1) Preparación <ol><li> Para empezar, el asiento del sujeto en una silla cómoda frente a una pantalla de ordenador. </li><li> Utilice una cinta métrica para asegurar la distancia entre la pantalla y nasion del sujeto es de 60 cm. </li><li> Finalmente, coloque una máscara que bloquea la luz en los ojos del sujeto. </li></ol> 2) La determinación del umbral fosfeno sobre V1/V2 <ol><li> Saber umbral fosfeno del sujeto será importante para explorar el comportamiento fosfénico más adelante en este protocolo. Para determinar este umbral, primero ajuste la máquina TMS al poder el 70%. </li><li> Sostenga la bobina por lo que la parte superior de la línea media en forma de 8 caras craneal. </li><li> Comience un solo pulso de estimulación en un punto cerca de 3 cm por encima del inion. </li><li> Después de cada pulso, pedir al paciente que informe de cualquier experiencia fosfénica. </li><li> Comenzar a mover la bobina en una cuadrícula pequeña búsqueda en torno a esta región (V1/V2). </li><li> Determine la ubicación en la estimulación provoca informes fosfeno coherente e inequívoca del sujeto. Se ha teorizado que la EMT no será capaz de obtener fosfenos en torno al 40% de la población, por lo que existe la posibilidad de que el sujeto no se informe de cualquier cosa. Si este es el caso, por desgracia, este experimento no funcionará. El tema debe ser permitido salir. </li><li> Una vez que el fosfénico &quot;punto caliente&quot; ha sido localizado, regula la potencia de TMS arriba y hacia abajo hasta que los fosfenos informes sometidos por exactamente 3 de 6 pulsos consecutivos. Este nivel es el sujeto V1/V2 umbral. Si está disponible, neuronavegación se pueden utilizar para lograr una mayor precisión espacial en la localización de la zona activa y la orientación V1/V2. </li></ol> 3) La determinación del umbral fosfénica el V5/MT + Complejo <ol><li> Utilizando el mismo procedimiento anterior y los parámetros, ahora vamos a determinar el umbral fosfeno sobre el V5/MT + complejo. Para encontrar esta región, comienzan en un punto de 3 cm dorsal y lateral de 5 cm de inion del sujeto. </li><li> Después de cada pulso, pedir al paciente que informe de cualquier experiencia fosfénica. </li><li> Una vez más, utilizando pulsos individuales, iniciar un patrón de búsqueda de la red pequeña hasta fosfenos inequívoca y uniforme son provocados. </li><li> Por último, regula la potencia de TMS arriba y hacia abajo hasta que los fosfenos informes sometidos por exactamente 3 de 6 pulsos consecutivos. Este nivel es V5/MT del sujeto + umbral complejo. De nuevo, si neuronavegación disponibles, se puede utilizar para lograr una mayor precisión espacial al localizar y llegar a los V5/MT + punto de acceso. </li></ol> 4) Determinar el comportamiento de referencia fosfeno <ol><li> Una vez que el umbral fosfeno se ha determinado para ambas áreas visuales, es necesario medir el comportamiento de referencia fosfénico. Para ello, primero retire el tema de la luz de bloqueo de la máscara. </li><li> A continuación, instruir al sujeto a mirar a una cruz de una fijación estable se presenta en el centro de la pantalla del ordenador durante 60 segundos. </li><li> Vuelva a colocar la máscara y, sobre el punto caliente V1/V2, generar un tren de pulsos individuales de 3 segundos a 120% V1/V2 umbral. </li><li> Espere cinco segundos y la conducta de otro tren. </li><li> Una vez más, esperar cinco segundos y realizar un tercer tren. </li><li> Después de que el tren de pulsos tercer single, pedir al paciente que describa las características del lugar y el motivo de cualquier provocado fosfenos. Esta será la línea de base. </li><li> Repita este mismo procedimiento en los V5/MT + punto caliente (no olvide restablecer el poder TMS al 120% del umbral de V5/MT +). </li></ol> 5) Condición número uno <ol><li> Una vez que la base phophenic se ha determinado, eliminar el tema de la luz de bloqueo de la máscara una vez más. </li><li> Instruir a los sujetos a mirar a una cruz fijación estable se presenta en el centro de la pantalla del ordenador durante 60 segundos. Esta vez, en lugar de una pantalla en blanco, vamos a incorporar una serie de puntos que se mueven en varios lugares alrededor de la cruz. <img src="/files/ftp_upload/2273/2273fig1.jpg" alt="Figura 1" /> Figura 1 Adaptación de los estímulos para una condición:. Movimiento de traslación simple. Todos los puntos se mueven coherentemente hacia la izquierda o la derecha. Asegúrese de que todos los puntos se mueven en la misma dirección. Este estímulo debe servir para generar la adaptación visual: un fenómeno en el que los cambios en la excitabilidad neuronal inducida por la exposición prolongada a un estímulo sirve para sesgar la percepción de los estímulos presentados posteriormente. </li><li> Después de 60 segundos, vuelva a colocar la máscara y, sobre el punto caliente V1/V2, generar un tren de pulsos solo por 3 segundos al 120% V1/V2 umbral. </li><li> Espere cinco segundos y repite dos veces más. </li><li> Después de tres trenes, pedir al paciente que informe la ubicación y las características de cualquier motivo provocado fosfenos. Si está disponible, el uso de un sistema de seguimiento de movimiento de los ojos en un cuarto oscuro puede ayudar a lograr una mayor precisión y el control cuantitativo de los movimientos oculares realizadas por los sujetos durante elicitations fosfeno. </li><li> Continúe esta secuencia hasta que three-train/report phospcomportamiento HeNe vuelve a la actividad de referencia. </li><li> Repita este procedimiento para la V5/MT + punto de acceso complejo. </li></ol> 6) Estado de número dos <ol><li> En esta segunda condición, repita el mismo procedimiento utilizado para la condición de uno. Esta vez, sin embargo, en lugar de presentar el tema con una serie de puntos que se mueven en una dirección singular, presente el tema con una serie de puntos de cada movimiento en su dirección cardinal relativa distancia de un punto central - similar a un &quot;estallido estrella&quot; . <img src="/files/ftp_upload/2273/2273fig2.jpg" alt="Figura 2" /> Figura 2 Adaptación de los estímulos para la condición de dos:. Movimiento radial. Puntos se mueven en los respectivos puntos cardinales ya sea hacia o desde un punto central. </li><li> Al igual que antes, después de 60 segundos, vuelva a colocar la máscara, llevar a cabo una serie de tres, los trenes umbral de 120% en V1/V2, pedir un informe de tema, y ​​continuará hasta el regreso de línea de base. </li><li> Repita el procedimiento para + V5/MT. </li></ol> 7) Diagrama <ol><li> Como una opción, después de cada estado (o después de todas las condiciones se han completado), pida a los sujetos a dibujar las regiones y la conducta motriz de cada serie de phopsphenes en un gráfico. Esto no es un paso integral, pero le proveerá de otro conjunto de datos interpretables. </li></ol> 8) Los resultados representativos La adaptación a los estímulos visuales motivo unidireccional debería generar un movimiento fosfénico idénticos en V1/V2. <img src="/files/ftp_upload/2273/2273fig3.jpg" alt="Figura 3" /> Figura 3. Ejemplos de fosfenos inducidos desde V1/V2 en el hemisferio derecho e izquierdo durante la condición de uno (basado en los dibujos de los sujetos). El V5/MT + fosfenos complejo también deberían verse afectadas, de tal manera que el fosfeno aparece ahora como una suma de la dirección del movimiento en el estímulo de la adaptación y el fosfeno línea de base. Sin embargo, la adaptación visual a la forma radial debería generar un movimiento estelar fosfénico idénticos desde el V5/MT + complejo, pero no debe cambiar la línea de base V1/V2. <img src="/files/ftp_upload/2273/2273fig4.jpg" alt="Figura 4" /> Figura 4. Ejemplos de fosfenos inducidos de la V5/MT + complejo en el hemisferio derecho e izquierdo durante la condición de dos (basado en los dibujos de los sujetos).

