Agradecemos a finales Rudy Limburg para fabricar el perímetro del brazo de cardán, Kimberly Jamison, Mateo McHenry, y Raju Metherate por prestarnos el equipo, y Almut Kelber y Kentaro Arikawa, para el estímulo. Este trabajo fue apoyado por una Fundación Nacional de Ciencia (NSF) Licenciado beca de investigación para KJM y NSF IOS-1.257.627 a ADB

Los autores declaran que no tienen intereses financieros en competencia.

registro intracelular puede ser una técnica difícil de dominar debido a los muchos pasos técnicos involucrados. Para los experimentos con éxito varios puntos importantes deben ser considerados. En primer lugar, es importante tener una mesa adecuadamente aislado vibratoriamente-en el que se realiza el experimento. Muchos investigadores utilizan mesas de aire, que separan completamente de la mesa de la base, dando el aislamiento de vibración superior. Nuestra configuración consiste en una mesa de mármol de espesor ...

Las propiedades eléctricas de las células tales como neuronas se observan mediante la medición de flujo de iones a través de las membranas celulares como un cambio en el voltaje o la corriente. Una variedad de técnicas electrofisiológicas han sido desarrollados para medir eventos bioeléctricos en las células. Las neuronas que se encuentran en los ojos de animales son accesibles y sus circuitos a menudo es menos compleja que en el cerebro, por lo que estas células buenos candidatos para el estudio electrofisiológico. Las aplicaciones más comunes de electrofisiología en el ojo incluyen el electrorretinograma (ERG) 1,2 y el registro intracelular de microelectrodos. ERG consiste en colocar un electrodo en o sobre el ojo de un animal, la aplicación de un estímulo de luz, y midiendo el cambio en la tensión como una suma de las respuestas de todas las células cercanas 3-6. Si uno está interesado específicamente en la caracterización de las sensibilidades espectrales de las células fotorreceptoras individuales, a menudo múltiples tipos de células responden de forma simultánea en diferentes puntos fuertes a un estímulo determinado; Por lo tanto,puede ser difícil de determinar las sensibilidades de los tipos celulares específicos a partir de datos ERG especialmente si hay varios tipos diferentes de células fotorreceptoras espectralmente similares en el ojo. Una posible solución es crear transgénico con el gen de Drosophila fotorreceptor (opsina) de interés se expresa en las células mayoría R1-6 en el ojo y después realizar ERGs 7. Las posibles desventajas de este método son que no a baja expresión de la proteína fotorreceptor 8, y el período de tiempo largo para la generación y detección de animales transgénicos. Para los ojos con un menor número de tipos de fotorreceptores espectralmente distintas, la adaptación del ojo con filtros de color puede ayudar con la reducción de la contribución de algunos tipos de células a la ERG, permitiendo de ese modo la estimación de máximos sensibilidad espectral 9. registro intracelular es otra técnica en la que una multa electrodo empala una célula y se aplica un estímulo. Los registros de electrodos sólo eso IndivLa respuesta de las células idual de manera que a grabar y analizar varias celdas individuales puede producir sensibilidades específicas de los diferentes tipos de células fisiológicamente 10-14. Aunque nuestro protocolo se centra en el análisis de sensibilidad espectral, los principios básicos de intracelular de grabación con electrodos afilados son modificables para otras aplicaciones. El uso de una preparación diferente de una muestra, por ejemplo, y el uso de electrodos de cuarzo afilados, se puede grabar de más profundo en el lóbulo óptico o de otras regiones en el cerebro, dependiendo de la pregunta que se hace. Por ejemplo, los tiempos de respuesta de las células fotorreceptoras individuales 15, la actividad de células en la óptica lóbulos 16 (lámina, médula o lobula 17), cerebro 18 o otros ganglios 19 también se pueden grabar con técnicas similares, o estímulos de color podría sustituirse con polarización 20 -22 o moción estímulos 23,24. Fototransducción, el proceso por el cual la luzla energía es absorbida y convertida en una señal electroquímica, es un antiguo rasgo común a casi todos los presentes el día 25 filos animales. El pigmento visual que se encuentra en las células fotorreceptoras y responsable de iniciar la fototransducción visual es la rodopsina. Rhodopsins en todos los animales se componen de una proteína opsina, un miembro de la familia 7 transmembrana G receptor acoplado a la proteína, y un cromóforo asociado que se deriva de la retina o una molécula similar 26,27. Opsin secuencia de aminoácidos y la estructura de cromóforo afectar la absorbancia de la rodopsina a diferentes longitudes de onda de luz. Cuando un fotón es absorbida por el cromóforo de la rodopsina se activa, iniciando una cascada de proteína G en la célula que en última instancia conduce a la apertura de los canales iónicos de membrana 28. A diferencia de la mayoría de las neuronas, las células fotorreceptoras se someten a posibles cambios graduales que se pueden medir como un cambio relativo en la amplitud de la respuesta con el cambio de estímulo luminoso. Normalmente, un dadotipo de fotorreceptor expresa sólo un gen de la opsina (aunque existen excepciones 8,10,29-31). la visión del color sofisticado, de la que se encuentra en muchos vertebrados y artrópodos, se consigue con un ojo complejo de cientos o miles de células fotorreceptoras cada que expresan uno o más tipos de vez en cuando rodopsina. La información visual se captura mediante la comparación de las respuestas sobre el mosaico fotorreceptor través de la señalización neural aguas abajo complejo en el ojo y el cerebro, dando como resultado la percepción de una imagen completa con el color y el movimiento. Después de medir las respuestas primas de una célula fotorreceptora a diferentes longitudes de onda de luz a través de registro intracelular, es posible calcular su sensibilidad espectral. Este cálculo se basa en el principio de univariante, que establece que la respuesta de una de las células fotorreceptoras depende del número de fotones que absorbe, pero no en las propiedades particulares de los fotones que absorbe 32. Cualquier fotón que es absorbed por rodopsina inducirá el mismo tipo de respuesta. En la práctica, esto significa que la amplitud de la respuesta en bruto de una célula aumentará ya sea debido a un aumento en la intensidad de la luz (más fotones para absorber), o a un cambio en la longitud de onda hacia su sensibilidad pico (mayor probabilidad de rodopsina de absorción de esa longitud de onda). Hacemos uso de este principio en relacionar las respuestas celulares a una intensidad conocida y la misma longitud de onda de respuestas a diferentes longitudes de onda y la misma intensidad pero la sensibilidad relativa desconocida. Los tipos de células a menudo se identifican por la longitud de onda a la que sus picos de sensibilidad. Aquí se muestra un método para el registro y análisis de sensibilidad espectral de los fotorreceptores en el ojo de una mariposa intracelular, con un enfoque en lo que este método sea más accesible para la comunidad científica en general. Aunque registro intracelular sigue siendo común en la literatura, particularmente con respecto a la visión de color en los insectos, hemos encontrado that descripciones de materiales y métodos son por lo general demasiado breve para permitir la reproducción de la técnica. Presentamos este método en el formato de vídeo con el fin de permitir su replicación más fácil. También describimos la técnica utilizando un equipo fácil de obtener y asequible. Nos dirigimos advertencias comunes que a menudo no son reportados, que ralentizan la investigación la hora de optimizar una nueva y compleja técnica.

