This work was supported by NIH R01 EY020533, WSU Startup Fund, and RPB grants.

Declaración de Ética: Los procedimientos que implican sujetos animales fueron aprobados por el Cuidado y Uso de Animales Comité Institucional (IACUC) de la Universidad Estatal de Wayne. 1. Preparación de la solución Experimental <ol><li> Prepare la solución de disección 1 día a 1 semana antes del experimento real. Use una solución tampón HEPES para la disección de la retina debido a su gran capacidad de amortiguación a temperaturas más bajas 16. Mezclar todos los productos químicos de la siguiente manera (en mM): 115 NaCl, 2,5 KCl, CaCl2 2,5, 1,0 MgCl2, 10 HEPES, 28 glucosa. Ajustar el pH a 7,4 con NaOH. Conservar la solución en el refrigerador hasta por 1 semana. </li><li> La solución de registro es medio de Ames, que es un líquido cefalorraquídeo artificial (LCRa) diseñado para preparaciones de la retina 17. Por el día del experimento, pesar polvo Ames &#39;(4,4 g por 500 ml de solución) en un tubo. Además, pesar NaHCO3 (0,95 g por 500 ml de solución) y mezclar con la Apolvo mes. </li><li> Prepare la solución de la pipeta intracelular para las grabaciones de patch clamp por el día del experimento. Mezclar todos los productos químicos de la siguiente manera (en mM): 111 K-gluconato, 1,0 CaCl2, 10 HEPES, 1,1 EGTA, 10 de NaCl, 1,0 MgCl 2, 5 ATP-Mg y 1,0 GTP-Na. Ajustar el pH a 7,2 con KOH. Se filtra la solución de la pipeta con un filtro de jeringa convencional. Haga ~ 500 ml de alícuotas y almacenarlos en un congelador o un congelador. </li></ol> 2. Preparación para el Día del Experimento <ol><li> La noche antes del experimento, coloca un ratón en una jaula (C57BL / 6J o antecedentes similares, 4-8 semanas de edad, macho) en un espacio oscuro de O / N adaptación a la oscuridad. </li><li> Preparación para la disección: <ol><li> Rellene disección solución (100 - 200 ml) en un vaso de vidrio, colóquelo sobre hielo, y la burbuja de oxígeno durante al menos 10 min. </li><li> Iniciar la oxigenación de la caja oscura para el almacenamiento de preparación de la retina. </li><li> Preparar cubreobjetos de plástico con rieles de engrase para rerebanada preparativos intes- y colocar cada uno de ellos en un plato de plástico de 35 mm. </li><li> Adjunte una nueva hoja de afeitar para un helicóptero (máquina de cortar el tejido). </li><li> Cortar una membrana de filtro en dos mitades. Esto es para el corte en lonchas de la retina. </li><li> Alinear las herramientas de disección y pipetas de transferencia cerca del microscopio de disección. El área de trabajo debe estar bien organizado para que cada herramienta se puede acceder fácilmente en la oscuridad. </li></ol></li><li> Preparación para las grabaciones de patch clamp: <ol><li> Disuelva el polvo de Ames y NaHCO3 en agua destilada (4,4 g Ames y NaHCO 3 0,95 g / 500 ml ddH 2 O). Medio de la burbuja el Ames &#39;con oxígeno mixta (95% O 2 y 5% de CO 2) durante al menos 30 min mientras se calienta a un nivel de temperatura de grabación (~ 35 ° C). Ajustar el pH a 7,4 con NaHCO 3 mientras que todavía burbujeante. </li><li> Descongelar la solución de la pipeta interna durante la disección. Después de que se haya descongelado, añadir 0,01% sulforodamina B para la tinción intracelular. </li></ol></li></ol> 3. Disección de retina <ol><li> En un cuarto oscuro, la eutanasia con el ratón utilizando dióxido de carbono y el neumotórax bilateral. Al cabo de 2 - 3 minutos de usar el dióxido de carbono, el ratón perder el conocimiento. </li><li> Cuando el ratón ya no responde a una pizca de cola, enuclear rápidamente los ojos y colocarlos en una solución de disección fría en un plato de plástico. Realice el neumotórax bilateral para asegurar la muerte del animal. Almacenar rápidamente el ojo en el plato en la caja oscura. Utilice un visor de infrarrojos para llevar a cabo el procedimiento en la oscuridad. NOTA: Como alternativa, isoflurano, la decapitación o dislocación cervical se pueden utilizar para la eutanasia. El método de la eutanasia debe seguir las directrices del Instituto Nacional de Salud (NIH) y / o el Comité de Cuidado y Uso de Animales institucional. </li><li> En una habitación oscura, ajuste el microscopio de disección y la iluminación infrarroja. Bajo el microscopio, transferir un ojo de su plato en un plato de plástico de 10 cm wITH la