Los autores desean agradecer a la Dra. Candace Hsieh para sugerencias y ayuda con técnicas de imagen y la Dra. Doris Wu por su experiencia en la anatomía del oído interno pollito durante la embriogénesis temprana. Este trabajo fue apoyado por NSF IOS-0642346, NIH T32-DC010775, NIH T32-GM008620, NIH R01-DC010796 y DOE GAANN P200A120165.

1. Preparar las herramientas de disección tras y Reactivos <ul><li> Líquido cefalorraquídeo artificial (LCRa; 130 mM NaCl, 3 mM de KCl, 1,2 mM KH 2 PO 4, 20 mM NaHCO 3, 3 mM de HEPES, 10 mM de glucosa, 2 mM CaCl 2, 1,3 mM MgSO 4) continuamente infundido con 95% de O 2/5% CO 2 a temperatura ambiente. Para la infusión, llene hasta 2/3 a 500 ml de boca ancha frasco Nalgene con un agujero perforado en la tapa. Tanque se fijan por medio de un tubo de vidrio vástago burbujeador, que penetra en el LCRa a través del agujero en la tapa del frasco. Ajustar la presión para obtener una corriente constante de burbujas que llegan a aproximadamente 1/3 del líquido, pero no son lo suficientemente fuerte como para causar salpicaduras sobre el borde. Sumergir 5 viales de vidrio ml (hasta 6) en el frasco Nalgene para separar las muestras individuales el uno del otro y de la turbulencia de flujo de gas. ACSF debe ser oxigenada durante al menos 20 min antes de cualquier tejido incubación. </li><li> Silicona pequeña placa de disección (35 mm x 10mm recubierto de Petri utilizando Sylgard élastomère Kit) con dos pernos de disección colocados en poliestireno hexagonal pequeño (47 mm inferior) pesan barco. </li><li> Dos pinzas de punta fina, una curva de punta de pinzas, un vaso de precipitados de 50 ml, y un contenedor para los residuos de tejido. </li><li> Sacó micropipeta de vidrio (1,2 OD / ID 0,9) rota a aproximadamente 20-50 micras abertura en la punta y se llena con rodamina dextrano amina (RDA, MW = 3.000, Invitrogen) en una solución de 6,25% con 0,4% Triton X-100 en PBS . Conecte mediante un tubo fino para picospritzer y estabilizar a micromanipulador. </li></ul> 2. Micro-disección de revelar Papilla basilar en 4,5 E8 <ol><li> Con pinzas curvas, corte E8 embrión justo debajo del tronco cerebral, colocando la cabeza directamente sobre la placa de disección recubierto de silicona dentro peso barco. </li><li> Con los pernos de disección, la posición de la cabeza con una vista ventral por lo que el oído del lado de interés es ligeramente visible, la cabeza ligeramente inclinada hacia dorsal y apuntando 10-30 grados fuera de center (Figura 2A). Inmediatamente fijar el cabezal con pernos. Si se desea una localización bilateral, el centro de la cabeza a los 0 grados, o una vista ventral completo por lo que ambas partes pueden tener acceso. Del mismo modo, para una inyección del lado izquierdo, asegurar la cabeza de -10 a -30 grados desde el centro de manera que el lado izquierdo es accesible. </li><li> Usando el vaso de precipitados de 50 ml, transferir 30 ml de LCRa oxigenada en el plato, sumergiendo completamente el tejido. </li><li> Pellizque suavemente hacia abajo y la cáscara de la mandíbula inferior con unas pinzas de punta fina, luego retire el párpado inferior del lado de los intereses. </li><li> Retire el tejido vascular suprayacente, incluyendo cualquier paladar restante y garganta. Estos tejidos blandos pueden despegarse revelar la superficie lisa de desarrollar condrocráneo con las arterias vertebrales distintivas en su superficie. En este punto, la característica masa otoconial blanco debe ser visible. </li><li> Diseccionar cuidadosamente la piel alrededor del meato externo, el cartílago de la articulación de la mandíbula, y el oído medio, mientras que en el dejandoner oído intacto. Utilice las pinzas para cortar suavemente lejos estas estructuras cartilaginosas. </li><li> Quite las arterias vertebrales y la acumulación de sangre mediante un movimiento de barrido suave con las puntas de las pinzas finas. La sangre puede oscurecer vista y la coagulación puede tapar la abertura de la pipeta de