Doy las gracias a Anthony Taylor para la formación inicial en la grabación en vivo intracelular y un Maxwell y David Brown para ayudar con el desarrollo inicial de la técnica de tinción intracelular. Doy las gracias a M. de plata en busca de ayuda con el macro collocalization. Muchos estudiosos con los que he colaborado siempre en perspectiva el desarrollo de esta técnica como R. Donga, Moritani M., Luo P., R. Ambalavanar. Esta técnica fue desarrollada con un apoyo considerable de las subvenciones del NIH DE10132, DE15386 y RR017971.

1. Preparación de los animales <ol><li> Anestesiar a ratas con pentobarbital sódico (50mg/kg IP) y colocarlo en una manta eléctrica. Afeitarse la piel que recubre el cráneo posterior con máquinas de cortar el animal. Comprobar que el animal para asegurar que un nivel quirúrgico de nivel de anestesia que se haya obtenido mediante la prueba de la ausencia de un reflejo de retirada y vocalización cuando los dedos queden atrapados, así como la ausencia de reflejo palpebral. Comprobar el nivel de anestesia cada 15 minutos y mantener un nivel quirúrgico de anesthetisa por medio de inyecciones de 15mg/kg de pentobarbital de sodio cada 45 minutos. </li><li> Utilice una técnica aséptica y hacer una incisión en la región inguinal, inmediatamente distal al pliegue formado por el abdomen y la cara interna del muslo y la inserción de una cánula (1mm de diámetro, Clay Adams) en la vena femoral y arteria femoral. Hacer una incisión en la región submandibular, reflejan los músculos infrahioideos. Haga una pequeña incisión en la tráquea e insertar una cánula (2 mm de diámetro) para permitir la ventilación. Tye una sutura alrededor del trachael cannual para asegurarlo en place.Monitor la presión arterial sistémica sistólica y diastólica, a través de la cánula arterial. Además de supervisar la retirada y el reflejo palpebral ahora monitor de presión arterial para evaluar el nivel de anestesia. Administrar anestesia adicional cuando sea necesario a través de la cánula venosa. </li><li> Lugar a la rata en un marco estereotáxico y realizar una craneotomía para exponer el cerebelo. Conecte la cánula traqueal a un ventilador de roedores. Ventilar al animal con un volumen de 2 cm 3 a una velocidad de 100/min con aire húmedo. Una presión positiva al final de la espiración de 1 cm H 2 O se debe mantener para evitar el colapso pulmonar. Cada 15 minutos hyperinflate los pulmones para prevenir la atelectasia. Luego, cubra la superficie del cerebro con aceite mineral caliente (30 ° C). Tenga cuidado de evitar el gran seno venoso situado directamente debajo de la unión entre los huesos parietal y interparietal. </li><li> Aplique el pegamento de cianoacrilato a una barra junto a un vibrador electromagnético y adjuntar la barra en el diastema de la mandíbula. Mover la mandíbula con señales de comando para el vibrador, ya sea una salida A / D o equipo generador de señal. </li><li> Coloque un electrodo de cloruro de plata siliver tierra debajo de la piel adyacente a la craneotomía. </li></ol> 2. Preparación de electrodos <ol><li> Fabricar microelectrodos de vidrio de cuarzo o aluminosilicato con un extractor de microelectrodos horizontal. </li><li> Rellene el microelectrodo con biotinamide 5-10% disuelto en 0,25 M KCl y 0,5 M tampón Tris-HCl (pH 7,6) o el 2% tetramethlyrhodamine en solución salina (pH 6). Compruebe la impedancia del electrodo con un probador de los electrodos. Para grabar desde los axones de gran diámetro que los electrodos con una impedancia de 60 Ω de 80 millones, de los axones y pequeñas interneuronas hacer los electrodos con una impedancia de 80 150MΩ. </li><li> Coloque el microelectrodo en la etapa de la cabeza del electrómetro. Visualizar el electrodo a través de un telescopio pequeño (20 veces con una retícula cerrada) que se fija en la posición detrás de la cabeza del animal. Usando el telescopio es importante porque permite a los electrodos que se stereotaxically posición con gran precisión. </li></ol> 3. Registro electrofisiológico y tinción intracelular <ol><li> Hacer una pequeña abertura en la piamadre y compensar en exceso la capacidad de retroalimentación de manera que cuando el electrodo toca el cerebro una señal de retroalimentación se produce. Esto permite la localización precisa de la superficie del cerebro. </li><li> Producen repetidos desplazamientos mandibulares y avanzar en el electrodo en el cerebro con un motor paso a paso. </li><li> Reconocer impalements neuronal de descensos en el potencial DC e identificar intrasomatic impalements por la actividad sináptica neuronal en curso. Zumbando el electrodo con una compensación excesiva de la capacidad o tocando rara vez producen una penetración celular de éxito. Debido a la alta impedancia de los electrodos, las respuestas neuronales son raramente observadas antes de la penetración. </li><li> Después de atravesar una neurona y la penetración se considera estable, caracterizar la respuesta neuronal utilizando la rampa, tenencia y movimiento de la mandíbula sinusoidal. 