Authors would like to thank Peter Mombaerts for the generous gift of OR-GFP mice; Anne Lefranc and the CSGA animal facility for excellent animal care. Funding was provided by CNRS through an ATIP and ATIP Plus grants, by Conseil R&#233;gional de Bourgogne (FABER and PARI grants), by Universit&#233; de Bourgogne (BQR program).

Este protocolo sigue las directrices de cuidado de los animales de la Université de Bourgogne y fue aprobado por el comité de ética de la Universidad de Bourgogne. 1. Los animales <ol><li> Utilice ratones O-IRES-tauGFP ingeniería genética disponibles en el Laboratorio Jackson. Estos ratones fueron desarrollados en el laboratorio del Dr. Peter Mombaerts &#39;con el fin de analizar la orientación axón y el desarrollo del sistema olfativo 19. Por ejemplo, la línea MOR23-IRES-tauGFP, código RS 006643, lleva el nombre de la cepa B6 oficial; 129P2- Olfr16 tm2Mom / MomJ; del mismo modo, la línea SR1-IRES-tauGFP, código RS 6717 lleva el nombre B6 oficial; 129P2- Olfr124 tm1Mom / MomJ. </li><li> En cuanto a la edad de los animales: para un mejor resultado del protocolo, utilizar animales entre 2 y 4 semanas de edad. En este grupo de edad, la disección es más fácil (huesos más blandos, más firme epitelio olfativo) y las perillas dendríticas son bigger comparar a los animales más viejos. </li></ol> 2. Preparación de electrodos y Soluciones <ol><li> Para las pipetas estimulantes: compra prepulled estimular pipetas. De lo contrario, preparar manualmente. <ol><li> El uso de una llama, doble seis 1 mm pipetas de vidrio en alrededor de 1 cm de la punta. Inserte estos seis pipetas más unas rectas 7 º en 1,5 cm tubo de aislamiento fortalecer por un ojal. </li><li> Termocontraíbles el tubo para mantener los barriles unidos juntos. Adjuntar un tubo de aislamiento adicional a la otra extremidad de los barriles. Tire de esta pipeta estimulante con un extractor de múltiples barril. </li><li> Añadir un poco de pegamento líquido blanco alrededor del ojal para fortalecer las puntas dobladas. Deje secar O / N. </li></ol></li><li> Preparar 1 o 2 litros de solución extracelular normal del timbre (en mM: NaCl 124, KCl 3, MgSO4 1,3, CaCl2 2, NaHCO3 26, NaH 2 PO 4 1,25, glucosa 15; pH 7.6 y 305 mOsm). Mantenga a 4 ° Chasta su uso. </li><li> Preparar solución madre intracelular (en mM): KCl 70, KOH 53, ácido metanosulfónico 30, EGTA 5, HEPES 10, sacarosa 70; pH 7,2 (KOH) y 310 mOsm. Mantenga a 4 ° C hasta su uso. Bueno para varias semanas. </li><li> Prepare la solución intracelular de grabación con nistatina extemporáneamente (a última hora) antes de experimento. <ol><li> Pesar 3 mg de nistatina, añadir 50 l de DMSO; vórtice 20 seg después sonicar 2-3 min hasta diluido por completo. </li><li> Añadir 20 l de solución de DMSO-nistatina en 5 ml de solución madre intracelular. Vortex 20 seg, después sonicar 3 min. Mantener esta solución a 4 ° C y proteger de la luz directa, nistatina es sensible a la luz. Esta solución se puede usar durante unas pocas horas. Vuelva a colocar todos los días. </li><li> Una vez que la preparación epitelio olfativo está bajo el microscopio, tomar parte de esta solución en una jeringa de 1 ml con una punta amarilla llama alargada para rellenar los electrodos; protegerlo de la luz directa. Una vez a temperatura ambiente, la solución de nistatinano es estable; sustituir la solución en la jeringa de 1 ml cada hora o mantenerlo en hielo. </li></ol></li><li> Tire de electrodos de grabación con un extractor para obtener cuello largo y pequeña propina (~ 2 m) con una resistencia de 15 a 20 mO con la solución de nistatina interna. </li><li> Prepare la solución odorante en 0,5 M en DMSO bajo campana de humos; alícuota y mantenerse a -20 ° C hasta su uso. Diluir odorante en solución de Ringer hasta la concentración deseada. Llenar la pipeta estimulante. </li></ol> 3. Preparación de epitelio olfativo Nota: los ratones O-IRES-tauGFP expresan la tauGFP bajo el control de la O promotor. En estos ratones, todas las neuronas que expresan el OR de interés están etiquetados con las buenas prácticas agrarias. Este protocolo está adaptado para RUP expresadas en todas las zonas. Sin embargo, disecciones y grabaciones son más fáciles para las RUP expresadas en la zona dorsal. <ol><li> Anestesiar al animal mediante la inyección de una mezcla de ketamina y xilazina (150 mg / kg y 10 mg / kg de DBOpeso y, respectivamente). Decapitación puede