inyección. </li><li> El conducto coclear con epitelio adyacente sensorial (papila basilar) y el ganglio acústico encuentra directamente debajo de la condrocráneo 6 y se puede visualizar como una forma alargada en forma de dedo saliente que se extiende desde el oído interno ventralmente, con la punta más distal (ápice) cerca de la anterior línea media 5. Puede haber una pequeña acumulación de sangre (roja) de la disección y / o calcificaciones otolíticos (blanco) en el vértice de la cóclea del conducto (Figura 2B) que puede ayudar en la visualización. En el vértice es la mácula lagenar, un parche sensorial cree que la función vestibular 7,8. </li></ol> 3. Etiquetado selectiva de CN VIII fibras auditivas <ol><li&gt; Coloque la pipeta de inyección cerca de la superficie del embrión en la región de la papila del conducto coclear y basilar. </li><li> Baje lentamente la micropipeta para perforar primero el cráneo. Esta región craneal es más delgada que las zonas circundantes y requiere menos fuerza para penetrar. La micropipeta ahora debe estar en el interior del conducto coclear. Opcional: una pequeña inyección de tinte trazado se puede hacer para ayudar en la visualización de la papila basilar. </li><li> Sin tener que reposicionar la micropipeta, seguir bajando hasta que sólo rompe la frontera profunda del conducto coclear (Figura 1). Realizar una inyección y repetir aquí en varias regiones a lo largo de la longitud de la papila basilar (figuras 2C, 2D), con exclusión de la punta más proximal donde la vestibular lagena se encuentra. Con la picospritzer establecido entre 10-30 psi, varias inyecciones se hacen. El volumen de fluido que se inyecta medio de contraste varía, y depende de la extensión de etiquetado deseado. Cada inyección debe dar lugar a un tinte fluorescente-lapunto Beled de aproximadamente 200 a 400 m de diámetro (Figura 2D). </li><li> Una vez que la inyección se ha completado, el uso de fórceps para seccionar la muestra por encima del tronco cerebral y el uso de una pipeta de transferencia para colocar la porción caudal en un pequeño frasco dentro del recipiente de LCRa continuamente se perfundieron con 95% O 2/5% CO 2. </li><li> Para cada embrión, 20-30 min para permitir la transferencia de colorantes, en función de la distancia del centro de trazado deseado. </li><li> Quitar el tejido y prepararse para el seccionamiento histológico. En el ejemplo que se muestra aquí, el tejido se sumerge en 4% de paraformaldehído (en 1X PBS) durante la noche sacarosa,% 30 (en 1X PBS) durante la noche. </li></ol> 4. Contratinción y Análisis <ol><li> Contratinción es útil para ayudar a orientar al observador a la ubicación anatómica, así como la identificación de regiones particulares de interés. Una tinción de contraste eficaz en esta preparación es immunolabeling neurofilamentos (figuras 1, 3B, 3E), que fuertemente lAbels axones y permite la demarcación de las vías axonales en el SNC. </li><li> Análisis debe ser realizado en toda la región etiquetada, como las proyecciones pueden extenderse varios cientos de micras o más a lo largo del eje rostrocaudal. Algunos ejemplos de los análisis básicos son: las tasas de error de orientación, el tiempo y / o cambios en los patrones de divergencia de proyección. Axones marcados RDA-se visualizan en el sistema nervioso periférico y central con alta resolución (Figuras 3A, 3D). </li></ol>

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No hay conflictos de intereses se declaran.

Estudios sobre el desarrollo temprano del nervio VIII se han limitado, en parte debido a la dificultad en la identificación de los axones embrionarias derivadas de los ganglios de distinta múltiple. Varios estudios han explorado las señales moleculares que guían auditivo y vestibular célula sensorial y destinos de células ganglionares durante el desarrollo temprano, 5,11,12 pero los procesos que regulan la inervación central tienen todavía por determinar. Informes de acústicos proyecciones de célula...