solución de disección enfriado. Continuamente burbuja de oxígeno a través de la tubería colocada en la parte inferior del plato de plástico. Ajustar el volumen de flujo por lo que no es demasiado baja, pero asegurarse de que no es lo suficientemente alta como para perturbar la disección. </li><li> Córnea y el cristalino eliminación: <ol><li> Hacer una incisión en la parte superior de la córnea con un cuchillo micro-quirúrgica. Sosteniendo el nervio óptico en el otro extremo del globo ocular evita que el ojo se mueva. Extienda el corte con un par de tijeras quirúrgicas. Recorta la córnea con algunos esclerótica alrededor. </li><li> Coge la lente con una pinza fina y tire lentamente el objetivo. Si el vítreo es demasiado pegajosa para quitar la lente, cortar el vítreo con un par de tijeras. </li><li> Una vez que se hace el ojo-taza, vierta suavemente solución de disección fría y oxigenada en el ocular con una pequeña pipeta de transferencia. NOTA: El globo ocular se desinfla rápidamente después se hace una incisión en la córnea. Es importante para mantener la forma del ojo como deformar el ojo durante este proprocedimiento daña la retina y causar desprendimiento de retina. </li></ol></li><li> La identificación de las partes dorsal y ventral de la retina: NOTA: Debido a la desigual distribución de la verde y la luz ultravioleta (UV) conos 18, la identificación de un área específica de la retina que es importante para las grabaciones de respuesta de luz. <ol><li> Identificar la línea que va a través del ocular de la retina que pasa cerca de la cabeza del nervio óptico. Esta línea pasa sobre todo a través de la parte ventral del nervio óptico; el lado incluyendo el nervio óptico es el lado dorsal, mientras que el otro es ventral 19. Ver este hito claramente bajo espectadores infrarrojos. </li><li> Dependiendo del propósito del experimento, hacer un corte en el lado innecesario. Esto será útil después de aislar la retina de la copa para el ojo. </li></ol></li><li> Eliminación Vítreo: <ol><li> Opcionalmente, se incuba el ojo-taza con hialuronidasa (0,5 mg / ml) durante 15 min. </li><li> Retire el vítreo con pinzas extra-finas. El humor vítreo es principalmenteunido a la parte inferior y el borde exterior del ojo-cup. Retire con cuidado sin tocar o meter la retina. Como el vítreo podría estar estrechamente unida a la ojera, retire con cuidado para evitar el desprendimiento de retina del ojo taza. Continúe tirando hasta que no sienta la tensión. NOTA: Este paso es especialmente importante para la disección de la retina del ratón; Sin embargo, no es un problema para las retinas de otras especies, tales como la rata o salamandra. </li></ol></li><li> Aislar la retina del ojo taza. Coge la esclerótica y pelar suavemente de la retina utilizando la parte trasera de fórceps. NOTA: Todo el montaje preparativos de la retina se puede hacer de la retina aislada. Hacer tres y cincuenta y siete cortes en los bordes y aplanar la retina, o cortar la retina en varios pedazos. </li><li> Recortar la retina y deseche el medio innecesario de la retina (paso 3.5). En el mismo plato de plástico, cortar la retina restante en dos piezas. Por cada pieza de la retina, cortar los bordes para hacer unalosa de la retina, y recortar los bordes plegables. </li><li> Transferir una losa de la retina en una placa de vidrio usando una pipeta de transferencia de gran tamaño (~ 2 ml). Aspirar el exceso de solución con una pequeña pipeta, usar un trozo de papel de filtro para aplanar la losa, y luego colocar rápidamente una pieza de la membrana de filtro en la parte superior del tejido. <ol><li> Utilizando una pequeña pipeta de transferencia, colocar una gota de solución en la disección de la membrana de filtro. Espere ~ 15 seg hasta que la solución se extiende sobre la membrana. Durante este tiempo, el tejido de la retina se adhieren a la membrana de filtro. </li><li> Verter solución de disección más fría debajo y alrededor del filtro. El tejido de la retina con la membrana de filtro flotará. Coge la membrana de filtro con una pinza y colocarla en la cámara de corte. Vierta la disección de solución sobre el tejido, y almacenar la preparación en la caja oscura. NOTA: El procedimiento descrito aquí es fundamental. Es importante para llevar a cabo estos pasos con rapidez para garantizar que los palos tejido