<table><tbody><tr><td>Butterfly pupae</td><td></td><td></td><td>Several local species available, need USDA permits for shipping. Carolina Bio Supply has several insect species that may be ordered within the U.S. without the need for additional permits</td></tr><tr><td>Large plastic cylinder</td><td></td><td></td><td>Any chamber that remains humidified will work</td></tr><tr><td>Insect pins, size 2</td><td>BioQuip</td><td>1208B2</td><td></td></tr><tr><td>100% Desert Mesquite Honey</td><td>Trader Joe&#039;s</td><td></td><td>Any honey or sucrose solution will work</td></tr><tr><td>Xenon Arc Lamp</td><td>Oriel Instruments</td><td>66003</td><td>Oriel is now a part of Newport Corporation</td></tr><tr><td>Universal Power Supply</td><td>Oriel Instruments</td><td>68805</td><td>Oriel is now a part of Newport Corporation</td></tr><tr><td>Optical Track</td><td>Oriel Instruments</td><td>11190</td><td>Oriel is now a part of Newport Corporation</td></tr><tr><td>Rail Carrier, Large (2x)</td><td>Oriel Instruments</td><td>11641</td><td>Oriel is now a part of Newport Corporation</td></tr><tr><td>Rail Carrier, Small (4x)</td><td>Oriel Instruments</td><td>11647</td><td>Oriel is now a part of Newport Corporation</td></tr><tr><td>Thread Adaptor, 8-32 Male to 1/4-20 Male, pack of 10</td><td>Newport Corporation</td><td>TA-8Q20-10</td><td></td></tr><tr><td>Optical Mounting Post, 1.0 in., 0.5 in. Dia. Stainless, 8-32 &amp;amp; 1/4-20 (5x)</td><td>Newport Corporation</td><td>SP-1</td><td></td></tr><tr><td>No Slip Optical Post Holder, 2 in., 0.5 in. Diameter Posts, 1/4-20 (5x)</td><td>Newport Corporation</td><td>VPH-2</td><td></td></tr><tr><td>Fixed lens mount, 50.8 mm</td><td>Newport Corporation</td><td>LH-2</td><td></td></tr><tr><td>Fixed lens mount, 25.4 mm</td><td>Newport Corporation</td><td>LH-1</td><td></td></tr><tr><td>Condenser lens assembly</td><td>Newport Corporation</td><td>60006</td><td></td></tr><tr><td>Convex silica lens, 50.8 mm</td><td>Newport Corporation</td><td>SPX055</td><td></td></tr><tr><td>Six Position Filter Wheel, x2</td><td>Newport Corporation</td><td>FW1X6</td><td></td></tr><tr><td>Filter Wheel Mount Hub</td><td>Newport Corporation</td><td>FWM</td><td></td></tr><tr><td>Concave silica lens, 25.4 mm</td><td>Newport Corporation</td><td>SPC034</td><td></td></tr><tr><td>Collimator holder</td><td>Newport Corporation</td><td>77612</td><td></td></tr><tr><td>Collimating beam probe</td><td>Newport Corporation</td><td>77644</td><td></td></tr><tr><td>Ferrule Converter, SMA Termination to 11 mm Standard Ferrule</td><td>Newport Corporation</td><td>77670</td><td>This adapter allows the fiber optic to fit into the collimator holder&amp;nbsp;</td></tr><tr><td>600 &amp;mu;m diameter UV-vis fiber obtic cable</td><td>Oriel Instruments</td><td>78367</td><td>Oriel is now a part of Newport Corporation</td></tr><tr><td>Shutter with drive unit</td><td>Uniblitz</td><td>100-2B</td><td></td></tr><tr><td>UV Fused Silica Metallic ND Filter, 0.1 OD</td><td>Newport</td><td>FRQ-ND01</td><td></td></tr><tr><td>UV Fused Silica Metallic ND Filter, 0.3 OD</td><td>Newport</td><td>FRQ-ND03</td><td></td></tr><tr><td>UV Fused Silica Metallic ND Filter, 0.5 OD</td><td>Newport</td><td>FRQ-ND05</td><td></td></tr><tr><td>UV Fused Silica Metallic ND Filter, 1.0 OD</td><td>Newport</td><td>FRQ-ND10</td><td></td></tr><tr><td>UV Fused Silica Metallic ND Filter, 2.0 OD</td><td>Newport</td><td>FRQ-ND30</td><td></td></tr><tr><td>UV Fused Silica Metallic ND Filter, 3.0 OD</td><td>Newport</td><td>FRQ-ND50</td><td></td></tr><tr><td>LS-1-Cal lamp</td><td>Ocean Optics</td><td>LS-1-Cal</td><td></td></tr><tr><td>Spectrometer</td><td>Ocean Optics</td><td>USB-2000</td><td></td></tr><tr><td>SpectraSuite Software</td><td>Ocean Optics</td><td></td><td></td></tr><tr><td>Interference bandpass filter, 300 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67749</td><td></td></tr><tr><td>Interference