<table border="1"><tbody><tr><td> Nombre del reactivo / material </td><td> Empresa </td><td> Número de Catálogo </td><td> Comentarios </td></tr><tr><td> Poliestireno Pesar Dish </td><td> Fisher Scientific </td><td> 02-202-101 </td><td></td></tr><tr><td> Placa de Petri de 35 x 10 mm </td><td> Fisher Scientific </td><td> 50820644 </td><td> Se utiliza para hacer plato de silicona disección </td></tr><tr><td> Kit de elastómero de silicona Sylgard </td><td> World Precision Instruments </td><td> SYLG184 </td><td> Escudo Petri plato para hacer la disección </td></tr><tr><td> Clavijas Dissection </td><td> Vario </td><td></td><td> Sostiene embrión en su lugar durante la disección </td></tr><tr><td> NaCl </td><td> Vario </td><td></td><td> parte de la receta LCRa </td></tr><tr><td> KCl </td><td> Vario </td><td></td><td> parte de la receta LCRa </td&gt; </tr><tr><td> KH 2 PO 4 </td><td> Vario </td><td></td><td> parte de la receta LCRa </td></tr><tr><td> NaHCO 3 </td><td> Vario </td><td></td><td> parte de la receta LCRa </td></tr><tr><td> Glucosa </td><td> Vario </td><td></td><td> parte de la receta LCRa </td></tr><tr><td> CaCl 2 </td><td> Vario </td><td></td><td> parte de la receta LCRa </td></tr><tr><td> MgSO 4 </td><td> Vario </td><td></td><td> parte de la receta LCRa </td></tr><tr><td> Contenedor para LCRa. Sugerir translúcido Nalgene de boca ancha jar, 500 ml (16 onzas) con tapa. </td><td> CPLabSafety </td><td> QP-PLC-03717 </td><td> Apertura taladro agujero en la parte superior de la tapa de vidrio del vástago bubling para penetrar líquido </td></tr><tr><td> Vacío 5 ml vial de vidrio o vial transparente comparable </td><td> American Pharmaceutical Partners, Inc </td><td> 6332300105 </td><td&gt; usa durante la incubación LCRa para mantener las muestras separadas unas de otras y de la corriente de burbujeo </td></tr><tr><td> Tanque de carbógeno (95% O 2/5% CO 2) conectado mediante un tubo a burbujeador </td><td> Vario </td><td></td><td> Conecte mediante un tubo de vidrio pelele madre para infusión en LCRa </td></tr><tr><td> Vidrio tallo burbujeador </td><td> Vario </td><td></td><td> Para infundir carbógeno en LCRa </td></tr><tr><td> Curva de punta de pinzas </td><td> World Precision Instruments </td><td> 501008 </td><td> Para quitar la cabeza de embrión de huevo </td></tr><tr><td> Dos pinzas de punta fina </td><td> World Precision Instruments </td><td> 501985 </td><td> Para los micro-disección </td></tr><tr><td> 50 ml Vaso </td><td> vario </td><td></td><td></td></tr><tr><td> Rodamina dextrano Amine (RDA) </td><td> Invitrogen </td><td> vario </td><td> Fluorescente axón trazador </td></tr><tr><td> Triton X-100 </td><td> ICN Biomedicals </td><td></td><td></td></tr><tr><td> Solución salina tamponada con fosfato, (PBS 1X) </td><td> Vario </td><td></td><td> Reactivo de laboratorio estándar </td></tr><tr><td> Pared Delgada capilares de vidrio, 1,2 OD, ID 0.9 de 4 &quot;(100 mm) de longitud </td><td> World Precision Instruments </td><td> TW120F-4 </td><td> Cargue con RDA. Cada capilar hace dos micropipetas de vidrio </td></tr><tr><td> Aguja / extractor de pipetas </td><td> David Kopf Instruments </td><td> Modelo 720 </td><td> Ajustes utilizados: Calor 16,4, 2,2 solenoide </td></tr><tr><td> Picospritzer </td><td> Parker Instrumentation </td><td> vario </td><td> Conecte mediante un tubo fino de vidrio micropipeta </td></tr><tr><td> Micromanipulador </td><td> Narishige </td><td> vario </td><td></td></tr><tr><td> Disección microscopio con fluorescencia </td><td> Vario </td><td></td><td></td&gt; </tr><tr><td> 4% de paraformaldehído </td><td> Vario </td><td></td><td> Reactivo de laboratorio estándar </td></tr><tr><td> anticuerpo anti-neurofilamentos, opcional </td><td> Millipore </td><td> AB1991 </td><td> Seguir el protocolo recomendado por el fabricante histológico </td></tr><tr><td> Criostato y materiales conexos para seccionar </td><td> Leica </td><td> vario </td><td></td></tr><tr><td> Microscopio de epifluorescencia para formación de imágenes </td><td> Zeiss, diversos </td><td></td><td></td></tr></tbody></table>