5 Mapa del campo receptivo de la neurona mediante el sondeo de la piel alrededor de la cabeza y la cavidad oral dentro de una sonda no conductores como un palo de madera. Si es relevante para el estudio, examinar la respuesta de la neurona a otros estímulos funcionalmente relevantes como la contracción muscular y los estímulos nocivos. 6 aferentes primarias nociceptivas receptores neuronales para responder a los estímulos nociceptivos. Anesthesetics generales, tales como el pentobarbital sódico no bloquean la conducción axonal en las neuronas aferentes primarias, sino que deprime la transmisión sináptica. Por lo tanto la conducción axonal en el axón neuronal aferente primaria se conserva. </li><li> Inyectar corriente continua (CC, 1-4NA) para un tiempo de inyección total de 15 70nA minutos. Monitor de la penetración del electrodo durante la inyección de corriente y suspender si el potencial de membrana se vuelve más positivo que-30mV.</li></ol> 4. Procesamiento de tejidos <ol><li> La eutanasia de los animales mediante una sobredosis de pentobarbital (140mg/kg IV) y perfusión vascular con un enjuague (0,9% NaCl 38 ° C que contiene 500 unidades de heparina y 2% 1ml xilocaína seguido de un 4% de paraformaldehído en tampón fosfato 0,1 M (pH 7,4) . </li><li> Quitar el cerebro y en la sección que el 50-100μm con un vibratome, ya sea en el plano frontal, sagital u horizontal. Secions se recogen flotando libre en PBS 25 ° C. </li><li> Proceso en el cerebro para DAB mediante la incubación de suero de cabra normal de 1-2% y 1% de Triton X-100 en PBS 0,01 M seguido de incubación en avidina biotina (1:50 Elite Vectastain). Reaccionar con secciones de níquel-DAB con H 2 O 2. Para el proceso de Texas Red, las secciones se incuban en complejo avidina-biotina (1:50) en PBS durante una noche a 4 ° C y luego se incuban en Texas el 4% DCS Red avidina en PBST a 4 ° C durante la noche. Contratinción secciones con un fluorescente Nissl mancha (NeuroTrace) 20 minutos a 23 ° C. </li><li> Si las neuronas se inyectó con rodamina, visualizar la neurona se inyecta directamente con un microscopio de fluorescencia (Ex 545nm). </li></ol> 5. La combinación de método con el etiquetado retrógrada, la inmunocitoquímica, la imagen confocal, análisis cuantitativo de colocalización El éxito de combinar esta técnica con otros métodos depende en gran medida el etiquetado intracelular buena. <ol><li> El método de visualizar aquí se puede combinar fácilmente con el etiquetado neuronal retrógrada. 7-10 Para ello, anestesiar al animal y utilizando una técnica aséptica, se inyecta un trazador neuronal como la peroxidasa de rábano picante (20% peroxidasa de rábano picante. VI Sigma, Sigma) y el 1% de germen de trigo aggultinated peroxidasa de rábano (Sigma) en las regiones objetivo periféricos como el músculo masetero. Este marcador neuronal se abordará en los axones periféricos y se transporta a través del transporte axonal de la motoneurona cuerpos celulares. Para el músculo masetero, 15μl de trazador se inyecta en el músculo con una microjeringa estéril de 10 microlitros. Los animales se colocan en un cojín de calefacción y control hasta que se recupere de la anestesia y luego se colocan en las jaulas de recuperación. Después de 24 horas, anestesiar a los animales y fisiológicamente caracterizan las neuronas y intracelularmente a las manchas. Luego perfundir el animal como se describió anteriormente y secciones proceso de tejido para detectar la presencia de HRP con tetrametilbenzidina (TMB) como cromógeno y tungstato sódico como el estabilizador y se intensificó con el cobalto. Se colocan en el 1% tungstato sódico disuelto en PBS 0,1 M (pH 6,5) y 0,0007% TMB disuelto en etanol absoluto y acetona a 15 ° C durante 20 min. Entonces reaccionan las secciones de tejido mediante la adición de 1,0 ml de 0,3% de H 2 O 2 por cada 100 ml de solución de incubación durante 60 minutos. Enjuague el tejido en PBS 0,1 M (pH 6,5) y el lugar en el 0,05% diaminobencidina (pH 7,4), el 0,02% de cobalto, y el 0,01% H 2 O 2 en PBS 0,1 M (pH 7,4) durante 10 minutos. a 37 ° C. Proceso de tejido para la visualización de la neurona inyectados con biotinamide como se describe y visualizar las relaciones entre la neurona sensorial y la etiqueta de una variedad de las motoneuronas. </li><li> La técnica se muestra aquí también pueden ser fácilmente combinados con immnocytochemistry. 