Este trabajo fue financiado por el INE y el NIMH.

1. VideoSwitcher 1,1. Conecte VideoSwitcher a un ordenador El VideoSwitcher (Figura 1) recibe de vídeo analógicas (VGA) entradas y se utiliza para conducir tubo de rayos catódicos (CRT) de color. Antes de utilizar el VideoSwitcher, asegúrese de que la tarjeta de gráficos de computadora tiene un puerto VGA (Figura 2A) o una interfaz visual digital (DVI-I) del puerto (Figura 2B) que transmite señales de vídeo analógicas y digitales. Un adaptador de DVI a VGA (Figura 2C) es necesario para conectar el puerto DVI-I de la VideoSwitcher. Apague el ordenador y el monitor y desconecte el monitor de la tarjeta de gráficos por ordenador. Conecte el VGA o el puerto DVI-I de la tarjeta de gráficos por ordenador al puerto de entrada de la VideoSwitcher utilizando el cable VGA proporcionado. Use un destornillador pequeño para asegurar las conexiones. Conecte el cable VGA del monitor al puerto de salida de la una VideoSwitcherª asegurar las conexiones. Conecte el VideoSwitcher a una fuente de alimentación mediante el adaptador de corriente suministrado. Encienda el ordenador y el monitor. 1,2. Dos modos de visualización del conmutador de vídeo El botón de interruptor en el VideoSwitcher permite al usuario cambiar atrás y hacia adelante en dos modos de visualización. En la modalidad de color, la VideoSwitcher no altera la señal de vídeo desde la tarjeta de gráficos por ordenador. En el modo de escala de grises, la pantalla se vuelve monocromática, con su luminancia determinada principalmente por la señal de vídeo del canal azul de la tarjeta gráfica, ligeramente afectado por la señal del canal rojo, y ninguna contribución de la canal verde. Esto es porque la red resistor en el interior del VideoSwitcher atenúa la señal del canal rojo por un factor de aproximadamente 128, y la señal del canal verde no contribuye en absoluto. La relación de las contribuciones de la azul und canales rojo a la salida del VideoSwitcher se llama azul a rojo relación (BRratio). El BRratio es propiedad de un conmutador de vídeo, independientemente de la configuración de la pantalla. Se puede determinarse experimentalmente para cada VideoSwitcher (véase 1,3). El modo de visualización también se puede cambiar mediante el envío de una señal especial por el canal verde de la tarjeta gráfica mediante una llamada a la función de Matlab: PsychVideoSwitcher (&#39;modo conmutado &quot;, whichScreen, ToGrayMode); donde ToGrayMode de 1 o 0 significa para cambiar a modo de escala de grises o color. La Figura 3 muestra el diagrama esquemático de un VideoSwitcher. 1,3. Mida BRratio El BRratio se puede medir para cada VideoSwitcher utilizando una rejilla de onda cuadrada tarea de detección. Los dos niveles de la onda cuadrada se asignan valores RGB tales como (0, 0, 40) y (x, 0, 39). Se puede ajustar el valor de x hasta que la rejilla de onda cuadradadesaparece, momento en el cual el valor de x es el BRratio --- la magnitud de la señal en el canal R que conduce a la intensidad producida por una unidad de señal en el canal B. Aunque se puede medir la BRratio electrónicamente, por ejemplo para medir la relación de las salidas de tensión de una VideoSwitcher en valores RGB de (0, 0, 255) y (255, 0, 0), el método psicofísico descrito aquí es muy simple y precisa. 