de la retina a los filtros de membrana y no se seque. </li></ol></li><li> El uso de un interruptor, cortar el tejido de la retina en rodajas. Alrededor de 200 micras de espesor es factible para los estudios de patch clamp. </li><li> Coloque un cubreobjetos de plástico con rieles de grasa en la cámara de corte. Transferir una rebanada de la retina en la parte superior de los carriles de grasa en un cubreobjetos de plástico. Girar la rebanada 90 ° de modo que la sección transversal es visible. Presione la membrana de filtro hacia abajo sobre el cubreobjetos, a continuación, cubrir los lados del papel de filtro con grasa. <ol><li> Después de la rebanada se inmoviliza sobre un cubreobjetos de plástico, agarrar el cubreobjetos y la transfiere a un plato de plástico de 35 mm. Vierta la solución de disección fría en la preparación de la retina y sumergir la preparación. Almacenar cada plato en la caja oscura, que debe ser oxigenada continuamente. NOTA: Todo el procedimiento que se debe hacer con cuidado para que la membrana de filtro no se deforma. De lo contrario, la rebanada de la retina se separa fácilmente de la membrana de filtro. </li></ol></li></ol><ol><li> Grabación de preparación: <ol><li> Prime los tubos de perfusión con medio de Ames. Permitir todas las burbujas que atraviesan al llenar el tubo. </li><li> Haga pipetas de registro patch clamp con un extractor. Para llenar una pipeta con la solución de la pipeta, baño en la parte trasera de una pipeta de 1 min hasta que se rellena la punta de la pipeta. A continuación, llenar ~ 1/3 de la pipeta usando una carga micro-pipeta. Guarde cada pipeta en una caja pipeta húmedo. </li><li> Encienda el equipo para patch clamp grabaciones; incluyendo la computadora, un amplificador, cámara CCD, y el microscopio. </li></ol></li><li> Apague la luz de la habitación. Coloque una rebanada preparación de la retina en la cámara de platina del microscopio utilizando un visor de infrarrojos. Después de que se inmoviliza, comenzar la perfusión continua. Ajuste la temperatura de perfusión a 33 a 37 ° C. </li><li> Ver la superficie rebanada con la cámara CCD. Centrarse en la ubicación de destino donde el objetivo cetipos ll residen. Seleccione una celda de aspecto saludable para la grabación de patch clamp (es decir, debe tener la superficie de aspecto suave y una buena forma de la célula). </li><li> Coloque una pipeta de grabación en un soporte de pipetas. Avanzar en la pipeta para la preparación rebanada. Cuando se está cerca de la rebanada preparación (~ 2 mm por encima), encontrar la punta de la pipeta con el microscopio. Una vez que la punta de la pipeta es visible bajo el microscopio, mover la punta hacia abajo, hacia la célula diana. </li><li> Ajuste el amplificador. Ajustar la pipeta a 0 mV / 0 pA (puesta a cero) y comenzar un pulso eléctrico continuo de ~ 5 mV a ~ 10 Hz. Compruebe la resistencia de la pipeta; resistencia ideal está entre 5 y 10 MΩ para la mayoría de las neuronas de la retina. </li><li> Comience soplando desde la punta. Utilice una boquilla, o utilizar una jeringa, para aplicar presión positiva cuando la pipeta se sumerge en la solución del baño. Continuamente soplar la solución interna hasta que la punta está en la superficie de la célula diana. <ol><li> Cuando la presión positiva hace un pequeño hoyuelo en el thsuperficie celular e, avanzar en la punta ligeramente y dejar de soplar. Compruebe la resistencia de la pipeta, que debería aumentar. Si está aumentando continuamente, déjelo y vigilar la resistencia hasta que llegue&gt; 1 GΩ (gigaseal). Si la resistencia no aumenta de forma espontánea, aplicar suavemente presión negativa hasta que se convierte en un gigaseal. </li></ol></li><li> Después de lograr la gigaseal, cambie el potencial de mantenimiento de -70 mV. A continuación, aplicar intermitentemente presión negativa para romper la membrana dentro de la punta de la pipeta. Cuando se realiza la configuración de célula completa, la resistencia de la pipeta puede estar entre 500 MΩ y 1 GΩ, y la corriente capacitiva se ve. A veces las corrientes postsinápticas espontáneas se pueden observar. </li><li> Anote la relación IV -80-40 mV. Los diferentes tipos de canales dependientes de voltaje se activan dependiendo del tipo de célula. </li><li> Evocó luz corrientes o tensiones sinápticas Record. </li></ol>