6 A modo de ejemplo, immunocytochemically proceso del cerebro para sinaptofisina para localizar con precisión las sinapsis en las neuronas marcadas (Fig. 3). Para ello, se incuban las secciones del cerebro de ratón anti-sinaptofisina anticuerpos (1:10.000) por 2 días a 4 ° C y luego se incuban en anti-ratón FITC (1:400) durante 1 hora a 23 ° C. </li><li> Neuronas marcadas con marcadores fluorescentes o tratados con imágenes fluorescentes son ideales para la visualización de imagen confocal. La Figura 3 es un ejemplo de una sección óptica obtenida con microscopía confocal a través de un axón Bouton. (Fig. 3). Generar animaciones de neuronas marcadas por la adquisición de varias secciones ópticas a través de la neurona marcada (Fig. 4). </li><li> Neuronas marcadas con este método se puede utilizar para el análisis cuantitativo de colocalización. Para ello, utilice una macro de software a disposición del público en relación con NIH Image (que se encuentra en http://phy.ucsf.edu/ ° idl / colocalization.htm). Localizar sinaptofisina en terminales de los axones individuales mediante la combinación de etiquetado intracelular con inmunocitoquímica sinaptofisina 6. </li></ol> 6. Los resultados representativos: Un resumen de los resultados representativos que se pueden obtener con este método se ilustra en la Figura 1. Esta sola neurona cerebral se registró electrofisiológico durante el movimiento de la mandíbula y, como se puede ver claramente, la respuesta de esta neurona (Figura 1 abajo a la izquierda, la luz azul) fue modulada durante el movimiento. Esta neurona se le inyectó después biotinamide caracterización electrofisiológica y posteriormente procesadas para su visualización. La neurona reconstruida (Figura 1 medio, verde) puede ser realted a un punto de referencia anatómica, en este caso el núcleo motor del trigémino designado (línea roja). Sobre la base de la respuesta neuronal durante el movimiento y la reconstrucción esta neurona puede ser identificado como una neurona aferente del huso muscular secundaria. La figura 2 muestra un ejemplo representativo de la respuesta fisiológica de la neurona durante el desplazamiento mandibular. La respuesta de la neurona se representa como frecuencia de disparo instantáneo. Tenga en cuenta que la respuesta neuronal imita el desplazamiento mandibular que indica que esta neurona en particular proporciona retroalimentación sensorial relacionada con la posición mandibular. Figura 3 es una imagen de gran aumento de un axón intracelularmente manchadas combinado con tinción para sinaptofisina y una mancha de Nissl. Tenga en cuenta la colocalización de sinaptofisina (amarillo) en el bouton axón. Figura 4 es una animación de una sola neurona, fisiológicamente caracterizan y etiquetado intracelular. <img src="/files/ftp_upload/2650/2650fig1.jpg" alt="Figura 1"/> Figura 1. Descripción del método. Arriba a la izquierda: el desplazamiento mandibular. Grabación de media intracelular (verde) de una sola neurona (amarillo). La morfología de esta neurona se reconstruyó después de la grabación intracelular y la inyección. Contorno de color rojo indica la ubicación del núcleo motor del trigémino. Abajo a la izquierda: la respuesta fisiológica de esta neurona durante el movimiento de la mandíbula. <img src="/files/ftp_upload/2650/2650fig2.jpg" alt="Figura 2"/> Figura 2. Respuesta fisiológica Representante de una neurona sensorial solo músculo grabado en vivo durante el movimiento de la mandíbula. Tenga en cuenta la similitud de la respuesta neuronal de los desplazamientos mandíbula. <img src="/files/ftp_upload/2650/2650fig3.jpg" alt="Figura 3"/> Figura 3. Arborización terminal axonal con botones sinápticos (hinchazón de color rojo) de una neurona sensorial intracelularmente manchadas de que respondió en los músculos de sondeo. Posterior tratamiento inmuno sinaptofisina muestra la localización de sinaptofisina en el bouton axonal (amarillo). El verde es un Nissl tinción fluorescente. Figura 4. Animación del huso muscular primaria axón neurona aferente cuya respuesta fisiológica fue grabado en vivo durante los movimientos mandibulares, el axón entonces intracelularmente manchadas y procesados ​​para su visualización. <a href="/files/ftp_upload/2650/collateral4.avi">Descargar un vídeo de alta resolución de la Figura 4 aquí</a> <a href="/files/ftp_upload/2650/medres.mov">Descargar un vídeo de resolución media de la Figura 4 aquí</a>