realizarse con unas tijeras afiladas para ratones jóvenes o con una guillotina roedor adecuadamente mantenido para los ratones de más edad. <ol><li> Usando anillo tijeras de disección hacen una incisión medial longitudinal a través de la piel dorsal. Quitar la piel tirando de él aparte. Usando las tijeras, corte la mandíbula inferior en la articulación de la mandíbula. Retire los dientes frontales superiores por un corte coronal paralela a los dientes. </li><li> Haga un corte coronal de la cabeza detrás de los ojos y mantener sólo la parte anterior de la cabeza. Sumergir en solución de Ringer helada para la disección en el ámbito. </li></ol></li><li> Diseccionar bajo el ámbito de aplicación: en la parte ventral, hacer un corte longitudinal a lo largo de la mandíbula superior / los dientes. Cortar los huesos dorsales longitudinalmente, siguiendo el lado dorso-lateral de la cavidad nasal. Retire la mayoría de los huesos y el paladar; transferir el tabique y el epitelio que se le atribuye en Ringer oxigenada a TA. </li><li> Para la disección definitiva: Justo antes de comenzar la grabaciónsesión, pelar lejos el epitelio del tabique subyacente con fórceps. Separe el epitelio con pinzas y con dos 4-5 mm cortes de tijera (uso microvannas tijeras) en la parte anterior del tabique, donde la adherencia es más fuerte. <ol><li> Retirar con cuidado el órgano vomeronasal por el corte a lo largo de su conexión dorsal al epitelio septal. Transferir el epitelio a una cámara de grabación con la capa de moco hacia arriba; mantenga plana en la cámara con un arpa. </li></ol></li><li> Instalar cámara bajo un microscopio vertical equipado con óptica de fluorescencia y una cámara sensible. Visualice la preparación en la pantalla del ordenador a gran aumento a través de un 40X objetivo de inmersión en agua (apertura numérica 0,8) y una ampliación 2X o 4X adicional alcanzado por una lente de aumento. Perfundir continuamente con Ringer oxigenada a TA (1-2 ml / min). </li></ol> 4. Grabación Sesión <ol><li> Búsqueda de células fluorescentes: excitar a la preparación a 480nm para EGFP, que emite luz en el rango de 530 a 550 nm; orientar una perilla dendríticas que es visible de forma fiable en la fluorescencia y bajo campo brillante. </li><li> Llenar electrodo con solución intracelular con nistatina; eliminar las burbujas golpeando suavemente sobre el electrodo. </li><li> Inserte electrodo sobre soporte del electrodo, aplique presión positiva en la pipeta; La resistencia debe ser 15-20 mO. </li><li> Traiga electrodo cerca de la célula; una vez que la resistencia alcanza ~ 40 mO, liberar la presión positiva y aplicar una ligera presión negativa para llegar a un gigaseal. </li><li> Una vez que se alcanza el sello, configure el potencial de membrana en alrededor de -75 mV; </li><li> Una vez que se abre la celda, proceder con protocolos de estimulación, tratamientos farmacológicos. Para medir la respuesta a una sola estimulación odorante, ficha 200-500 mseg de la actividad espontánea, para estimular 500 mseg y medir la respuesta de la célula para un máximo de 10 seg 13. Para los tratamientos farmacológicos, perfundir los agentes farmacológicos enla concentración deseada 20. </li></ol> Análisis 5. Datos <ol><li> Analizar las corrientes provocados por la estimulación odorante como sigue: la amplitud máxima, el tiempo de subida (tiempo necesario para alcanzar el 90% de la amplitud máxima, en mseg), la corriente total (área bajo la curva en PAS), el tiempo en el 50% (tiempo entre el inicio y el desplazamiento de la respuesta al 50% de la amplitud máxima, en mseg). <ol><li> El uso de las corrientes de pico de transducción (amplitud máxima alcanzada) a diferentes concentraciones, dibujar y ajustar una curva dosis-respuesta utilizando la ecuación de Hill: I = Imax / (1 ​​+ (K medio / C) n), donde I representa la corriente máxima IMAX la respuesta máxima a concentraciones de saturación, K1 / 2 la concentración a la que se llegó a la mitad de la respuesta máxima, C la concentración de odorante y n el coeficiente de Hill. </li></ol></li><li> Analizar las grabaciones de potencial de membrana en pinza de corriente para la despolarización máxima, la total potencial suscitó (área bajo la curva en mVsec); registro de actividad clavar espontánea durante 30 seg a 1 min grabaciones; registro de la excitabilidad través picos provocados por la inyección de corrientes (10.5 Pa). </li></ol>