bandpass filter, 310 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67752</td><td></td></tr><tr><td>Interference bandpass filter, 320 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67754</td><td></td></tr><tr><td>Interference bandpass filter, 330 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67756</td><td></td></tr><tr><td>Interference bandpass filter, 340 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65614</td><td></td></tr><tr><td>Interference bandpass filter, 350 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67757</td><td></td></tr><tr><td>Interference bandpass filter, 360 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67760</td><td></td></tr><tr><td>Interference bandpass filter, 370 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67761</td><td></td></tr><tr><td>Interference bandpass filter, 380 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67762</td><td></td></tr><tr><td>Interference bandpass filter, 390 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67763</td><td></td></tr><tr><td>Interference bandpass filter, 400 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65732</td><td></td></tr><tr><td>Interference bandpass filter, 410 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65619</td><td></td></tr><tr><td>Interference bandpass filter, 420 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65621</td><td></td></tr><tr><td>Interference bandpass filter, 430 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65622</td><td></td></tr><tr><td>Interference bandpass filter, 440 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67764</td><td></td></tr><tr><td>Interference bandpass filter, 450 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65625</td><td></td></tr><tr><td>Interference bandpass filter, 460 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67765</td><td></td></tr><tr><td>Interference bandpass filter, 470 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65629</td><td></td></tr><tr><td>Interference bandpass filter, 480 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65630</td><td></td></tr><tr><td>Interference bandpass filter, 492 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65633</td><td></td></tr><tr><td>Interference bandpass filter, 500 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65634</td><td></td></tr><tr><td>Interference bandpass filter, 510 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65637</td><td></td></tr><tr><td>Interference bandpass filter, 520 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65639</td><td></td></tr><tr><td>Interference bandpass filter, 532 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65640</td><td></td></tr><tr><td>Interference bandpass filter, 540 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65642</td><td></td></tr><tr><td>Interference bandpass filter, 550 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65644</td><td></td></tr><tr><td>Interference bandpass filter, 560 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67766</td><td></td></tr><tr><td>Interference bandpass filter, 570 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67767</td><td></td></tr><tr><td>Interference bandpass filter, 580 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65646</td><td></td></tr><tr><td>Interference bandpass filter, 589 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65647</td><td></td></tr><tr><td>Interference bandpass filter, 600 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65648</td><td></td></tr><tr><td>Interference bandpass filter, 610 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65649</td><td></td></tr><tr><td>Interference bandpass filter, 620 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65650</td><td></td></tr><tr><td>Interference bandpass filter, 632 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65651</td><td></td></tr><tr><td>Interference