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	<li>Cuellar, C. A., Tapia, J. A., Juarez, V., Quevedo, J., Linares, P., Marinez, L., Manjarrez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation+of+sinusoidal+electrical+waves+along+the+spinal+cord+during+a+fictive+motor+task.">Propagation of sinusoidal electrical waves along the spinal cord during a fictive motor task.</a> J. Neurosci. 29, 798-810 (2010).</li><li>Frigon, A., Gossard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+specialized+rhythm-generating+mechanisms+in+the+adult+mammalian+spinal+cord.">Evidence for specialized rhythm-generating mechanisms in the adult mammalian spinal cord.</a> J. Neurosci. 30, 7061-7071 (2010).</li><li>Wang, W., Chan, S. S., Heldman, D. A., Moran, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+cortical+representation+of+hand+translation+and+rotation+during+reaching.">Motor cortical representation of hand translation and rotation during reaching.</a> J. Neurosci. 30, 958-962 (2010).</li><li>Ma, C., He, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+investigating+cortical+control+of+stand+and+squat+in+conscious+behavioral+monkeys.">A method for investigating cortical control of stand and squat in conscious behavioral monkeys.</a> J. Neurosci. 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Neurol. 406, 384-401 (1999).</li><li>Hassani, O. K., Henny, P., Lee, M. G., Jones, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+neurons+intermingled+with+orexin+and+MCH+neurons+in+the+lateral+hypothalamus+discharge+maximally+during+sleep.">GABAergic neurons intermingled with orexin and MCH neurons in the lateral hypothalamus discharge maximally during sleep.</a> Eur. J. Neurosci. 32, 448-457 (2010).</li><li>Inokawa, H., Yamada, H., Matsumoto, N., Muranishi, M., Kimura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Juxtacellular+labeling+of+tonically+active+neurons+and+phasically+active+neurons+in+the+rat+striatum.">Juxtacellular labeling of tonically active neurons and phasically active neurons in the rat striatum.</a> Neuroscience. 168, 395-404 (2010).</li></ol>