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The authors declare that they have no competing financial interest.

La capacidad de este protocolo para controlar correctamente las propiedades de OSN saludables depende en gran medida de la calidad de la preparación. Por lo tanto, los pasos de disección son críticos. En primer lugar, es crítico que prestar atención a la calidad (pH, osmolaridad), la oxigenación y la temperatura del medio de disección (pero no congelado enfriado con hielo). En segundo lugar, la manipulación del epitelio con herramientas de disección debe ser tan limitado como sea posible para evitar daños. Por...

Las neuronas sensoriales olfativas (OSN) representan el primer paso de la percepción olfativa. Situado en el epitelio olfativo que recubre la cavidad nasal en roedores, transforman la información química de los odorantes en los potenciales de acción se ha enviado a través de su axón al cerebro. Para entender mejor los mecanismos de codificación olfativas, es necesario para caracterizar las propiedades de transducción y de membrana de OSN. Hasta hace poco, la mayoría de las técnicas utilizadas para caracterizar las propiedades de OSN mamíferos se llevaron a cabo en disociado OSN 1-4. El proceso de disociación utiliza varios (es decir, enzimas) procesos mecánicos y químicos para liberar a los OSNs de su entorno. Estos procesos provocan un bajo número de células disponibles para las grabaciones. Este número bajo puede ser aún más crítico en el caso de células GFP marcado. La disociación también elimina los locales interacciones célula a célula entre OSN y otras células del epitelio olfativo que pueden mejorar survivcol y la modulación de las propiedades OSN. Con el fin de eludir el procedimiento de disociación, una preparación intacta fue desarrollado 5. Cada OSN expresa un receptor olfativo (OR) seleccionado de una gran familia multigénica 6. Hay ~ 1000 RUP expresadas en el epitelio olfativo principal en el ratón. Debido a la gran cantidad de O en los animales de tipo salvaje, las posibilidades para grabar OSN que expresan el mismo o son muy bajas. Para superar estas limitaciones, los ratones de genes dirigidos están disponibles en el que todos OSN expresan un identificadas o están etiquetados con una proteína fluorescente 7-9. Estos OSN marcadas se usan para hacer el análisis funcional en preparaciones disociadas 7,10,11 con los inconvenientes mencionados anteriormente. Una preparación epitelio intacto 5 de ratones genéticamente etiquetados por lo tanto, evita estos problemas. Permite el seguimiento de la actividad de OSN expresando RUP definidas con precisión en un entorno tan cerca en vivo posible. Además, patch-clamp grabaciones de OSN también permiten un análisis preciso de las propiedades de membrana, vía de transducción de la farmacología, ligando / O interacciones. Todos estos temas casi no se pueden analizar usando grabaciones extracelulares. Se utilizó esta técnica para controlar las respuestas de OSN que expresan los receptores de olor SR1 y MOR23 12,13. La viabilidad de la técnica fue confirmado por otros grupos en MOR23 expresan OSN 14, así como en otras regiones ultraperiféricas que expresan neuronas 15,16. El seguimiento de una población definida de OSN puede conducir al análisis de sus propiedades en muchos contextos diferentes, tales como el desarrollo 14, el envejecimiento 17, plasticidad inducida odorante 18, y el papel de las variaciones en la secuencia del receptor de odorizantes de olor en la codificación de 15. Así, este protocolo proporciona una poderosa herramienta para controlar las propiedades funcionales de OSN definidos a nivel de la membrana.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td colspan="4">heavy equipment</td>
		</tr>
		<tr>
			<td>vibration table with Faraday cage</td>
			<td>TMC</td>
			<td>63-500 SERIES</td>
			<td>required : isolates the recording system from vibrations induced by the environment (movements of experimenter, vibrations of equipment such as fans for cooling computers, etc); can also be purchased with a Faraday cage, or equipped by a custom made Faraday cage; this cage is recommended to avoid electric noise from the environment</td>
		</tr>
		<tr>
			<td colspan="4">optics</td>
		</tr>
		<tr>
			<td>microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td>upright microcope equipped with epifluorescence; fixed or moving stage depending on the user&#39;s preference</td>
		</tr>
		<tr>
			<td>objectives</td>
			<td>Olympus</td>
			<td>LUMPLFL40XW</td>
			<td>at least 2 objectives required: a 4x or 10x for coarse approach to the cell; and a 40x immersion long distance example Olympus LUMPLFL40XW/IR/0,8/WD:3.3 MM</td>
		</tr>
		<tr>
			<td>magnifier</td>
			<td>Olympus</td>
			<td>U-TVCAC</td>
			<td>ABSOLUTELY REQUIRED: placed in the light path between the objective and the camera; allows to magnify the image on the screen in order to reach precisely the knob with the recording electrode</td>
		</tr>
		<tr>
			<td>camera</td>
			<td>Olympus</td>