bandpass filter, 640 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65653</td><td></td></tr><tr><td>Interference bandpass filter, 650 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65655</td><td></td></tr><tr><td>Interference bandpass filter, 660 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67769</td><td></td></tr><tr><td>Interference bandpass filter, 671 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65657</td><td></td></tr><tr><td>Interference bandpass filter, 680 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67770</td><td></td></tr><tr><td>Interference bandpass filter, 690 nm&amp;nbsp;</td><td>Edmund Optics</td><td>65659</td><td></td></tr><tr><td>Interference bandpass filter, 700 nm&amp;nbsp;</td><td>Edmund Optics</td><td>67771</td><td></td></tr><tr><td>Faraday cage</td><td></td><td></td><td>Any metal structure will work that can be grounded and that fits the experimental setup.</td></tr><tr><td>Stereomicroscope, 6X, 12X, 25X, 50X magnification</td><td>Wild Heerbrugg</td><td>Wild M5</td><td>Any Stereomicroscope will do</td></tr><tr><td>Microscope stand with swinging arm and heavy base</td><td>McBain Instruments</td><td></td><td>Any heavy base with arm will do</td></tr><tr><td>Cardan arm</td><td></td><td></td><td>Custom built, See &lt;strong&gt;Figure 4&lt;/strong&gt;</td></tr><tr><td>Fiber-lite high intensity illuminator</td><td>Dolan-Jenner</td><td>MI-150</td><td>For lighting specimen</td></tr><tr><td>Fiber-lite goose-neck light guide</td><td>Dolan-Jenner</td><td>EEG 2823</td><td>Any goose-neck light guide will do</td></tr><tr><td>Marble table</td><td></td><td></td><td></td></tr><tr><td>Raised wooden table</td><td></td><td></td><td>Hole should be cut through this table so that the sandbox can rest on the marble table underneath</td></tr><tr><td>Wooden box filled with sand</td><td></td><td></td><td>custom built, any box with sand</td></tr><tr><td>Manipulator</td><td>Carl Zeiss - Jena</td><td></td><td></td></tr><tr><td>Electrode holder</td><td></td><td></td><td></td></tr><tr><td>Specimen stage</td><td></td><td></td><td></td></tr><tr><td>Alligator clip wires for grounding</td><td></td><td></td><td></td></tr><tr><td>Insulated copper wire</td><td></td><td></td><td></td></tr><tr><td>Silver wire, 0.125 mm diameter</td><td>World Precision Instruments</td><td>AGW0510</td><td></td></tr><tr><td>BNC cables</td><td></td><td></td><td></td></tr><tr><td>Preamplifier with headstage</td><td>Dagan Corporation</td><td>IX2-700</td><td></td></tr><tr><td>Humbug Noise reducer</td><td>Quest Scientific</td><td>Humbug</td><td></td></tr><tr><td>Oscilloscope, 30 MHz, 2 CH, Dual Trace, Alt-triggering, without probe</td><td>EZ Digital</td><td>os-5030</td><td></td></tr><tr><td>BNC T-adapter</td><td></td><td></td><td></td></tr><tr><td>Powerlab hardware 2/20</td><td>ADI instruments</td><td>ML820</td><td></td></tr><tr><td>Labchart software</td><td>ADI instruments</td><td>Chart 5</td><td></td></tr><tr><td>10 MHz Pulse Generator</td><td>BK Precision</td><td>4030</td><td></td></tr><tr><td>Glass pipette puller</td><td>Sutter Instruments</td><td>P-87</td><td></td></tr><tr><td>Borosillicate glass capillaries with filament</td><td>World Precision Instruments</td><td>1B120F-4</td><td></td></tr><tr><td>Potassium chloride, 3 M</td><td></td><td></td><td></td></tr><tr><td>Slotted plastic tube</td><td></td><td></td><td></td></tr><tr><td>Low melting temperature wax</td><td></td><td></td><td></td></tr><tr><td>Soldering Iron</td><td>Weller</td><td></td><td></td></tr><tr><td>Platform with ball-and-socket magnetic base</td><td>Hama photo and video</td><td></td><td></td></tr><tr><td>Double edge carbon steel, breakable razor blade</td><td>Electron Microscopy Sciences</td><td>72004</td><td></td></tr><tr><td>Vaseline</td><td></td><td></td><td></td></tr><tr><td>Microsoft Excel</td><td>Microsoft</td><td></td><td></td></tr></tbody></table>