Los experimentos con animales se realizaron de acuerdo con las guias y la regulación se establece en la Guía para el Cuidado y Uso de Animales de Laboratorio (NIH Publicación N º 86-23, revisado en 1985) y la Universidad de Maryland Cuidado de los Animales y el empleo Comisión.

El método se ilustra aquí es una poderosa técnica que aporta datos importantes en la función de neuronas individuales y cómo la respuesta de las neuronas individuales contribuye a los circuitos neuronales. 9 Este conocimiento es fundamental para entender la función sensorio. La mayor fortaleza de esta técnica es que permite la determinación de un gran número de parámetros de una neurona como la fisiología, la morfología y la morfología sináptica y la distribución. Cuando se combina con otras t?...

<table border="1"><tbody><tr><th> Nombre del reactivo o equipo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios </th></tr><tr><td> vibrador electromagnético </td><td> Ling Sistemas Dinámicos </td><td> V101 </td><td></td></tr><tr><td> generador de señal </td><td> Sistemas de retroalimentación </td><td> PFG605 </td><td> capaz de producir la señal de salida trapezoidal </td></tr><tr><td> electrodo de vidrio </td><td> Sutter Instrumentos </td><td> AF100-68-10 </td><td> con filamento </td></tr><tr><td> electrodo extractor </td><td> Sutter Instrumentos </td><td> Modelo P-2000 y P-80 </td><td></td></tr><tr><td> biotinamide </td><td> Vector Laboratories </td><td> SP-1120 </td><td> se almacenan a 4 ° C </td></tr><tr><td> Texas Red avidina DCS </td><td> Vector Laboratories </td><td> A-2016 </td><td></td></tr><tr><td> tetramethlyrhodamine </td><td> Las sondas moleculares </td><td> D-3308 </td><td> 3000 peso molecular, la lisina puede arreglar </td></tr><tr><td> ratón de anticuerpos anti-sinaptofisina </td><td> Chemicon </td><td> MAB5258 </td><td></td></tr><tr><td> fluorescentes Nissl mancha </td><td> Neurotrace, sondas moleculares </td><td> N-21480 </td><td></td></tr><tr><td> electrodo probador </td><td> Winston Electronics </td><td> BL-1000-B </td><td> para medir la impedancia del electrodo </td></tr><tr><td> electrómetro </td><td> Axon Instruments </td><td> Axoprobe 1A, 2B Axoclamp </td><td></td></tr></tbody></table>

physiological, morphological and neurochemical characterization of neurons modulated by movement