<ol>
	<li>Pascual-Leone, A., Davey, M., Wassermann, E. M., Rothwell, J., Puri, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Transcranial Magnetic Stimulation. , (2002).</li><li>Walsh, V., Pascual-Leone, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Transcranial Magnetic Stimulation: A Neurochronometrics of Mind. , (2005).</li><li>Silvanto, J., Muggleton, N. G., Cowey, A., Walsh, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+adaptation+reveals+state-dependent+effects+of+transcranial+magnetic+stimulation.">Neural adaptation reveals state-dependent effects of transcranial magnetic stimulation.</a> European Journal of Neuroscience. 25, 1874-1881 (2007).</li><li>Silvanto, J., Pascual-Leone, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State-Dependency+of+Transcranial+Magnetic+Stimulation.">State-Dependency of Transcranial Magnetic Stimulation.</a> Brain Topography. 21, 1-10 (2008).</li><li>Silvanto, J., Cattaneo, Z., Battelli, L., Pascual-Leone, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baseline+cortical+excitablility+determines+whether+TMS+disrupts+or+facilitates+behavior.">Baseline cortical excitablility determines whether TMS disrupts or facilitates behavior.</a> Journal of Neurophysiology. 99, 2725-2730 (2008).</li><li>Silvanto, J., Muggleton, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+the+validity+of+the+TMS+state-dependency+approach:+targeting+functionally+distinct+motion-selective+neural+populations.">Testing the validity of the TMS state-dependency approach: targeting functionally distinct motion-selective neural populations.</a> Neuroimage. 40, 1841-1848 (2008).</li><li>Tootell, R. B., Rappas, J. B., Kwong, K. K., Malach, R., Born, R. T., Brady, T. J., Rosen, B. R., Belliveau, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+human+MT+and+related+visual+cortical+areas+using+magnetic+resonance+imaging.">Functional analysis of human MT and related visual cortical areas using magnetic resonance imaging.</a> Journal of Neuroscience. 15, 3215-3230 (1995).</li><li>Singh, K. D., Smith, A. T., Greenlee, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+frequency+and+direction+sensitivities+of+human+visual+areas+measured+using+fMRI.">Spatiotemporal frequency and direction sensitivities of human visual areas measured using fMRI.</a> Neuroimage. 12, 550-564 (2000).</li><li>Rutchmann, R. M., Schrauf, M., Greenlee, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+activation+during+dichoptic+presentation+of+optic+flow+stimuli.">Brain activation during dichoptic presentation of optic flow stimuli.</a> Exp Brain Res. 134, 533-537 (2000).</li><li>Morrone, M. C., Tosetti, M., Montanaro, D., Fiorentini, A., Cioni, G., Burr, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cortical+area+that+responds+specifically+to+optic+flow+revealed+by+fMRI.">A cortical area that responds specifically to optic flow revealed by fMRI.</a> Nat. Neuroscience. 3, 1322-1328 (2000).</li></ol>