			<td>DP72</td>
			<td>a good camera is required to see the neurons in fluorescence as well as in bright field; the controlling software is simple and allows to take pictures and do live camera image to monitor the approach of the electrode to the cell. An ultrasensitive camera is not necessary</td>
		</tr>
		<tr>
			<td>filters</td>
			<td>Olympus/Chroma</td>
			<td></td>
			<td>depending on the fluorescent protein used in the mice; example for GFP: excitation : BP460-490: emission: HQ530/50m</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">recording electrodes /system</td>
		</tr>
		<tr>
			<td>amplifier</td>
			<td>HEKA</td>
			<td>EPC10 USB</td>
			<td>monitors the currents flowing through the recording electrode and also controls the puffing by sending a TTL signal to the spritzer; the EPC10 setup is controled by computer</td>
		</tr>
		<tr>
			<td>software</td>
			<td>HEKA</td>
			<td>Patchmaster</td>
			<td>controls the amplifier during the experiment</td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Sutter</td>
			<td>MP225</td>
			<td>precision micromanipulator, allows precise movements down to 1/1Oth of a micrometer; this model is very stable; avoid hydraulic manipulators that may drift</td>
		</tr>
		<tr>
			<td>electrode puller</td>
			<td>sutter</td>
			<td>P97</td>
			<td>with a FT345-B wide trough filament;&#160; to prepare recording pipets of about 2&#181;m diameter with a long tip to reach the cells; the resistance should be 15 to 20Mohm with perforated patch clamp solution</td>
		</tr>
		<tr>
			<td>glass</td>
			<td>sutter</td>
			<td>BF120-69-10</td>
			<td>in our recording conditions, this glass is ideal for recording pipets</td>
		</tr>
		<tr>
			<td>recording chamber</td>
			<td>warner instruments</td>
			<td>RC-26G</td>
			<td>a chamber is needed to set the preparation under the microscope. To maintain the preparation in the center of the chamber, a net/anchor should be used.</td>
		</tr>
		<tr>
			<td colspan="4">stimulation</td>
		</tr>
		<tr>
			<td>glass</td>
			<td>WPI</td>
			<td>TW100F-4</td>
			<td>attached in groups of 7, these pipettes are used to prepare prepulled stimulating pipettes</td>
		</tr>
		<tr>
			<td>multibarrel puller</td>
			<td>MDI</td>
			<td>PMP-107-Z</td>
			<td>by association of pull and twist, this puller allows us to prepare puffing electrodes with 7 barrels</td>
		</tr>
		<tr>
			<td>precision pressure injector&#160;</td>
			<td>Toohey Company</td>
			<td>P/N T25-1-900 Single Channel&#160;&#160;&#160;</td>
			<td>this precision pressure injector&#160; controls the pressure ejected in the multibarrel puller; it is controlled manually or by the amplifier by a 5V&#160; TTLs</td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Narishige</td>
			<td>YOU-1</td>
			<td>a coarse manipulator is enough to bring the puffing electrode close to the recording site</td>
		</tr>
		<tr>
			<td>tubings</td>
			<td>N/A</td>
			<td></td>
			<td>tygon tubing to bring the pressure from the puffer to the puffing pipette</td>
		</tr>
		<tr>
			<td colspan="4">solutions/perfusion/chemicals</td>
		</tr>
		<tr>
			<td>vacuum pump</td>
			<td>gardner denver</td>
			<td>300 series</td>
			<td>a vibrating membrane pump is more quiet and efficient than other types of pumps</td>
		</tr>
		<tr>
			<td>perfusion system</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>gravity perfusion system with polyethlylen tubing to bring in and out the external solution from the recording chamber</td>
		</tr>
		<tr>
			<td>nystatin</td>
			<td>Sigma-Aldrich</td>
			<td>N3503</td>
			<td>mandatory to perpare internal solution for perforated patch clamp</td>
		</tr>
		<tr>
			<td>DIMETHYL SULFOXIDE</td>
			<td>Sigma-Aldrich</td>
			<td>D5879</td>
			<td>used to disolve nystatin for internal solution for perforated patch&#160;</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9625</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P4504</td>
			<td>intracellular/extracellular solution</td>
		</tr>
		<tr>
			<td>Calcium chloride di hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C7902</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic monohydrate (NaH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate (MgSO4 7H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>63140</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S6297</td>
			<td>extracellular solution</td>
		</tr>
		<tr>
			<td>EGTA (Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid)</td>
			<td>Sigma-Aldrich</td>
			<td>E3889</td>
			<td>internal solution</td>
		</tr>
		<tr>
			<td>Potassium hydroxyde</td>
			<td>Sigma-Aldrich</td>
			<td>P1767</td>
			<td>internal solution</td>
		</tr>
		<tr>
			<td>MethylSulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>47,135-6</td>
			<td>intracellular solution</td>
		</tr>
		<tr>
			<td>Hepes-Na</td>
			<td>Sigma-Aldrich</td>
			<td>H7006</td>
			<td>intracellular solution</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td>intracellular solution</td>
		</tr>
	</tbody>
</table>

