlado por un ordenador. Si se utiliza un estimulador bombilla halógena óptico, la intensidad de prueba flash y longitud de onda puede ser controlado por un conjunto de filtros de densidad neutra y estrechos filtros de interferencia de banda, respectivamente. Prueba de duración del flash pueden ser controlados por ordenador basadas en persianas. </li><li> Opcional: para mantener la eficienciala supresión del componente glial de la foto-con BaCl 2 durante todo el día, cambiar la solución de electrodo inferior de vez en cuando (por ejemplo, antes de la prueba cada retina) por una purga con una jeringa de plástico llena con solución fresca. </li></ol> 5. Los resultados representativos Soluciones <ol><li> Retina del ratón perfusión solución: 112,5 mM de NaCl, 3,6 mM de KCl, 2,4 mM de MgCl 2, 1,2 mM de CaCl 2, HEPES 10 mM (pH 7,2), 20 mM NaHCO 3, 3 mM de Na succinato, 0,5 mM de Na glutamato, 0,02 mM EDTA, y 10 mM de glucosa. Además, la solución se complementa con 2 mM de L-glutamato y 10 mM de DL-AP-4 para bloquear componentes de mayor orden de la fotorrespuesta 10,11, y con MEM 0,1% vitaminas y soluciones MEM aminoácidos (Sigma) para mejorar la retina viabilidad. </li><li> Ratón solución electrodo: 140 mM de NaCl, 3,6 mM de KCl, 2,4 mM de MgCl 2, 1,2 mM de CaCl 2, 3 mM HEPES (pH 7,4, NaOH). La solución en el menor elementoespacio ctrode también contiene 2 mM de L-glutamato para bloquear la transmisión sináptica 10 y, además, 10 mM de BaCl 2 para suprimir el componente glial de la fotorrespuesta 3,12. </li><li> Retina solución de incubación: se disuelven 272 mg de medio L-15 (Sigma) y 20 mg de BSA en 20 ml de agua desionizada. Final L-15 y las concentraciones de BSA son 13,6 mg / ml y 1 mg / ml, respectivamente. Ajustar el pH a 7,4 con HCl 1 M. </li></ol><img alt="Figura 1." src="/files/ftp_upload/3424/3424fig1.jpg" /> Figura 1. Diagrama esquemático que muestra el aislamiento de la retina del ratón para transretinal grabaciones ERG. <img alt="Figura 2." src="/files/ftp_upload/3424/3424fig2.jpg" /> Figura 2. Fotografía de la cámara de perfusión y de electrodos de registro con sus titulares. Las flechas rojas indican la dirección del flujo de perfusión. <img alt="Figura 3." src="/files/ftp_upload/3424/3424fig3.jpg"/&gt; Figura 3. Esquema del montaje experimental para la transretinal grabaciones del ERG. Las flechas rojas indican la dirección del flujo de perfusión. <img alt="Figura 4." src="/files/ftp_upload/3424/3424fig4.jpg" /> Figura 4 resultados representativos:. Registrado familia de ratón la barra impulsadas por las respuestas transretinal. Photoresponses fueron filtrada de paso bajo a 30 Hz (8-Bessel), digitaliza a 1 kHz y almacenadas en un ordenador para su posterior análisis. Trazos mostrados corresponden a las medias de las respuestas de 5-6 a intensidades de luz tenue y las respuestas de 2-3 en la intensidad de luz de saturación. <img alt="Figura 5." src="/files/ftp_upload/3424/3424fig5.jpg" /> Figura 5: Los resultados representativos. Familia registrada de ratón de cono impulsadas por las respuestas transretinal. Photoresponses fueron filtrada de paso bajo a 30 Hz (8-Bessel), digitaliza a 1 kHz y almacenadas en un ordenador para su posterior análisis. Trazas se muestran en la AVERAGes de las respuestas de 5-10 a intensidades de luz tenue y las respuestas de 3-5 en la intensidad de luz de saturación.