Este experimento se en el corazón de la dependencia estatal. Las neuronas en V1/V2 se cree que corresponden con el movimiento simple, direccionales 7,8 Por lo tanto, la adaptación a los estímulos motivo unidireccional aumento de la excitabilidad de las neuronas registro de este movimiento -. Como tales, deben ser los primeros en reaccionar a la TMS pulso. El V5/MT + complejo también consta de neuronas en sintonía con el movimiento de traslación simple, por lo tanto, los fosfenos inducidos de esta regió...

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<td>Light Blocking Eye Mask</td>
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<td>Stimuli Developed &amp; Presented on Computer using Adobe Photoshop</td>
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<td>Any Single Pulse Capable TMS Device</td>
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state-dependency effects on tms:  a look at motive phosphene behavior

La estimulación magnética transcraneal (TMS) es un neurostimulatory no invasiva y la técnica neuromoduladores que de forma transitoria o duradera modular la excitabilidad cortical (aumento o disminución de la misma) a través de la aplicación de pulsos localizados campo magnético. 1,2 En el ámbito de la EMT, el término dependencia del Estado se refiere a la condición inicial, de referencia de la región neural específica para la estimulación. Como se puede deducir, los efectos de la TMS puede (y) varían en función de la susceptibilidad y capacidad de respuesta primaria de la zona cortical específica. 3,4,5 En este experimento, vamos a examinar el concepto de dependencia del Estado a través de la estimulación y la experiencia subjetiva de los fosfenos motivo. Fosfenos son percibidas visualmente destellos de pequeñas luces provocadas por los pulsos electromagnéticos de la corteza visual. Estas pequeñas luces puede asumir distintas características en función del tipo de la corteza visual está siendo estimulado. En este estudio, vamos a tener como objetivo fosfenos motivo que provocó a través de la estimulación de la V1/V2 y las regiones visuales V5/MT + complejo 6.

Watch this Scientific Journal Video about State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior at JoVE.com

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

En este artículo, se examinan los efectos de la dependencia estatal visual relevante en TMS inducida presentaciones motivo fosfénico.

La dependencia estatal Efectos sobre la EMT: Una mirada a la conducta motriz fosfeno

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

state-dependency-effects-on-tms-a-look-at-motive-phosphene-behavior

Research

JoVE Journal

Neuroscience

10.5K vistas.  Beth Israel Deaconess Medical Center. En este artículo, se examinan los efectos de la dependencia estatal visual relevante en TMS inducida presentaciones motivo fosfénico.