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The authors have nothing to disclose.

Good recordings can only be achieved with good retinal preparations and well-designed patch clamp setups. Although all the steps described above are important, the discussion highlights some critical steps both for the dissection and recordings. 
For dissection, two things are especially important: cooling and oxygenation. After enucleating the eye, quickly remove the front part of the eye in a dissecting chamber with oxygen-bubbled, cooled dissecting solution, and pour cold solution into the ...

The retina is one of the unique parts of the nervous system. As an accessible part of the brain, its synaptic architecture has been well characterized. In addition, the functions of this neural network can be examined with a physiological stimulus: light. If the retinal tissue is isolated in a dark room with appropriate procedures, neurons in the tissue will respond to light. This preparation has been used to study visual signal processing and elucidate various synaptic mechanisms and neural network functions, as well as disease mechanisms. 
Light responses in retinal neurons have been recorded for decades. Early studies used sharp electrodes to make intracellular recordings from mudpuppy retinal neurons1. In the 1980s, the patch clamp technique was invented2, and soon became a popular method among vision researchers3,4. Single cell recordings from lower vertebrates, including mudpuppy and fish retinal neurons, were popular methods that contributed to the elucidation of visual signal processing mechanisms5,6. 
After genetic mutation techniques were developed, the mouse retina became a more popular model for vision researchers7-9. The mammalian retina is more attractive than that of lower vertebrates because it is evolutionarily closer to the human retina, and there is an opportunity to use disease models. However, mouse retinal cells are small and fragile10, and making retinal preparations and conducting patch clamp recordings in a dark room is challenging. As technology has improved, diverse approaches have become available to study visual signaling mechanisms such as imaging studies11 and the electroretinogram (ERG)12. Nevertheless, single cell recording with the patch clamp method is still important because it is highly temporally and spatially sensitive compared to other methods. Therefore, we have continuously conducted patch clamp recordings and improved our methods to investigate visual signal processing in mouse retinal slice preparations13-15.
In this video tutorial, the protocols are presented with important tips. Good recordings can only be achieved with good preparation. Practicing animal dissection and building a sturdy patch clamp rig will enable most researchers to achieve successful recordings.