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General properties of the response.</a> Vision Res. 9, 1435-1442 (1969).</li><li>Vinberg, F., Koskelainen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+sets+the+physiological+value+of+the+dominant+time+constant+of+saturated+mouse+rod+photoresponse+recovery.">Calcium sets the physiological value of the dominant time constant of saturated mouse rod photoresponse recovery.</a> PLoS One. 5, e13025-e13025 (2010).</li><li>Bolnick, D. A., Walter, A. E., Sillman, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barium+suppresses+slow+PIII+in+perfused+bullfrog+retina.">Barium suppresses slow PIII in perfused bullfrog retina.</a> Vision Res. 19, 1117-1119 (1979).</li></ol>

Los autores no tienen nada que revelar.

El método de los bastones, los conos y las grabaciones basadas en transretinal-ERG se ha descrito anteriormente se está convirtiendo en una poderosa herramienta para la investigación de la función de los fotorreceptores de ratón en el tipo salvaje y animales modificados genéticamente. Además de la caracterización de las propiedades de fácil fotorrespuesta básicos, esta técnica simple proporciona estabilidad durante gran respuesta de larga duración experimentos realizados en preparaciones retina cercana al in...

<table border="1"><tbody><tr><td> Nombre del Material </td><td> Tipo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentario </td></tr><tr><td> DL-AP-4 </td><td></td><td> Biociencia Tocris </td><td> 0101 </td><td></td></tr><tr><td> Todos los demás reactivos </td><td></td><td> Sigma-Aldrich </td><td></td><td></td></tr><tr><td> Capilares de vidrio </td><td></td><td> World Precision Instruments </td><td> TW100-4 </td><td> Para hacer electrodos </td></tr><tr><td> Papel de filtro </td><td> HARG </td><td> Millipore </td><td> HABG01300 </td><td></td></tr><tr><td> Fotómetro </td><td></td><td> UDT Instruments </td><td> S350 </td><td> Para la calibración de la luz </td></tr><tr><td> Sensor de silicio radiométrica </td><td></td&gt; <td> UDT Instruments </td><td> 221 </td><td> Para la calibración de la luz </td></tr><tr><td> Anti-vibración de mesa </td><td></td><td> Técnico Manufacturing Corporation </td><td> TMC 78-239-02R, TMC63-26012-01 </td><td> Para reducir al mínimo el ruido mecánico </td></tr><tr><td> Compresor de aire Pantera P 15TC </td><td></td><td> Werther Internacional </td><td> P 15TC </td><td> Conectado a la mesa anti-vibración </td></tr><tr><td> Estereomicroscopio </td><td></td><td> LEICA </td><td> MZ9.5 </td><td> Para la disección del ratón ocular </td></tr><tr><td> Convertidores de imagen por infrarrojos </td><td></td><td> BEMeyers </td><td> ProwlerTM </td><td> Ligado a estereomicroscopio </td></tr><tr><td> Amplificador diferencial DP-311 </td><td></td><td> Warner Instruments </td><td> DP-311 </td><td></td></tr><tr><td> Axon Digidata 1440A digitalizador </td><td>; </td><td> Molecular Devices </td><td> 1440A </td><td></td></tr><tr><td> Dual Channel de 8 polos del filtro </td><td></td><td> Krohn-HITE Corporación </td><td> 3382 </td><td></td></tr><tr><td> Resistencia de cerámica </td><td></td><td> TE Conectividad </td><td> CGS SBCHE618RJ </td><td> Para recalentar la solución de perfusión </td></tr><tr><td> Termopar </td><td> T </td><td> Physitemp Instruments </td><td> TI-18 </td><td></td></tr><tr><td> Monitor de temperatura </td><td></td><td> Omega </td><td> DPi32 </td><td> Conectado a un termopar </td></tr><tr><td> LED de 505 nm </td><td></td><td> TT Electronics / Optek Technology </td><td> Digi-Key P / N 365-1185-ND </td><td> Para aplicar destellos de luz de prueba o de blanqueo </td></tr><tr><td> Pluma de cauterización </td><td></td><td> Bovie </td><td> AA25 </td><td> Para marcar la parte dorsaldel globo ocular ratón </td></tr><tr><td> pCLAMP 10 Electrofisiología de Adquisición de Datos y Análisis </td><td></td><td> Molecular Devices </td><td></td><td></td></tr></tbody></table>