1,4. Muestra de calibración Para la mayoría de los monitores, la luminancia de la pantalla no se incrementa linealmente con el nivel de píxel gris especificado por la tarjeta de gráficos por ordenador. La relación puede ser descrito como: <img src="/files/ftp_upload/3312/3312eq1.jpg" alt="Ecuación 1" /> donde L máx, L min y γ se determinó con un procedimiento de corrección gamma, ya sea mediante el uso de fotómetros o por una combinación de procedimientos psicofísicos y mediciones fotométricas 7,8. Gamma es la calibración Important para producir contrastes precisos en una pantalla, sino que debe realizarse para cada dispositivo de visualización. Con un fotómetro, uno simplemente puede mostrar un cuadro uniforme, grande en graylevels píxeles diferentes (por ejemplo, de 0 a 255), medir la luminancia del centro de la plaza, y se ajustan los resultados con la ecuación 1 para obtener γ. El procedimiento implica la creación de psicofísica, en un área de la superficie de visión, lo más uniforme posible una mezcla (en espacio y tiempo) de cantidades iguales de cero y llena de intensidad de los píxeles y que determinan el valor graylevel de píxeles en un área homogénea adyacente ( en la que todos los pixeles tienen la misma intensidad) que produce una coincidencia psicofísica a la luminancia de la zona mixta-píxel. El primer partido determina el valor graylevel durante 1/2 de la luminancia máxima. Una mezcla de pixeles con intensidades de luminancia de 1/2 y 1 se utiliza para determinar el valor 3/4, y este procedimiento se repite hasta siete valores fROM 1/8 a 7/8 han sido determinadas. Estas coincidencias se repiten, y diversos controles de consistencia se realizan, tales como la verificación de que una mezcla de 3/4 y 1/4 partidos de 1/2. En este procedimiento, tanto el B y los niveles de R de la zona homogénea se ajustan, y U = B + R / BRratio. Los resultados se pueden encajar con la Ecuación 1 para obtener γ. Después de obtener los valores de L min, max L y γ, se puede establecer la luminancia de fondo L 0 = (L + L max min) / 2, la relación entre el contraste de píxeles c (U) y el nivel de gris de píxeles U es: <img src="/files/ftp_upload/3312/3312eq2.jpg" alt="Ecuación 2" /> Podemos reescribir la ecuación para encontrar el nivel requerido de grises para un contraste que se utiliza: <img src="/files/ftp_upload/3312/3312eq3.jpg" alt="Ecuación 3" /> donde U es normalmente un número no entero. El valor RGB del píxel correspondiente es: R = BRratio * [U-int (U)], G = 0, B =int (U), donde int () toma el valor entero de su entrada. 1,5. Demostración de una rejilla de onda sinusoidal El efecto del aumento de escala de grises de resolución puede demostrarse mediante la comparación de dos rejillas de onda sinusoidal. La rejilla de la izquierda utiliza la capacidad total de la VideoSwitcher con cada intensidad de los píxeles especificado por el R apropiado y los valores de B. La rejilla de la derecha es el mismo que el de la izquierda, excepto que sólo la U redondeado se utiliza como valor B de cada píxel, imitando la salida de un dispositivo de visualización de 8-bits. 2. RTbox 2,1. Conecte el RTbox a un ordenador Conecte el RTbox (Figura 4) a un puerto USB de un ordenador con el cable suministrado. Si es necesario, descargue el controlador del dispositivo desde el <a href="http://www.ftdichip.com/Drivers/VCP.htm">http://www.ftdichip.com/Drivers/VCP.htm</a> . Instale el controlador siguiendo las instrucciones de la página web. 2,2. El uso de la RTcaja para medir el tiempo de respuesta El RTbox puede utilizarse de dos maneras diferentes. Si el programa del usuario puede volver confiables tiempos de inicio del estímulo, tales como el tiempo devuelto por la pantalla (&#39;Flip&#39;) y PsychPortAudio en Psychtoolbox [8], el RTbox puede ser usado como un teclado. Debido a los tiempos del comienzo del estímulo y los tiempos de RTbox botón se basan los relojes de la computadora host y RTbox, respectivamente, debemos