<table><tbody><tr><td>Mice (28-60 days old, male)</td><td>Jackson laboratory</td><td>C57BL/6J strain</td><td></td></tr><tr><td>Ames&#039; medium powder</td><td>Sigma</td><td>A1420</td><td>excellent</td></tr><tr><td>Stereo microscope</td><td>Nikon</td><td>SMZ745</td><td>excellent</td></tr><tr><td>Dissecting tool: forceps</td><td>Dumont</td><td>#4, #5, #55</td><td>excellent</td></tr><tr><td>Dissecting tool: scissors</td><td>Roboz</td><td>RS-5605</td><td>excellent</td></tr><tr><td>Dissecting tool: surgery knife</td><td>Surgistar</td><td>7514</td><td>excellent</td></tr><tr><td>Razor blade (for chopper)</td><td>EMS</td><td>71970</td><td>excellent</td></tr><tr><td>Chopper</td><td>handmade</td><td></td><td></td></tr><tr><td>Infrared viewer</td><td>Night Owl Optics</td><td>NOBG1</td><td>It shows bright view. Focusing small objects is an issue.</td></tr><tr><td>Puller</td><td>Sutter</td><td>P-1000</td><td>excellent; makes consistent size pipettes.</td></tr><tr><td>Dark box</td><td>Pelican</td><td>dark box</td><td>excellent</td></tr><tr><td>Patch clamp system</td><td>Scientifica</td><td>slice scope 2000</td><td>Excellent setup. Most key components are included in one package. Micromanipulators are excellent.</td></tr><tr><td>Amplifier</td><td>Molecular Devices</td><td>multiclamp 700B</td><td>Excellent and easy control.</td></tr><tr><td>Acquiring software</td><td>Molecular Devices</td><td>pClamp software</td><td>Excellent and easy control.</td></tr><tr><td>Light source (LED)</td><td>Cool LED</td><td>pE-2 4 channel system</td><td>Excellent</td></tr><tr><td>CCD camera</td><td>Q-imaging</td><td>Retiga 2000</td><td>Excellent</td></tr><tr><td>Faraday cage</td><td>handmade</td><td></td><td></td></tr></tbody></table>

recording light-evoked postsynaptic responses in neurons in dark-adapted, mouse retinal slice preparations using patch clamp techniques

La retina es la puerta de entrada al sistema visual. Para entender los mecanismos de procesamiento de señales visuales, investigamos las funciones de redes neuronales de la retina. Neuronas de la retina en la red forman parte de numerosos subtipos. Más de 10 subtipos de células bipolares, células ganglionares, células amacrinas y han sido identificados por los estudios morfológicos. Múltiples subtipos de neuronas de la retina se cree que codificar distintas características de señalización visual, tales como el movimiento y el color, y formar múltiples vías neurales. Sin embargo, los papeles funcionales de cada neurona en el procesamiento de señal visual no se entienden completamente. El método de patch clamp es útil para abordar esta cuestión fundamental. Aquí, un protocolo para registrar las respuestas sinápticas luz evocadas en las neuronas de la retina de ratón utilizando grabaciones de patch clamp en condiciones adaptadas a la oscuridad se proporciona. Los ojos del ratón son adaptado a la oscuridad O / N, y el tramo preparativos de la retina son disecados en un cuarto oscuro utilizando iluminación infrarroja y los espectadores. La luz infrarroja noactivar fotorreceptores de ratón y así preserva su capacidad de respuesta luz. Patch clamp se utiliza para registrar las respuestas de luz evocadas en las neuronas de la retina. Se inyecta un tinte fluorescente durante las grabaciones para caracterizar los subtipos morfológicos neuronales. Este procedimiento nos permite determinar las funciones fisiológicas de cada neurona en la retina del ratón.

Una rodaja de preparación representativo se muestra en la Figura 1. La preparación rebanada está en una posición recta, mostrando fotorreceptores a las células ganglionares en una superficie plana, y no hay desprendimiento del papel de filtro. Si se inclina una rebanada, sólo una parte de la preparación está en foco, lo que hace difícil identificar una célula apropiada para la sujeción del parche. Para las grabaciones, es importante seleccionar una buena soma, que generalmente tiene una super...