El papel de las neuronas individuales y su función en los circuitos neuronales es fundamental para comprender los mecanismos neuronales de las funciones motoras y sensoriales. La mayoría de las investigaciones de los mecanismos sensoriomotores se basan en el examen de cualquiera de las neuronas, mientras que un animal es de 1,2 estática o grabar la actividad neuronal extracelular durante un movimiento de 3,4. Aunque estos estudios han proporcionado la base fundamental para la función sensorio, o bien no evaluar la información funcional que se produce durante un movimiento o están limitados en su capacidad para caracterizar el fenotipo de la anatomía, la fisiología y la neuroquímica de las neuronas. Una técnica se muestra aquí, que permite una amplia caracterización de las neuronas individuales en un movimiento en vivo. Esta técnica puede ser utilizada no sólo para estudiar las neuronas aferentes primarias, sino también para caracterizar a las motoneuronas y las interneuronas sensorio. Inicialmente la respuesta de una sola neurona se registra utilizando métodos electrofisiológicos en los diversos movimientos de la mandíbula, seguida por la determinación del campo receptivo de la neurona. Un trazador neuronal luego se inyecta en forma intracelular de la neurona y el cerebro se procesa para que la neurona se puede visualizar con microscopía de luz, de electrones o confocal (Fig. 1). La morfología detallada de la neurona se caracteriza se reconstruye de modo que la morfología neuronal se puede correlacionar con la respuesta fisiológica de la neurona (Figs. 2,3). En esta comunicación los detalles importantes claves y consejos para la implementación exitosa de esta técnica se proporcionan. Una valiosa información adicional puede ser determinada por la neurona en estudio mediante la combinación de este método con otras técnicas. Etiquetado neuronal retrógrada puede ser usado para determinar las neuronas con las que la sinapsis de neuronas marcadas, lo que permite la determinación detallada de los circuitos neuronales. Inmunocitoquímica se puede combinar con este método para examinar los neurotransmisores dentro de la neurona y la etiqueta para determinar los fenotipos químicos de las neuronas con las que la etiqueta sinapsis neuronal. La neurona etiqueta también puede ser procesado para microscopía electrónica para determinar las características ultraestructurales y microcircuitos de las neuronas etiquetados. En general, esta técnica es un poderoso método para caracterizar completamente las neuronas durante el movimiento en vivo lo que permite visión precisa del papel de la neurona en funciones sensoriales y motrices.

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Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

Una técnica es descrita para cuantificar la respuesta in vivo fisiológicas de las neuronas de mamíferos durante el movimiento y correlacionar la fisiología de las neuronas con morfología neuronal, fenotipo neuroquímicos y microcircuitos sináptica.

Caracterización fisiológicas, morfológicas y neuroquímicas de las neuronas modulada por el Movimiento

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

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13.7K vistas.  University of Maryland. Una técnica es descrita para cuantificar la respuesta in vivo fisiológicas de las neuronas de mamíferos durante el movimiento y correlacionar la fisiología de las neuronas con morfología neuronal, fenotipo neuroquímicos y microcircuitos sináptica.

Video: Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

Eye Movement Monitoring of Memory

Explicit (often verbal) reports are typically used to investigate memory (e.g. "Tell me what you remember about the person you saw at the bank yesterday."), however such reports can often be unreliable or sensitive to response bias 1, and may be unobtainable in some participant populations. Furthermore, explicit reports only reveal when information has reached consciousness and cannot comment on when memories were accessed during processing, regardless of whether the information is subsequently accessed in a conscious manner. Eye movement monitoring (eye tracking) provides a tool by which memory can be probed without asking participants to comment on the contents of their memories, and access of such memories can be revealed on-line 2,3. Video-based eye trackers (either head-mounted or remote) use a system of cameras and infrared markers to examine the pupil and corneal reflection in each eye as the participant views a display monitor. For head-mounted eye trackers, infrared markers are also used to determine head position to allow for head movement and more precise localization of eye position. Here, we demonstrate the use of a head-mounted eye tracking system to investigate memory performance in neurologically-intact and neurologically-impaired adults. Eye movement monitoring procedures begin with the placement of the eye tracker on the participant, and setup of the head and eye cameras. Calibration and validation procedures are conducted to ensure accuracy of eye position recording. Real-time recordings of X,Y-coordinate positions on the display monitor are then converted and used to describe periods of time in which the eye is static (i.e. fixations) versus in motion (i.e., saccades). Fixations and saccades are time-locked with respect to the onset/offset of a visual display or another external event (e.g. button press). Experimental manipulations are constructed to examine how and when patterns of fixations and saccades are altered through different types of prior experience. The influence of memory is revealed in the extent to which scanning patterns to new images differ from scanning patterns to images that have been previously studied 2, 4-5. Memory can also be interrogated for its specificity; for instance, eye movement patterns that differ between an identical and an altered version of a previously studied image reveal the storage of the altered detail in memory 2-3, 6-8. These indices of memory can be compared across participant populations, thereby providing a powerful tool by which to examine the organization of memory in healthy individuals, and the specific changes that occur to memory with neurological insult or decline 2-3, 8-10.