perforated patch-clamp recording of mouse olfactory sensory neurons in intact neuroepithelium: functional analysis of neurons expressing an identified odorant receptor

Analizando las respuestas fisiológicas de las neuronas sensoriales olfativas (OSN) cuando son estimuladas con ligandos específicos es fundamental para entender la base de comportamientos-olfativas impulsada y su modulación. Estas propiedades de codificación dependen en gran medida de la interacción inicial entre las moléculas de olor y el receptor olfatorio (OR) expresados ​​en las OSN. El espectro de la identidad, la especificidad y el ligando de la O expresado son críticos. La probabilidad de encontrar el ligando de la O expresado en una OSN elegido al azar dentro del epitelio es muy baja. Para hacer frente a este reto, este protocolo utiliza genéticamente ratones etiquetados que expresan la proteína fluorescente GFP bajo el control del promotor de RUP definidos. OSNs se encuentran en un epitelio apretado y organizada que recubre la cavidad nasal, con las células vecinas influir en su maduración y función. Aquí se describe un método para aislar un epitelio olfatorio intacto y grabar a través de patch-clamp grabaciones las propiedades de OSN correoXpressing receptores de olor definidas. El protocolo permite que uno para caracterizar propiedades de la membrana OSN mientras se mantiene la influencia del tejido vecino. Análisis de los resultados de patch-clamp produce una cuantificación precisa de ligando / O interacciones, las vías de transducción y la farmacología, las propiedades de codificación OSN y de su modulación a nivel de la membrana.

El resultado de este protocolo depende de la calidad de la disección. Estos pasos de disección deben ser cortos (menos de 10 a 15 min) y preciso (es decir, para evitar daños del epitelio). La Figura 1 ilustra cómo una preparación ideal se parece a diferentes niveles de aumento. Con una ampliación baja en campo claro los diferentes tipos de células (tales como perillas de OSN, el apoyo a las células) son distinguibles (Figura 1). En el nivel de ampliación más alta, nor...