sincronizar los dos relojes. Esto se hace con una llamada a la función RTBox (&#39;clear&#39;) antes de cada inicio de estímulo para borrar los eventos no deseados en el búfer. El tiempo de respuesta puede ser simplemente mide como t resp = t - inicio de t, donde tonset es el estímulo de regresar de el programa de usuario, y t es el tiempo el botón que devuelve la llamada a la función [t, un botón] = RTBox (tiempo de espera). Esto es porque el controlador RTBox convierte automáticamente el tiempo desde el momento botón RTbox para alojar tiempo de ordenador. Si el programa del usuario no puedevolver confiables tiempos de inicio de estímulo, señales de disparo que indican inicios de estímulo debe ser proporcionada a la RTbox. Una solución es utilizar un sensor de luz para detectar el inicio del estímulo o un parche de luz que se sincroniza con el estímulo. La salida del sensor de luz está conectado al puerto de la luz RTbox. Una segunda solución consiste en utilizar la salida del disparador de la VideoSwitcher. El gatillo puede ser conectado directamente al puerto de impulso de la RTbox. Una tercera solución es conectar la señal de audio del ordenador al puerto de sonido de la RTbox. Para generar un disparador de inicio del estímulo de la VideoSwitcher, que &quot;encienda&quot; el canal verde de los píxeles en la parte central de la primera imagen del estímulo visual. Debido a que no contribuye a la red de resistencias, la señal en el canal verde es invisible para el sujeto. Una llamada a la función [t, un botón] = RTBox (&quot;pulso&quot;, tiempo de espera) devuelve el tiempo de respuesta, donde &quot;pulso&quot; instruye al RTbox para calcular t<html> él tiempo de respuesta relativa al gatillo. La llamada de función se utiliza el mismo para el gatillo de sonido. Para los desencadenantes del sensor de luz, la llamada a la función es [t, un botón] = RTBox (&#39;luz&#39;, tiempo de espera). 3. Integrar VideoSwitcher y RTbox Hemos programado un programa de demostración que usa el VideoSwitcher para controlar la visualización, la RTbox para recoger el tiempo de respuesta precisa, y la RTbox para enviar el código de evento y las respuestas a un sistema ERP. El programa se puede descargar desde <a href="http://lobes.usc.edu/videoswitcher/VideoSwitcherRTBoxERP_demo.m">http://lobes.usc.edu/videoswitcher/VideoSwitcherRTBoxERP_demo.m</a> . 4. Los resultados representativos <img src="/files/ftp_upload/3312/3312fig1.jpg" alt="Figura 1" /> Figura 1. Imagen de un conmutador de vídeo. Cuenta con entrada de VGA y puertos de salida (dos mujeres), un botón para cambiar entre dos modos de visualización, y un puerto de salida de disparo. CONTENIDO &quot;&gt; <img src="/files/ftp_upload/3312/3312fig2.jpg" alt="Figura 2" /> Figura 2. Fotos de VGA (A) y DVI-I (B) y los puertos. Un DVI-I a VGA (C) <img src="/files/ftp_upload/3312/3312fig3.jpg" alt="Figura 3" /> Figura 3. El esquema de un conmutador de vídeo. El multiplexor tiene dos modos de entrada, controlados por un nivel de tensión. Cuando está en el modo mostrado en el esquema, los canales RGB de un monitor de recibir la misma señal, por lo que la pantalla está en escala de grises. La tensión de conmutación es generada por un chip de CPLD, controlada por señales en el G-canal y el botón del interruptor. El gatillo también es controlado por la entrada en el G-canal. <img src="/files/ftp_upload/3312/3312fig4.jpg" alt="Figura 4" /> Figura 4. Imagen de un RTbox. Tiene un puerto USB-B, un puerto de entrada de la luz, un puerto de entrada de pulso / de sonido, un puerto de entrada de pulsador externo, y un puerto de salida TTL.