determination of photoreceptor cell spectral sensitivity in an insect model from in vivo intracellular recordings

registro intracelular es una técnica poderosa utilizada para determinar cómo una sola célula puede responder a un estímulo dado. En la investigación de la visión, el registro intracelular ha sido históricamente una técnica común utilizada para estudiar la sensibilidad de las células fotorreceptoras individuales a diferentes estímulos luminosos que se sigue utilizando hoy en día. Sin embargo, sigue habiendo una escasez de metodología detallada en la literatura para los investigadores que desean replicar experimentos registro intracelular en el ojo. Aquí se presentan los insectos como modelo para examinar la fisiología del ojo de manera más general. células fotorreceptoras de insectos se encuentran cerca de la superficie del ojo y por lo tanto son fáciles de alcanzar, y muchos de los mecanismos implicados en la visión se conservan a través de filos animal. Se describe el procedimiento básico para la grabación intracelular in vivo de las células fotorreceptoras en el ojo de una mariposa, con el objetivo de hacer que esta técnica sea más accesible a los investigadores con poca experiencia previa en el correolectrophysiology. Introducimos el equipo básico necesario, cómo preparar una mariposa en vivo para la grabación, la forma de insertar un microelectrodo de vidrio en una sola celda, y, finalmente, el procedimiento de grabación en sí. También se explica el análisis básico de datos de respuesta primas para la determinación de la sensibilidad espectral de distintos tipos de células. Aunque nuestro protocolo se centra en la determinación de la sensibilidad espectral, otros estímulos (por ejemplo, luz polarizada) y variaciones del método son aplicables a esta configuración.