Video: State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

El dispositivo de TMS NeuroStar: Realización de la FDA aprobó el Protocolo para el tratamiento de la depresión

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Investigación

Neurociencias

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1&deg;C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

La criopreservación de tejido cortical Bloques para la generación de cultivos neuronales altamente enriquecido

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

Studying the Integration of Adult-born Neurons

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the hippocampal dentate gyrus throughout life. Previous reports have shown that adult hippocampal neurogenesis is associated with diverse brain disorders, including epilepsy, schizophrenia, depression and anxiety (1). Deciphering the process of normal and aberrant adult-born neuron integration may shed light on the etiology of these diseases and inform the development of new therapies.
SGZ adult neurogenesis mirrors embryonic and post-natal neuronal development, including stages of fate specification, migration, synaptic integration, and maturation. However, full integration occurs over a prolonged, 6-week period. Initial synaptic input to adult-born SGZ dentate granule cells (DGCs) is GABAergic, followed by glutamatergic input at 14 days (2). The specific factors which regulate circuit formation of adult-born neurons in the dentate gyrus are currently unknown.
Our laboratory uses a replication-deficient retroviral vector based on the Moloney murine leukemia virus to deliver fluorescent proteins and hypothesized regulatory genes to these proliferating cells. This viral technique provides high specificity and resolution for analysis of cell birth date, lineage, morphology, and synaptogenesis.
A typical experiment often employs two or three viruses containing unique label, transgene, and promoter elements for single-cell analysis of a desired developmental process in vivo. The following protocol describes a method for analyzing functional newborn neuron integration using a single green (GFP) or red (dTomato) fluorescent protein retrovirus and patch-clamp electrophysiology.

A way to study the integration of newborn dentate granule cells in adult animals is described. This technique uses an engineered retrovirus to label newborn neurons, followed by electrophysiological recordings to determine in vivo functional integration.

El estudio de la integración de los adultos nacidos en las neuronas

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

Las investigaciones sobre alteraciones de la función del circuito del hipocampo después de una lesión cerebral traumática leve

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

Un procedimiento para el implante Cámara Espinal para Longitudinal<em&gt; En Vivo</em&gt; Imágenes de la médula espinal de ratón

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

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 ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Uso de los canales lineales de agarosa para estudiar<em&gt; Drosophila</em&gt; Comportamiento de las larvas de arrastre

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

Combinando Optogenetics con microRNAs artificiales para caracterizar los efectos de Gene caída en la función presináptica en circuitos neuronales intactas

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them water-soluble with the ability to reversibly enter and exit membrane lipid bilayers. These styryl dyes are relatively non-fluorescent in aqueous medium, but insertion into the outer leaflet of the plasma membrane causes a &#62;40X increase in fluorescence. In neuronal synapses, FM dyes are internalized during SV endocytosis, trafficked both within and between SV pools, and released with SV exocytosis, providing a powerful tool to visualize presynaptic stages of neurotransmission. A primary genetic model of glutamatergic synapse development and function is the Drosophila neuromuscular junction (NMJ), where FM dye imaging has been used extensively to quantify SV dynamics in a wide range of mutant conditions. The NMJ synaptic terminal is easily accessible, with a beautiful array of large synaptic boutons ideal for imaging applications. Here, we compare and contrast the three ways to stimulate the Drosophila NMJ to drive activity-dependent FM1-43 dye uptake/release: 1) bath application of high [K+] to depolarize neuromuscular tissues, 2) suction electrode motor nerve stimulation to depolarize the presynaptic nerve terminal, and 3) targeted transgenic expression of channelrhodopsin variants for light-stimulated, spatial control of depolarization. Each of these methods has benefits and disadvantages for the study of genetic mutation effects on the SV cycle at the Drosophila NMJ. We will discuss these advantages and disadvantages to assist the selection of the stimulation approach, together with the methodologies specific to each strategy. In addition to fluorescent imaging, FM dyes can be photoconverted to electron-dense signals visualized using transmission electron microscopy (TEM) to study SV cycle mechanisms at an ultrastructural level. We provide the comparisons of confocal and electron microscopy imaging from the different methods of Drosophila NMJ stimulation, to help guide the selection of future experimental paradigms.

Synaptic vesicle (SV) cycling is the core mechanism of intercellular communication at neuronal synapses. FM dye uptake and release are the primary means of quantitatively assaying SV endo- and exocytosis. Here, we compare all the stimulation methods to drive FM1-43 cycling at the Drosophila neuromuscular junction (NMJ) model synapse.

Tinte de FM ciclismo en la sinapsis: comparación de potasio alto despolarización, eléctrico y Channelrhodopsin estimulación

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Efectos de Neurotrauma inducido ráfaga en roedor a presión media de las arterias cerebrales

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...