Eye movement monitoring (or eye tracking) reveals where in space the eyes linger, when and for how long. Here, we demonstrate how eye tracking can be used to investigate the integrity of memory in multiple participant populations, without requiring verbal, or otherwise explicit, reports.

Ojo vigilancia de los movimientos de la Memoria

Explicit (often verbal) reports are typically used to investigate memory (e.g. "Tell me what you remember about the person you saw at the bank ...

Investigación

Neurociencias

Coherence between Brain Cortical Function and Neurocognitive Performance during Changed Gravity Conditions

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and focused on psychological aspects. Until now, objective observation of neurophysiological parameters has not been carried out - undoubtedly because the technical and methodological means have not been available -, investigations into the neurophysiological effects of weightlessness are in their infancy (Schneider et al. 2008).
While imaging techniques such as positron emission tomography (PET) and magnetic resonance imaging (MRI) would be hardly applicable in space, the non-invasive near-infrared spectroscopy (NIRS) technique represents a method of mapping hemodynamic processes in the brain in real time that is both relatively inexpensive and that can be employed even under extreme conditions. The combination with electroencephalography (EEG) opens up the possibility of following the electrocortical processes under changing gravity conditions with a finer temporal resolution as well as with deeper localization, for instance with electrotomography (LORETA).
Previous studies showed an increase of beta frequency activity under normal gravity conditions and a decrease under weightlessness conditions during a parabolic flight (Schneider et al. 2008a+b). Tilt studies revealed different changes in brain function, which let suggest, that changes in parabolic flight might reflect emotional processes rather than hemodynamic changes. However, it is still unclear whether these are effects of changed gravity or hemodynamic changes within the brain. Combining EEG/LORETA and NIRS should for the first time make it possible to map the effect of weightlessness and reduced gravity on both hemodynamic and electrophysiological processes in the brain. Initially, this is to be done as part of a feasibility study during a parabolic flight. Afterwards, it is also planned to use both techniques during medium- and long-term space flight.
It can be assumed that the long-term redistribution of the blood volume and the associated increase in the supply of oxygen to the brain will lead to changes in the central nervous system that are also responsible for anaemic processes, and which can in turn reduce performance (De Santo et al. 2005), which means that they could be crucial for the success and safety of a mission (Genik et al. 2005, Ellis 2000).
Depending on these results, it will be necessary to develop and employ extensive countermeasures. Initial results for the MARS500 study suggest that, in addition to their significance in the context of the cardiovascular and locomotor systems, sport and physical activity can play a part in improving neurocognitive parameters. Before this can be fully established, however, it seems necessary to learn more about the influence of changing gravity conditions on neurophysiological processes and associated neurocognitive impairment.

The effect of weightlessness and hypergravity on both hemodynamic and electrophysiological processes in the brain is going to be followed during parabolic flight by EEG and NIRS techniques. 
A feasibility study of a more complex experiment, which is planned to carry out during medium- and long-term space flight.

La coherencia entre la función cerebral cortical y rendimiento neurocognitivo en condiciones de gravedad ha cambiado

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and ...