transretinal erg recordings from mouse retina: rod and cone photoresponses

Hay dos clases distintas de formación de la imagen-los fotorreceptores de la retina de los vertebrados: los bastones y conos. Los bastones son capaces de detectar fotones individuales de luz, mientras que los conos de funcionar de forma continua en las cambiantes condiciones de luz brillante. La absorción de luz por la A-Rod y específicos de cono pigmentos visuales en los segmentos externos de fotorreceptores desencadena una cascada de fototransducción que finalmente conduce al cierre de los nucleótidos cíclicos-bloqueados los canales de la membrana plasmática y la hiperpolarización de la célula. Este cambio inducido por la luz en la membrana de actuales y potenciales pueden ser registrados como una foto-, ya sea por 1,2 de electrodos de aspiración clásica técnica de grabación o grabaciones transretinal electrorretinograma (ERG) de retinas aisladas con componentes farmacológicamente bloqueados respuesta postsináptica 3-5. Este último método permite drogas accesibles duraderos grabaciones de fotorreceptores de ratón y es particularmente útil para obtener photoresponses estables from los conos de ratones escasos y frágiles. En el caso de los conos, estos experimentos se puede realizar tanto en las condiciones adaptados a la oscuridad y la iluminación intensa siguiente que blanquea prácticamente todos pigmento visual, para supervisar el proceso de recuperación de cono de fotosensibilidad en adaptación a la oscuridad 6,7. En este video, vamos a mostrar cómo llevar a cabo de vara y M / L de cono-impulsadas por las grabaciones de transretinal adaptada a la oscuridad retina del ratón. Grabaciones Rod se llevó a cabo utilizando retina de tipo salvaje (C57BL / 6) ratones. Por simplicidad, las grabaciones de cono se obtendrá a partir modificado genéticamente varilla transducina α-subunidad extractor (Tα - / -) ratones que carecen de barra de señalización 8.

Watch this Scientific Journal Video about Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses at JoVE.com

Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

Se describe un método relativamente simple de transretinal electrorretinograma (ERG) para la obtención de grabaciones photoresponses conos y bastones de la retina del ratón intacto. Este enfoque se aprovecha del bloque de la transmisión sináptica de fotorreceptores para aislar sus respuestas de luz y grabar usando electrodos colocados sobre el terreno a través de la aislada plana montada en la retina.

Transretinal Grabaciones ERG de retina del ratón: Rod y el Cono Photoresponses

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light ...

transretinal-erg-recordings-from-mouse-retina-rod-cone

Research

JoVE Journal

Neuroscience

17.7K vistas.  Washington University School of Medicine. Se describe un método relativamente simple de transretinal electrorretinograma (ERG) para la obtención de grabaciones photoresponses conos y bastones de la retina del ratón intacto. Este enfoque se aprovecha del bloque de la transmisión sináptica de fotorreceptores para aislar sus respuestas de luz y grabar usando electrodos colocados sobre el terreno a través de la aislada plana montada en la retina.

Video: Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

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 ...

Investigación

Neurociencias

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

Registros electrofisiológicos de la ruta de fibra gigante de D. melanogaster</em

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

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 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 ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

Evaluación de la Vascular regeneración en el SNC Utilizando la retina del ratón

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 &#181;m thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 &#181;m is discarded before 200 &#181;m of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 &#176;C. Pieces of outer layer are incubated in 500 &#181;l of Ringer&#39;s solution with 2 units of activated papain for 20 min at 37 &#176;C. The reaction is stopped by adding 500 &#181;l 10% fetal calf serum (FCS) in Dulbecco&#39;s Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 &#181;l of DMEM and seeded at 1 x 105 cells/cm2. The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.

Neural retina of a mouse aged 8 days is on top of a 4% gelatin block. After isolation of the photoreceptor layer (200 &#181;m) by vibratome, the photoreceptors are seeded after mechanical and enzymatic dissociation for culture. The photoreceptor layer can be used for molecular, biochemical analyses or transplantation.