selective tracing of auditory fibers in the avian embryonic vestibulocochlear nerve

El embrión de pollo es un modelo ampliamente utilizado para el estudio de las proyecciones de células ganglionares centrales y periféricas. En el sistema auditivo, el etiquetado selectivo de los axones en el nervio auditivo craneal VIII aumentaría el estudio del desarrollo central de circuito auditivo. Este enfoque es un reto porque varios órganos sensoriales del oído interno contribuir al nervio VIII 1. Además, los marcadores que distinguir confiablemente auditivo frente a los grupos vestibulares de los axones a la aviar nervio VIII aún no se han identificado. Vías auditivas y vestibular no pueden distinguirse funcionalmente en los embriones tempranos, como sensoriales evocados por las respuestas no están presentes antes de los circuitos se forman. Centralmente se proyectan axones del nervio VIII han sido localizados en algunos estudios, pero auditivo etiquetado axón fue acompañado por el etiquetado de otros componentes nerviosos VIII 2,3. Aquí se describe un método para la anterógrada trazado del ganglio acústico para selectivamente label axones del nervio auditivo dentro VIII desarrollo. En primer lugar, después de la disección parcial de la región cefálica anterior de un embrión de pollo de 8-día sumergido en fluido cerebroespinal artificial oxigenado, el conducto coclear es identificado por puntos de referencia anatómicos. A continuación, una micropipeta fina tira de cristal está posicionado para inyectar una pequeña cantidad de rodamina dextrano amina en la región profunda del conducto y adyacente donde las células ganglionares de la acústicos se encuentran. Dentro de los treinta minutos después de la inyección, los axones auditivos se remontan centralmente en el cerebro posterior y más tarde se puede visualizar después de la preparación histológica. Este método proporciona una herramienta útil para los estudios de desarrollo de periféricos al centro de formación de circuito auditivo.

Los componentes del nervio VIII y la anatomía del propio nervio son complejas y enrevesadas (Figuras 1, 3). Por selectivamente rastreo de fibras que surgen a partir de células ganglionares de la acústicos, los segmentos del nervio VIII, así como los aferentes primarios auditivas dentro del tronco encefálico puede ser limpiamente trazado y distinguirse de sus homólogos vestibulares (Figuras 2, 3). Del mismo modo, esta técnica podría ser utilizada para estudiar proyecciones perif?...

Watch this Scientific Journal Video about Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve at JoVE.com

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

Aquí describimos una técnica de microdisección seguida de la inyección de tinte fluorescente en el ganglio de la acústica de los primeros embriones de pollo para el seguimiento selectivo de las fibras auditivas axones en el nervio y cerebro posterior.

Rastreo Selectivo de Fibras auditivo en el nervio vestíbulo coclear aviar embrionarias

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

selective-tracing-auditory-fibers-avian-embryonic-vestibulocochlear

Research

JoVE Journal

Neuroscience

9.1K vistas.  University of California, Irvine. Aquí describimos una técnica de microdisección seguida de la inyección de tinte fluorescente en el ganglio de la acústica de los primeros embriones de pollo para el seguimiento selectivo de las fibras auditivas axones en el nervio y cerebro posterior.