Para muchos elementos de la configuración de la grabación, una descripción escrita no proporciona suficiente detalle. La Figura 1 es un esquema de los componentes involucrados en el sistema de grabación completa. En la Figura 2, los espectros se trazan para la luz blanca y cada filtro de interferencia para dar una idea de por qué se necesita un factor de corrección y lo que se necesita para calcular esta corrección. La figura 3 muestra fotos y un diagram...

Watch this Scientific Journal Video about Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings at JoVE.com

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

La técnica electrofisiológica de registro intracelular se demuestra y se utiliza para determinar las sensibilidades espectrales de las células fotorreceptoras individuales en el ojo compuesto de una mariposa.

Determinación de los fotorreceptores de la célula de sensibilidad espectral en un modelo de insectos<em&gt; En Vivo</em&gt; registros intracelulares

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular ...

determination-photoreceptor-cell-spectral-sensitivity-an-insect-model

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

11.3K vistas.  University of California, Irvine.  La técnica electrofisiológica de registro intracelular se demuestra y se utiliza para determinar las sensibilidades espectrales de las células fotorreceptoras individuales en el ojo compuesto de una mariposa. 

Video: Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

La disección y la Cultura de las neuronas comisurales de médula espinal embrionaria

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Investigación

Neurociencias

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

Una sola unidad En vivo Grabaciones de la quiasma óptico de la rata

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

Caracterización electrofisiológica de las buenas prácticas agrarias que expresan las poblaciones de células en la retina intacta

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Marcaje fluorescente retrógrada Permite dirigida extracelular sola unidad de grabación de neuronas identificadas<em&gt; En vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Método electrofisiológico para registrar las respuestas de voltaje intracelular<em&gt; Drosophila</em&gt; Los fotorreceptores y interneuronas a la luz Estímulos<em&gt; En Vivo</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

Método electrofisiológico para grabaciones de abrazadera de voltaje de células enteras<em&gt; Drosophila</em&gt; Fotorreceptores

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Desensitization and Recovery of Crayfish Photoreceptors Upon Delivery of a Light Stimulus

A method to study desensitization and recovery of crayfish photoreceptors is presented. We performed intracellular electrical recordings of photoreceptor cells in isolated eyestalks using the discontinuous single electrode-switched voltage-clamp configuration. First, with a razor blade we made an opening in the dorsal cornea to get access to the retina. Thereafter, we inserted a glass electrode through the opening, and penetrated a cell as reported by the recording of a negative potential. Membrane potential was clamped at the photoreceptor's resting potential and a light-pulse was applied to activate currents. Finally, the two light-flash protocol was employed to measure current desensitization and recovery. The first light-flash triggers, after a lag period, the transduction ionic current, which after reaching a peak amplitude decays towards a desensitized state; the second flash, applied at varying time intervals, assesses the state of the light-activated conductance. To characterize the light-elicited current, three parameters were measured: 1) latency (the time elapsed between light flash delivery and the moment in which current achieves 10% of its maximum value); 2) peak current; and 3) desensitization time constant (exponential time constant of the current decay phase). All parameters are affected by the first pulse.
To quantify recovery from desensitization, the ratio p2/p1 was employed versus time between pulses. p1 is the peak current evoked by the first light-pulse, and p2 is the peak current evoked by the second pulse. These data were fitted to a sum of exponential functions. Finally, these measurements were carried out as function of circadian time.