Vibratome Seccionamiento retina del ratón para preparar cultivos de fotorreceptores

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Imagen Ca<sup&gt; 2 +</sup&gt; Dinámica de Terminales cono fotorreceptor axón de la retina del ratón

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Where You Cut Matters: A Dissection and Analysis Guide for the Spatial Orientation of the Mouse Retina from Ocular Landmarks

Accurately and reliably identifying spatial orientation of the isolated mouse retina is important for many studies in visual neuroscience, including the analysis of density and size gradients of retinal cell types, the direction tuning of direction-selective ganglion cells, and the examination of topographic degeneration patterns in some retinal diseases. However, there are many different ocular dissection methods reported in the literature that are used to identify and label retinal orientation in the mouse retina. While the method of orientation used in such studies is often overlooked, not reporting how retinal orientation is determined can cause discrepancies in the literature and confusion when attempting to compare data between studies. Superficial ocular landmarks such as corneal burns are commonly used but have recently been shown to be less reliable than deeper landmarks such as the rectus muscles, the choroid fissure, or the s-opsin gradient. Here, we provide a comprehensive guide for the use of deep ocular landmarks to accurately dissect and document the spatial orientation of an isolated mouse retina. We have also compared the effectiveness of two s-opsin antibodies and included a protocol for s-opsin immunohistochemistry. Because orientation of the retina according to the s-opsin gradient requires retinal reconstruction with Retistruct software and rotation with custom code, we have presented the important steps required to use both of these programs. Overall, the goal of this protocol is to deliver a reliable and repeatable set of methods for accurate retinal orientation that is adaptable to most experimental protocols. An overarching goal of this work is to standardize retinal orientation methods for future studies.

This protocol provides a comprehensive dissection and analysis guide for the use of deep ocular landmarks, s-opsin immunohistochemistry, Retistruct, and custom code to accurately and reliably orient the isolated mouse retina in anatomical space.

Donde usted cortar materias: Una disección y guía de análisis para la orientación espacial de la Retina de ratón de hitos Ocular

Accurately and reliably identifying spatial orientation of the isolated mouse retina is important for many studies in visual neuroscience, including the ...

Noninvasive EEG Recordings from Freely Moving Piglets

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel telemetric electroencephalography system in combination with standard self-adhesive hydrogel electrodes. The piglets are calmed down without the use of sedatives. After their release into the pigpen, the piglets behave normally&#8212;they drink and sleep in the same cycle as their siblings. Their sleep phases are used for the EEG recordings.

Here, we present a protocol to record telemetric electroencephalograms (EEGs) from freely moving piglets directly in the pigpen without the use of a sedative, making it possible to record typical EEG patterns during non-REM sleep, like spindle bursts.

Grabaciones de EEG no invasivo se mueva libremente lechones

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel ...

Visualizing Axonal Growth Cone Collapse and Early Amyloid &#946; Effects in Cultured Mouse Neurons

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the brains of AD patients, these do not improve memory functions. Since A&#946; aggregates in the brain before the appearance of memory impairments, targeting A&#946; may be inefficient for treating AD patients who already exhibit memory deficits. Therefore, downstream signaling due to A&#946; deposition should be blocked before AD development. A&#946; induces axonal degeneration, leading to the disruption of neuronal networks and memory impairments. Although there are many studies on the mechanisms of A&#946; toxicity, the source of A&#946; toxicity remains unknown. To help identify the source, we propose a novel protocol that uses microscopy, gene transfection, and live cell imaging to investigate early changes caused by A&#946; in axonal growth cones of cultured neurons. This protocol revealed that A&#946; induced clathrin-mediated endocytosis in axonal growth cones followed by growth cone collapse, demonstrating that inhibition of endocytosis prevents A&#946; toxicity. This protocol will be useful in studying the early effects of A&#946; and may lead to more efficient and preventative AD treatment.

Here a protocol to investigate the early effects of amyloid-&#946; (A&#946;) in the brain is presented. This shows that A&#946; induces clathrin-mediated endocytosis and collapse of axonal growth cones. The protocol is useful in studying early effects of A&#946; on axonal growth cones and may facilitate prevention of Alzheimer's disease.

Visualizar el colapso del cono de crecimiento Axonal y temprano efectos β amiloidea en las neuronas de ratón cultivadas

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the ...

Transretinal Grabaciones ERG de retina del ratón: Rod y el Cono Photoresponses

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