Watch this Scientific Journal Video about Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques at JoVE.com

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

Grabación Luz evocó-Respuestas postsinápticos en las neuronas en adaptadas oscuro, Preparaciones ratón retina Slice Uso de técnicas Patch Clamp

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

recording-light-evoked-postsynaptic-responses-neurons-dark-adapted

Research

JoVE Journal

Neuroscience

8.4K vistas.  Wayne State University School of Medicine. We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

Video: Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

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

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

Una preparación aislada de retina para registrar la respuesta de la luz a partir de semillas etiquetadas las células ganglionares retinianas

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents.  The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

De células enteras grabaciones de la Luz evocados excitatorios de las corrientes sinápticas en la porción de retina

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

Biología

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4.
A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.
Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.

Simultáneas registros de células de fotorreceptores y las neuronas de segundo orden en una rebanada preparación retina Anfibios

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic network, electrophysiological recordings are frequently used to study connections among individual neurons. We have optimized a flat-mount preparation for patch clamp recording of genetically marked neurons in both GCL (ganglion cell layer) and INL (inner nuclear layer) of mouse retinas. Recording INL neurons in flat-mounts is favored over slices because both vertical and lateral connections are preserved in the former configuration, allowing retinal circuits with large lateral components to be studied. We have used this procedure to compare responses of mirror-partnered neurons in retinas such as the cholinergic starburst amacrine cells (SACs).

This protocol demonstrates how to perform whole-cell patch clamp recording on retinal neurons from a flat-mount preparation.

Patch Clamp grabación de células amacrinas Starburst en un plano de montaje Preparación de Deafferentated retina del ratón

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

Preparación de cortes horizontales de ratón adulto Retina de Estudios electrofisiológicos

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

Preparaciones y protocolos para todo celular parche abrazadera de neuronas Tectal de Xenopus laevis

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

Light-Evoked Postsynaptic Responses in Neurons of Mouse Retinal Slices

Source: Hellmer, C. B., et al. <a href="https://app.jove.com/v/52422">Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques.</a> J. Vis. Exp. (2015).
This video demonstrates the recording of light-evoked post-synaptic responses in a dark-adapted mouse retinal tissue slice preparation. The retinal slice is placed in a recording chamber, and a recording pipette is used to establish electrical continuity with a retinal ganglion cell. Light pulses are applied to generate excitatory post-synaptic potentials in the ganglion cells, which are recorded by the pipette.

Respuestas postsinápticas evocadas por la luz en neuronas de cortes de retina de ratón

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Experimental ...

JoVE Encyclopedia of Experiments

Neurophysiology

Imaging and Optical Techniques

Photostimulation and Whole-Cell Patch Clamp Recordings of Neurons in Mouse Hippocampal Slices

Source: Richevaux, L., et al. <a href="https://app.jove.com/v/59534">In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices.</a> J. Vis. Exp. (2019)
The video demonstrates a protocol to study synaptic interactions and neuronal responses in a transfected mouse hippocampal brain slice. It involves illuminating long-range sensory neurons to trigger action potentials and recording the resulting excitatory post-synaptic potentials in the recorded neuron using the whole-cell patch-clamp technique.

Fotoestimulación y registros de pinza de parche de células enteras de neuronas en cortes de hipocampo de ratón

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Solutions for acute slice ...

Electrophysiology Techniques

Optogenetic Stimulation and Electrophysiological Recording of Synaptic Events in Rat Brain Slices

Source: Kinnavane, L., et al. <a href="https://app.jove.com/v/63077">Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex.</a> J. Vis. Exp. (2022)
This video demonstrates the procedure for studying synaptic efficiency in the lateral entorhinal cortex (LEC) by optogenetically stimulating medial prefrontal cortex (mPFC) axons and recording the responses of pyramidal neurons in the LEC. This method combines patch-clamp recordings with synaptic activation by light stimulation to study synaptic plasticity.

Estimulación optogenética y registro electrofisiológico de eventos sinápticos en cortes de cerebro de rata

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Electrophysiology and ...