Video: Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

Preparación y tejido de fibras inmunotinción ratón nerviosas sensoriales que inervan la piel y los huesos de extremidades

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

Investigación

Neurociencias

A Lightweight, Headphones-based System for Manipulating Auditory Feedback in Songbirds

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and sensorimotor plasticity1. Consistent sensory perturbations result in compensatory changes in motor output, reflecting changes in feedforward motor control that reduce the experienced feedback error. By quantifying how different sensory feedback errors affect human behavior, prior studies have explored how visual signals are used to recalibrate arm movements2,3 and auditory feedback is used to modify speech production4-7. The strength of this approach rests on the ability to mimic naturalistic errors in behavior, allowing the experimenter to observe how experienced errors in production are used to recalibrate motor output.
Songbirds provide an excellent animal model for investigating the neural basis of sensorimotor control and plasticity8,9. The songbird brain provides a well-defined circuit in which the areas necessary for song learning are spatially separated from those required for song production, and neural recording and lesion studies have made significant advances in understanding how different brain areas contribute to vocal behavior9-12. However, the lack of a naturalistic error-correction paradigm - in which a known acoustic parameter is perturbed by the experimenter and then corrected by the songbird - has made it difficult to understand the computations underlying vocal learning or how different elements of the neural circuit contribute to the correction of vocal errors13.
The technique described here gives the experimenter precise control over auditory feedback errors in singing birds, allowing the introduction of arbitrary sensory errors that can be used to drive vocal learning. Online sound-processing equipment is used to introduce a known perturbation to the acoustics of song, and a miniaturized headphones apparatus is used to replace a songbird's natural auditory feedback with the perturbed signal in real time. We have used this paradigm to perturb the fundamental frequency (pitch) of auditory feedback in adult songbirds, providing the first demonstration that adult birds maintain vocal performance using error correction14. The present protocol can be used to implement a wide range of sensory feedback perturbations (including but not limited to pitch shifts) to investigate the computational and neurophysiological basis of vocal learning.

We describe the design and assembly of miniaturized headphones suitable for replacing a songbird&#x2019;s natural auditory feedback with a manipulated acoustic signal. Online sound processing hardware is used to manipulate song output, introduce real-time errors in auditory feedback via the headphones, and drive vocal motor learning.

Un peso ligero, Auriculares basada Sistema de Manipulación de retroalimentación auditiva en Songbirds

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

Estimulación Optogenética del nervio auditivo

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

Condicional transsynaptic genética Seguimiento en el cerebro de embriones de ratón

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

Laser-guided Neuronal Tracing In Brain Explants

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake through electroporation in brain explants with targeted laser illumination and matching filter goggles during the procedure. The described technique of in vitro electroporation by itself yields relatively good visual control for targetting certain areas of the brain. By combining it with laser excitation of fluorescent genetic markers and their read-out through band-passing filter goggles, which can pick up the emissions of the genetically labeled cells/nuclei and the fluorescent tracing dye, a researcher can substantially increase the accuracy of injections by finding the area of interest and controlling for the dye-spread/uptake in the injection area much more efficiently. In addition, the laser illumination technique allows to study the functionality of a given neurocircuit by providing information about the type of neurons projecting to a certain area in cases where the GFP expression is linked to the type of transmitter expressed by a subpopulation of neurons.

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

Neuronal Tracing en explantes cerebrales guiadas por láser

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

Proyección de imagen tridimensional y análisis de las mitocondrias en las fibras de nervio humano Intraepidermal

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Las fibras serotoninérgicos proyección de imagen en la médula espinal de ratón El uso de la claridad / Técnica CÚBICO