A protocol for the study of desensitization and sensitivity recovery of crayfish photoreceptors as a function of circadian time is presented.

Desensibilización y recuperación de fotorreceptores de cangrejo de río tras la entrega de un estímulo ligero

A method to study desensitization and recovery of crayfish photoreceptors is presented. We performed intracellular electrical recordings of photoreceptor ...

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is initiated by photon absorption-inducing isomerization of the chromophore, promoting conformational changes of the opsin and resulting in a second dark-stable photopigment state (metarhodopsin, M). Investigation of this bi-stable photopigment using random mutagenesis requires simple and robust methods for screening mutant flies. Therefore, several methods for measuring reductions in functional photopigment levels have been designed. One such method exploits the charge displacements within the photopigment following photon absorption and the huge amounts of photopigment molecules expressed in the photoreceptors. This electrical signal, named the early receptor potential (or early receptor current), is measured by a variety of electrophysiological methods (e.g., electroretinogram and whole-cell recordings) and is linearly proportional to functional photopigment levels. The advantages of this method are the high signal-to-noise ratio, direct linear measurement of photopigment levels, and independence of phototransduction mechanisms downstream to rhodopsin or metarhodopsin activation. An additional electrophysiological method called prolonged depolarizing afterpotential (PDA) exploits the bi-stability of Drosophila photopigment and the absorption-spectral differences of fly R and M pigment states. The PDA is induced by intense blue light, converting saturating amounts of rhodopsin to metarhodopsin, resulting in the failure of light-response termination for an extended time in darkness, but it can be terminated by metarhodopsin to rhodopsin conversion using intense orange light. Since the PDA is a robust signal that requires massive photopigment conversion, even small defects in the biogenesis of the photopigment lead to readily detected abnormal PDA. Indeed, defective PDA mutants led to the identification of novel signaling proteins important for phototransduction.

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

We present a protocol to electrophysiologically characterize bi-stable photopigments: (i) exploiting the charge displacements within the photopigment molecules following photon-absorption and their huge amount in the photoreceptors, and (ii) exploiting the absorption-spectra differences of rhodopsin and metarhodopsin photopigment states. These protocols are useful to screen for mutations affecting bi-stable photopigment systems.&#160;

Métodos electrofisiológicos para medir los niveles de fotopigmento en fotorreceptores de Drosophila

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is ...

In Vivo Intracellular Recording of Photoreceptor Cell Spectral Sensitivity in a Butterfly

Source: McCulloch, K. J., et al.. <a href="https://appjove.unicartagenaproxy.elogim.com/v/53829">Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings.</a> J. Vis. Exp. (2016)
This video demonstrates the procedure for in vivo intracellular recording of photoreceptor cells in the eye of a butterfly and determining the spectral sensitivity of individual cell types. This technique enables the study of sensitivities of individual photoreceptor cells to different light stimuli.

Registro intracelular in vivo de la sensibilidad espectral de las células fotorreceptoras en una mariposa

1. Specimen Prep and Recording Procedure

	Prepare the Specimen
	
		Affix an individual butterfly inside a small plastic tube with hot wax so the head is ...

JoVE Encyclopedia of Experiments

Neurophysiology

Imaging and Optical Techniques

Electrophysiological Recording of Voltage Responses of Drosophila Retinal Photoreceptors to Light Stimuli

Source: Juusola, M., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/54142">Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo.</a> J. Vis. Exp. (2016).
This video outlines the protocol for electrophysiological recording of photoreceptor activity in the eye of an immobilized Drosophila, enabling precise measurement of voltage changes in response to light stimuli.

Registro electrofisiológico de las respuestas de voltaje de los fotorreceptores de la retina de Drosophila a los estímulos luminosos

1. Recording from R1-R6 Photoreceptors 

	Always be grounded when operating the microelectrode amplifier (for example by touching the metal surface of the ...

Determinación de los fotorreceptores de la célula de sensibilidad espectral en un modelo de insectos

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