<ol>
	<li>Li, X., Lu, Z. -. L., Xu, P., Jin, J., Zhou, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+high+gray-level+resolution+monochrome+displays+with+conventional+computer+graphics+cards+and+color+monitors.">Generating high gray-level resolution monochrome displays with conventional computer graphics cards and color monitors.</a> Journal of Neuroscience Methods. 130 (1), 9-18 (2003).</li><li>Pelli, D. G., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+control+of+contrast+on+microcomputer+displays.">Accurate control of contrast on microcomputer displays.</a> Vision Research. 31, 1337-1350 (1991).</li><li>Li, X., Liang, Z., Kleiner, M., Lu, Z. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RTbox:+A+device+for+highly+accurate+response+time+measurements.">RTbox: A device for highly accurate response time measurements.</a> Behavior Research Methods. 42 (1), 212-225 (2010).</li><li>Lu, Z. L., Sperling, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second-order+reversed+phi.">Second-order reversed phi.</a> Perception Psychophysics. 61, 1075-1088 (1999).</li><li>Colombo, E., Derrington, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+calibration+of+CRT+monitors.">Visual calibration of CRT monitors.</a> Displays. 22, 87-95 (2001).</li></ol>

No hay conflictos de interés declarado.

El conmutador de vídeo ofrece un método compacto, de bajo costo, y fácil de usar para generar la resolución de alta luminosidad muestra utilizando convencionales tarjetas de gráficos por ordenador y monitores analógicos de color. Se conecta entre las salidas analógicas de la tarjeta gráfica y el monitor. También puede cambiar entre los modos de pantalla monocromática y cromática, proporcionando mayor comodidad. Su canal de disparo permite a los investigadores para sincronizar otros equipos a pantallas visuale...

enabling high grayscale resolution displays and accurate response time measurements on conventional computers

Sistemas de visualización basados ​​en gráficos por ordenador las tarjetas convencionales son capaces de generar imágenes con resolución de 8 bits escala de grises. Sin embargo, la mayoría de los experimentos en la investigación de la visión requieren pantallas con más de 12 bits de resolución de luminancia. Hay varias soluciones disponibles. Bit + + 1 y 2 DataPixx utilizar la interfaz visual digital (DVI) salida de las tarjetas gráficas y de alta resolución (14 o 16-bit) convertidores de digital a analógico para manejar los dispositivos analógicos de pantalla. El 3 VideoSwitcher descrito aquí combina señales de vídeo analógicas de los canales rojo y azul de las tarjetas gráficas con diferentes pesos utilizando una red resistente pasivo de 4 y un circuito activo para proporcionar señales idénticas de video para los tres canales de monitores a color. El método proporciona una manera económica para permitir a pantallas de alta resolución monocromática utilizando tarjetas gráficas y monitores analógicos convencionales. También puede proporcionar señales de disparo que se pueden utilizar para marcarinicios de estímulo, lo que hace que sea fácil de sincronizar visuales con las grabaciones fisiológicas o mediciones de tiempo de respuesta. Aunque los teclados y ratones de ordenador se utilizan con frecuencia en la medición de los tiempos de respuesta (TR), la exactitud de estas mediciones es muy baja. El RTbox es un hardware especializado y software para medidas precisas de RT. Conectado al ordenador host a través de una conexión USB, el conductor del RTbox es compatible con todos los sistemas operativos convencionales. Utiliza un reloj del microprocesador y de alta resolución para registrar las identidades y el calendario de eventos de botón, que se almacenan hasta que el ordenador central los recupera. Los eventos de botón registradas no se verán afectados por la incertidumbre de tiempo o sesgos potenciales asociados con la transmisión y procesamiento de datos en el ordenador anfitrión. El almacenamiento asíncrono simplifica enormemente el diseño de los programas de usuario. Existen varios métodos para sincronizar los relojes de la RTbox y el Comput de acogidaer. El RTbox también puede recibir disparadores externos y ser usado para medir RT con respecto a los eventos externos. Tanto VideoSwitcher y RTbox están disponibles para los usuarios a comprar. La información relevante y muchos programas de demostración se puede encontrar en <a href="http://lobes.usc.edu/">http://lobes.usc.edu/</a> .