Watch this Scientific Journal Video about Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Perforado grabación de patch-clamp de ratón neuronas sensoriales olfativas en neuroepitelio Intacto: Análisis funcional de las neuronas que expresan un odorante del receptor Identificado

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

perforated-patch-clamp-recording-mouse-olfactory-sensory-neurons

Research

JoVE Journal

Neuroscience

26.6K vistas.  Université de Bourgogne. Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Video: Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and other social interactions. The electroolfactogram (EOG) recording is an informative, easy to conduct, and reliable method to assay olfactory function at the level of the olfactory epithelium. Since the 1956 description of the EOG by Ottoson in frogs1, the EOG recording has been applied in many vertebrates including salamanders, rabbits, rats, mice, and humans (reviewed by Scott and Scott-Johnson, 2002, ref. 2). The recent advances in genetic modification in mice have rekindled interest in recording the EOG for physiological characterization of olfactory function in knock-out and knock-in mice. EOG recordings have been successfully applied to demonstrate the central role of olfactory signal transduction components3-8, and more recently to characterize the contribution of certain regulatory mechanisms to OSN responses9-12.
Odorant detection occurs at the surface of the olfactory epithelium on the cilia of OSNs, where a signal transduction cascade leads to opening of ion channels, generating a current that flows into the cilia and depolarizes the membrane13. The EOG is the negative potential recorded extracellularly at the surface of the olfactory epithelium upon odorant stimulation, resulting from a summation of the potential changes caused by individual responsive OSNs in the recording field2. Comparison of the amplitude and kinetics of the EOG thus provide valuable information about how genetic modification and other experimental manipulations influence the molecular signaling underlying the OSN response to odor. 
Here we describe an air-phase EOG recording on a preparation of mouse olfactory turbinates. Briefly, after sacrificing the mouse, the olfactory turbinates are exposed by bisecting the head along the midline and removing the septum. The turbinate preparation is then placed in the recording setup, and a recording electrode is placed at the surface of the olfactory epithelium on one of the medial turbinates. A reference electrode is electrically connected to the tissue through a buffer solution. A continuous stream of humidified air is blown over the surface of the epithelium to keep it moist. The vapor of odorant solutions is puffed into the stream of humidified air to stimulate the epithelium. Responses are recorded and digitized for further analysis.

The electroolfactogram (EOG) recording is an informative, easy-to-conduct, and reliable way of assessing olfactory function at the level of the olfactory epithelium. This protocol describes a recording setup, mouse tissue preparation, data collection, and basic data analysis.

Las respuestas de los análisis de las neuronas sensoriales olfativas ratón Utilizando la grabación Electroolfactogram Aire fase

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and ...

Investigación

Neurociencias

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

Flash fotólisis de compuestos enjaulados en los cilios de las neuronas sensoriales olfativas

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

Inducidas Odorante-respuestas registradas de neuronas olfativas del receptor utilizando la técnica de aspiración con una pipeta

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

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 ...

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be performed in an intact brain preparation where the neural circuitry of the CNS remains intact. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with green fluorescent protein for identification in the intact brain. Fish have multiple populations of GnRH neurons, and their functions are dependent on their location in the brain and the GnRH gene that they express1 . We have focused our demonstration on GnRH3 neurons located in the terminal nerves (TN) associated with the olfactory bulbs using the intact brain of transgenic medaka fish (Figure 1B and C). Studies suggest that medaka TN-GnRH3 neurons are neuromodulatory, acting as a transmitter of information from the external environment to the central nervous system; they do not play a direct role in regulating pituitary-gonadal functions, as do the well-known hypothalamic GnRH1 neurons2, 3 .The tonic pattern of spontaneous action potential firing of TN-GnRH3 neurons is an intrinsic property4-6, the frequency of which is modulated by visual cues from conspecifics2 and the neuropeptide kisspeptin 15. In this video, we use a stable line of transgenic medaka in which TN-GnRH3 neurons express a transgene containing the promoter region of Gnrh3 linked to enhanced green fluorescent protein7 to show you how to identify neurons and monitor their electrical activity in the whole brain preparation6.

In this video, we will demonstrate how to record electrical activity from identified single neurons in a whole brain preparation, which preserves complex neural circuits. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with a fluorescent protein for identification in the intact brain preparation.

Registro de la actividad eléctrica de las neuronas identificadas en el cerebro intacto de peces transgénicos

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be ...

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

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

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

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

El aislamiento de las neuronas sensoriales de<em&gt; Aplysia californica</em&gt; Se Patch clamp grabaciones de Corrientes glutamatérgicos

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

This article describes three nervous system preparations using leeches: intracellular recording from neurons in ventral ganglia, culturing neurons from ventral ganglia, and recording from a patch of innervated skin to map sensory fields.

Intracelular de grabación, Sensorial Asignación de campos, y el cultivo de neuronas identificadas en el Leech, Hirudo medicinalis</i

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Todo el montaje de Immunolabeling olfativa del receptor de las neuronas en el<em&gt; Drosophila</em&gt; Antena

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

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

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

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. ...

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 ...