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory bowel diseases and diabetes. Here, we describe a protocol for the in vitro characterization of the electrophysiological properties of colonic afferents in rats. The colorectum, with the intact pelvic ganglion (PG) attached, is removed from the rat; superfused with carbogenated Krebs solution in the recording chamber; and cannulated at the oral and anal ends to allow for distension. A fine nerve bundle emanating from the PG is identified, and the multiunit afferent nerve activity is recorded using a suction electrode. Distension of the colonic segment elicits gradual increases in multiunit discharge. A principal component analysis is conducted to differentiate the low-threshold, the high-threshold, and the wide-dynamic range afferent fibers. Chemical sensitivity of colonic afferents can be studied through the bath or intraluminal administration of test compounds. This protocol can be modified for application to other species, such as mice and guinea pigs, and to study the differences in the electrophysiological properties of thoracolumbar/hypogastric and lumbosacral/pelvic afferents of the descending colon in normal and pathological conditions.

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Abnormal sensory function underlies visceral pain and other symptoms of functional and inflammatory bowel diseases. A protocol for the electrophysiological recording of the colonic afferent nerves in an ex vivo rat colorectum preparation is presented here.

In Vitro Caracterización de las propiedades electrofisiológicas de las fibras aferentes colónicas en ratas

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory ...

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating the respiratory diaphragm. In spinal cord slices, phrenic motor neurons cannot be identified from other motor neurons on morphological or biochemical criteria. We provide the description of procedures for visualizing phrenic motor neuron cell bodies in mice, following intrapleural injections of cholera toxin subunit beta (CTB) conjugated to a fluorophore. This fluorescent neuroanatomical tracer has the ability to be caught up at the diaphragm neuromuscular junction, be carried retrogradely along the phrenic axons and reach the phrenic cell bodies. Two methodological approaches of intrapleural CTB delivery are compared: transdiaphragmatic versus transthoracic injections. Both approaches are successful and result in similar number of CTB-labeled phrenic motor neurons. In conclusion, these techniques can be applied to visualize or quantify the phrenic motor neurons in various experimental studies such as those focused on the diaphragm-phrenic circuitry.

Here, we describe a protocol for identifying phrenic motor neurons in mice after intrapleural delivery of fluorophore conjugated cholera toxin subunit beta. Two techniques are compared to inject the pleural cavity: transdiaphragmatic versus transthoracic approaches.

Seguimiento Neuroanatomical retrógrada frénico de neuronas de Motor en ratones

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic ...

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the diagnosis still relies only on clinical signs of motor involvement that appear later on in the disease course, when most of the dopaminergic neurons are already lost. Hence, there is a strong need for a biomarker that can identify patients at the beginning of disease or at the risk of developing it. Over the last few years, skin biopsy has proved to be an excellent research and diagnostic tool for peripheral nerve diseases such as small fiber neuropathy. Interestingly, a small fiber neuropathy and alpha synuclein (&#945;Syn) neural deposits have been shown by skin biopsy in PD patients. Indeed, skin biopsy has the great advantage of being an easily accessible, minimally invasive and painless procedure that allows the analysis of peripheral nervous tissue prone to the pathology. Moreover, the possibility of repeating the skin biopsy in the course of the follow-up of the same patient allows studying the longitudinal correlation with the disease progression. We set up a standardized reliable protocol to investigate the presence of &#945;Syn aggregates in skin nerve fibers of the PD patient. This protocol involves few short fixation steps, a cryotome sectioning and then a free-floating immunofluorescence double-staining with two specific antibodies: anti Protein Gene Product 9.5 (PGP9.5) to mark the cutaneous nerve fibers and anti 5G4 for detecting &#945;Syn aggregates. It is a versatile, sensitive and easy to perform protocol that can also be applied for targeting other proteins of interest in skin nerves. The ability to mark &#945;Syn aggregates is another step forward to the use of skin biopsy as a tool for establishing a pre-mortem histopathological diagnosis of PD.

Here, we present a protocol for a free-floating indirect immunofluorescence assay on skin biopsy sections that allows for the identification of disease specific conformation variants of alpha synuclein involved in Parkinson disease and multiple proteins of the peripheral nervous system.

Dirigido a agregados de alfa sinucleína en fibras nerviosas periféricas cutáneas por el ensayo de inmunofluorescencia de flotación libre

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the ...