Watch this Scientific Journal Video about Enabling High Grayscale Resolution Displays and Accurate Response Time Measurements on Conventional Computers at JoVE.com

Enabling High Grayscale Resolution Displays and Accurate Response Time Measurements on Conventional Computers

Hardware de la computadora convencional no puede generar los estímulos visuales con suficientemente alta en escala de grises tiempos de resolución y la medida de respuesta con la suficiente precisión. Se describe cómo utilizar el VideoSwitcher para producir pantallas de alta resolución monocromática, y la RTbox para medir los tiempos de respuesta con una alta precisión en el hardware de ordenador convencional.

Habilitación de alta resolución en escala de grises Muestra y medidas precisas de tiempo de respuesta en los ordenadores convencionales

Display systems based on conventional computer graphics cards are capable of generating images with 8-bit gray level resolution. However, most experiments ...

enabling-high-grayscale-resolution-displays-accurate-response-time

Research

Education

JoVE Core

JoVE Journal

Electrical Engineering

Neuroscience

Unit 1

Transient and Steady-state Response Analysis

SCIENTISTS IN ACTION

9.3K vistas.  The Ohio State University. Hardware de la computadora convencional no puede generar los estímulos visuales con suficientemente alta en escala de grises tiempos de resolución y la medida de respuesta con la suficiente precisión. Se describe cómo utilizar el VideoSwitcher para producir pantallas de alta resolución monocromática, y la RTbox para medir los tiempos de respuesta con una alta precisión en el hardware de ordenador convencional.

Video: Enabling High Grayscale Resolution Displays and Accurate Response Time Measurements on Conventional Computers

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

Un ensayo Cortar organotípicos de alta resolución de imágenes a intervalos de tiempo de la migración neuronal en el cerebro postnatal

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

Investigación

Neurociencias

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

Un enfoque rápido para imágenes de fluorescencia de alta resolución en rodajas de cerebro semi-gruesa

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

De alta resolución Magnética Funcional por imágenes de los métodos de resonancia cerebro medio humano

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

Imágenes de alta resolución en vivo del comportamiento de las células en el desarrollo de neuroepitelio

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

En Vivo electrofisiológicos mediciones sobre los nervios ciáticos de ratones

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

La detección de ARNm axón localizado en las secciones cerebrales Uso de alta resolución<em&gt; In Situ</em&gt; La hibridación

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

De alta resolución de imagen de resonancia magnética estructural del subcorteza Humano<I&gt; En Vivo</I&gt; Y Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

Resolución de alta Análisis Cuantitativo Inmunoelectrónica de Receptores de Membrana en la retina cinta sinapsis

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and cell-cell interactions in the CNS in real-time. Previously, studying these cell-specific responses after injury often required complicated setups or the transfer of cells or animals into different, non-physiological environments, confounding immediate and short-term analysis. For example, drug-mediated ablation approaches often lack the specificity that is required to study single-cell responses and immediate cell-cell interactions. Similarly, while high-power pulsed laser ablation approaches provide very good control and tissue penetration, they require specialized equipment that can complicate real-time visualization of cellular responses. The refined UV laser ablation approach described here allows researchers to stress or kill an individual cell in a dose- and time-dependent manner using a conventional confocal microscope equipped with a 405-nm laser. The method was applied to selectively ablate a single neuron within a dense network of surrounding cells in the zebrafish spinal cord. This approach revealed a dose-dependent response of the ablated neurons, causing the fragmentation of cellular bodies and anterograde degeneration along the axon within minutes to hours. This method allows researchers to study the fate of an individual dying cell and, importantly, the instant response of cells-such as microglia and astrocytes-surrounding the ablation site.

Targeted manipulations to cause directed stress or death in individual cells have been relatively difficult to accomplish. Here, a single-cell-resolution ablation approach to selectively stress and kill individual cells in cell culture and living animals is described based on a standard confocal UV laser.

Desencadenar el estrés celular y la muerte Uso convencional UV láser confocal Microscopía

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Incremento dependiente del tiempo en la respuesta de la red a la estimulación de cultivos de células neuronales en matrices de microelectrodos

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...