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

Determinación rápida del umbral nociceptivo térmico en ratas diabéticas

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

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 ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

La detección de palmitoilación proteínas en neuronas del hipocampo cultivadas por inmunoprecipitación e Intercambio Acyl-Biotina (ABE)

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique known as electron probe microanalysis (EPMA) are described. Intramitochondrial calcium is a particular focus because of the critical role that mitochondrial calcium overload plays in neurodegenerative diseases. The method is based on the analysis of X-rays generated in an electron microscope (EM) by interaction of an electron beam with the specimen. In order to maintain the native distribution of diffusible elements in electron microscopy specimens, EPMA requires &#34;cryofixation&#34; of tissue followed by the preparation of ultrathin cryosections. Rapid freezing of cultured cells or organotypic slice cultures is carried out by plunge freezing in liquid ethane or by slam freezing against a cold metal block, respectively. Cryosections nominally 80 nm thick are cut dry with a diamond knife at ca. -160 &#176;C, mounted on carbon/pioloform-coated copper grids, and cryotransferred into a cryo-EM using a specialized cryospecimen holder. After visual survey and location mapping at &#8804;-160 &#176;C and low electron dose, frozen-hydrated cryosections are freeze-dried at -100 &#176;C for ~30 min. Organelle-level images of dried cryosections are recorded, also at low dose, by means of a slow-scan CCD camera and subcellular regions of interest selected for analysis. X-rays emitted from ROIs by a stationary, focused, high-intensity electron probe are collected by an energy-dispersive X-ray (EDX) spectrometer, processed by associated electronics, and presented as an X-ray spectrum, that is, a plot of X-ray intensity vs. energy. Additional software facilitates: 1) identification of elemental components by their &#34;characteristic&#34; peak energies and fingerprint; and 2) quantitative analysis by extraction of peak areas/background. This paper concludes with two examples that illustrate typical EPMA applications, one in which mitochondrial calcium analysis provided critical insight into mechanisms of excitotoxic injury and another that revealed the basis of ischemia resistance.

This paper describes the application of cryoanalytical electron microscopy to the quantitative measurement of total calcium content and distribution at subcellular resolution in physiologically defined biological specimens.

Medición de calcio total en neuronas por Sonda de Electrones X-ray microanálisis

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

Lineage-reprogramación de células derivadas de pericitos del cerebro humano adulto en neuronas inducidas

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

Electrofisiológico y Caracterización Morfológica de microcircuitos neuronales en el infarto agudo rodajas de cerebro Usando Emparejados patch-clamp grabaciones

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

Evaluación funcional y morfológica de Diafragma Inervación por frénico neuronas motoras

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing patterns. The inclusion of biocytin/neurobiotin in the recording electrode permits post-hoc recovery of morphological details, which are necessary to determine the dendritic arborization and the regions targeted by the axons of the recorded neurons. However, given the presence of morphologically similar neurons with distinct neurochemical identities and functions, immunohistochemical staining for cell-type-specific proteins is essential to definitively identify neurons. To maintain network connectivity, brain sections for physiological recordings are prepared at a thickness of 300 &#181;m or greater. However, this thickness often hinders immunohistological postprocessing due to issues with antibody penetration, necessitating the resectioning of the tissue. Resectioning of slices is a challenging art, often resulting in the loss of tissue and morphology of the cells from which electrophysiological data was obtained, rendering the data unusable. Since recovery of morphology would limit data loss and guide in the selection of neuronal markers, we have adopted a strategy of recovering cell morphology first, followed by secondary immunostaining. We introduce a practical approach to biocytin filling during physiological recordings and subsequent serial immunostaining for the recovery of morphology, followed by the restaining of sections to determine the neurochemical identity. We report that sections that were filled with biocytin, fixed with paraformaldehyde (PFA), stained, and coverslipped can be removed and restained with a second primary antibody days later. This restaining involves the removal of the coverslip, the washing of sections in a buffer solution, and the incubation of primary and secondary antibodies to reveal the neurochemical identity. The method is advantageous for eliminating data loss due to an inability to recover morphology and for narrowing down the neurochemical markers to be tested based on morphology.

This protocol presents a method for the morphological recovery of neurons patched during electrophysiological recordings using biocytin filling and subsequent immunohistochemical postprocessing. We show that thick biocytin-filled sections that were stained and coverslipped can be restained with a second primary antibody days&#160;or months later.

La inmunotinción de las secciones llenas de Biocitina y se procesaron para neuroquímicos Marcadores

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing ...