1. Determinar el umbral del motor en reposo: <ol class="lalpha"><li> Para empezar, el asiento de su sujeto en una silla cómoda. </li><li> A continuación, se aplican electrodos EMG de superficie sobre el músculo inerosseus primera dorsal de la mano derecha del sujeto. (INSERTAR Figura 1) Si usted está teniendo problemas para localizar este músculo, pedir al paciente que realiza un movimiento de pellizco o para mover su / su índice de ida y vuelta - esto debería hacer que el músculo más fácil de localizar. </li><li> Coloque un electrodo de masa en la parte carnosa del antebrazo del sujeto. </li><li> Determinar la ubicación de la corteza izquierda del sujeto motor. Aunque esta área es ligeramente diferente para cada uno, por lo general, será de alrededor de 3,5 cm de rostrolateral craneal vértice. No tenga miedo de explorar una pequeña red en torno a esta región y ajustar el ángulo de la estimulación hasta encontrar el motor &quot;caliente&quot;. </li><li> Una vez que hayas encontrado M1, a partir de 50% de potencia, empezar la estimulación de pulso único de esta región y medir los diputados al Parlamento Europeo resultante evocado. </li><li> Ajustar la potencia hacia arriba o hacia abajo hasta que son capaces de producir un eurodiputado de más de 50 mV en exactamente 5 de cada 10 pulsos consecutivos. Este nivel es el umbral del sujeto motor en reposo. </li></ol> 2. Determinar el umbral motor activo <ol class="lalpha"><li> Utilizando el mismo electrodo de configuración como se describió anteriormente, pedir al paciente que su índice pizca / su derecha y el pulgar con un 20% de presión. </li><li> Mientras que el tema ejerce en este puesto pellizcos, comenzará un solo pulso de estimulación en un 50% más de potencia M1 y medir los diputados al Parlamento Europeo resultante. </li><li> Ajustar la potencia hacia arriba o hacia abajo hasta que son capaces de producir un eurodiputado de más de 200 mV en exactamente 5 de cada 10 pulsos consecutivos. Este nivel es el umbral del sujeto activo del motor. </li></ol> 3. Determinar de referencia Mep <ol class="lalpha"><li> Para determinar de un sujeto de referencia amplitud MEP, estimular motor hotspot en el umbral de motor 120% de descanso con 10 pulsos individuales. </li><li> Medir el MEP después de cada pulso. </li><li> La media de los valores MEP de tres lotes de 10 pulsos. </li></ol> 4. Establecer los parámetros de TMS <ol class="lalpha"><li> Estaremos utilizando estándares Theta-Burst parámetros: tres pulsos a 50 Hz a una intensidad del 80% del umbral motor activo de intervalos de 200 ms. </li><li> Para la administración intermitente de TBS, generar un tren de estimulación de dos segundos (en los parámetros antes mencionados), seguido de ocho segundos de estimulación. Repita para 190 segundos (un total de 600 impulsos). ITB se ha demostrado que facilitan la actividad cortical. </li><li> Para la administración continua Theta-Burst, generar un tren de estimulación continua (en los parámetros antes mencionados) durante 47 segundos (un total de 600 impulsos). CTBS se ha demostrado que inhiben la actividad cortical. </li></ol> 5. Tms estimulación <ol class="lalpha"><li> Para este protocolo, administrar o ITBS o CTBS en el anterior punto de acceso establecido motor M1. </li></ol> 6. Determinar después de la estimulación del MEP <ol class="lalpha"><li> Después de TBS, como antes, estimular el motor hotspot en el umbral de motor 120% de descanso con 10 de un solo pulso. </li><li> Promedio del lote de 10 niveles de MEP. </li><li> Recoge 10 nuevos impulsos promedio por lotes a los 5 minutos, 10 minutos, 20, 30, 40, 50, 60, 75, 90, 105 y 120 minutos post-estimulación. </li></ol> 7. Resultados representante Cuando se utiliza un patrón de ITB, se debe notar un aumento en los valores de MEP por un período que refleja el estado natural del sujeto de la plasticidad. Del mismo modo, cuando se utiliza un patrón de CTBS, usted debe notar una disminución en los valores MEP durante un período que refleja el estado natural del sujeto de la plasticidad. <img src="/files/ftp_upload/2272/2272fig1.jpg" alt="Figura 1" /> Figura 1. Esquema de colocación de los electrodos para la medida del MEP. <img src="/files/ftp_upload/2272/2272fig2.jpg" alt="Figura 2" /> Figura 2. Esquema del patrón de estimulación ITB. <img src="/files/ftp_upload/2272/2272fig3.jpg" alt="Figura 3" /> Figura 3. Esquema del patrón de estimulación CTBS.

<ol>
	<li>Pfeiffer, B. E., Huber, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+state+of+synapses+in+fragile+x+syndrome.">The state of synapses in fragile x syndrome.</a> Neuroscientist. 15, 549-567 (2009).</li><li>O'Donnel, W. T., Warren, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+decade+of+molecular+studies+of+fragile+x+syndrome.">A decade of molecular studies of fragile x syndrome.</a> Annual Review of Neuroscience. 25, 315-338 (2002).</li><li>Tassone, F., Hagerman, R. J., Taylor, A. K., Mills, J. B., Wood, S., Gane, L. W., Hagerman, P. 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> American Journal of Human Genetics. 65, A493-A493 (1999).</li><li>Hagerman, R. J., Miller, L. J., McGrath-Clarke, J., Riley, K., Goldson, E., Harris, S. W., Simon, J., Church, K., Bonnell, J., Ognibene, T. C., McIntosh, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+stimulants+on+electrodermal+studies+in+fragile+x+syndrome.">Influence of stimulants on electrodermal studies in fragile x syndrome.</a> Microscopy Research and Technique. 57, 168-173 (2002).</li><li>Miller, L. J., McIntosh, D. N., McGrath, J., Shyu, V., Lampe, M., Taylor, A. K., Tassone, F., Neitzel, K., Stackhouse, T., Hagerman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrodermal+response+to+sensory+stimuli+in+individuals+with+fragile+x+syndrome:+a+preliminary+report.">Electrodermal response to sensory stimuli in individuals with fragile x syndrome: a preliminary report.</a> American Journal of Medical Genetics. 83, 268-279 (1999).</li><li>Hagerman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fragile+X:+perspectives+from+birth+through+aging.">Fragile X: perspectives from birth through aging.</a> Biological Psychiatry. 57, 73s-73s (2005).</li><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>Thickbroom, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcranial+magnetic+stimulation+and+synaptic+plasticity:+experimental+framework+and+human+models.">Transcranial magnetic stimulation and synaptic plasticity: experimental framework and human models.</a> Experimental Brain Research. 180, 583-593 (2007).</li><li>Huang, Y. Z., Edwards, M. J., Rounis, E., Bhatia, K. P., Rothwell, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theta+burst+stimulation+of+the+human+motor+cortex.">Theta burst stimulation of the human motor cortex.</a> Neuron. 45, 201-206 (2005).</li><li>Oberman, L., Eldaief, M. C., Gautam, S., Fecteau, S., Tormos, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+Cortical+Plasticity+in+Adults+with+Asperger's+Syndrome.">Abnormal Cortical Plasticity in Adults with Asperger's Syndrome.</a> , .</li></ol>

Sabiendo que la duración prolongada de la EMT secuelas es un reflejo de la plasticidad neuronal atípico, los estudios de este tipo podría servir para crear mejores y más precisos protocolos para el diagnóstico de autismo y trastornos relacionados. Del mismo modo, con más datos, podemos ser capaces de encontrar marcadores biológicos temporal para otras patologías neurológicas - como el síndrome de X frágil. En este caso, aunque aún no hay una opción de recuperación terapéutica,...

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tms: using the theta-burst protocol to explore mechanism of plasticity in individuals with fragile x syndrome and autism

Síndrome X Frágil (FXS), también conocido como síndrome de Martin-Bell, es una anomalía genética se encuentra en el cromosoma X 1,2 individuos que sufren de anormalidades FXS pantalla en la expresión del gen FMR1 -. Una proteína necesaria para el desarrollo típico, de los nervios sanos. 3 Los datos recientes han sugerido que la pérdida de esta proteína puede causar la corteza que se hiperexcitables afectando los patrones generales de la plasticidad neuronal. 4,5 Además, X frágil muestra una fuerte comorbilidad con autismo: de hecho, el 30% de los niños con SXF son diagnosticados con autismo, y 2 - 5% de los niños autistas sufren de FXS 6. Estimulación Magnética Transcraneal (a neurostimulatory no invasiva y la técnica neuromoduladores que transitoria o duradera modular la excitabilidad cortical mediante la aplicación de pulsos de campo magnético localizado 7,8) representa un método único de explorar la plasticidad y las manifestaciones del SXF en las personas afectadas. Más específicamente, Theta-Burst estimulación (TBS), un protocolo de estimulación específica que se muestra para modular la plasticidad cortical con una duración de hasta 30 minutos después de terminar la estimulación en poblaciones sanas, ya ha demostrado ser una herramienta eficaz en la exploración de la plasticidad anormal 9,10. Estudios recientes han demostrado los efectos de la última TBS considerablemente mayor en los individuos en el espectro autista -. Hasta 90 minutos 11 Este prolongado efecto de duración sugiere una anomalía en el estado del cerebro plasticidad natural en los individuos autistas - similar a la hiperexcitabilidad inducida por la frágil Síndrome X. En este experimento, utilizando un solo motor de impulsos potenciales evocados (MEP) como nuestro punto de referencia, vamos a explorar los efectos de los intermitentes y continuas TBS sobre la plasticidad cortical en personas que sufren de FXS y los individuos en el espectro autista.

Watch this Scientific Journal Video about TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism at JoVE.com

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

En este artículo examinamos los efectos de la Theta-Burst estimulación EMT en la plasticidad cortical en personas que sufren de síndrome de X frágil y los individuos en el espectro autista.

TMS: Uso del protocolo Theta-Burst para explorar Mechasnism de plasticidad en los individuos con el síndrome de X frágil y el autismo

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

tms-using-theta-burst-protocol-to-explore-mechanism-plasticity

Research

JoVE Journal

Neuroscience

16.8K vistas.  Beth Israel Deaconess Medical Center. En este artículo examinamos los efectos de la Theta-Burst estimulación EMT en la plasticidad cortical en personas que sufren de síndrome de X frágil y los individuos en el espectro autista.

Video: TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

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

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

Análisis morfológico de Drosophila Larvas dendritas neurona sensorial periférica y axones Con mosaicos genéticos

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval body wall contains several cell types that are models to study cell and tissue morphogenesis. For example tracheae are used to examine tube morphogenesis1, and the dendritic arborization (DA) sensory neurons of the Drosophila larva have become a primary system for the elucidation of general and neuron-class-specific mechanisms of dendritic differentiation2-5 and degeneration6.
The shape of dendrite branches can vary significantly between neuron classes, and even among different branches of a single neuron7,8. Genetic studies in DA neurons suggest that differential cytoskeletal organization can underlie morphological differences in dendritic branch shape4,9-11. We provide a robust immunological labeling method to assay in vivo microtubule organization in DA sensory neuron dendrite arbor (Figures 1, 2, Movie 1). This protocol illustrates the dissection and immunostaining of first instar larva, a stage when active sensory neuron dendrite outgrowth and branching organization is occurring 12,13.
In addition to staining sensory neurons, this method achieves robust labeling of microtubule organization in muscles (Movies 2, 3), trachea (Figure 3, Movie 3), and other body wall tissues. It is valuable for investigators wishing to analyze microtubule organization in situ in the body wall when investigating mechanisms that control tissue and cell shape.

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

To understand how complex cell shapes, such as neuronal dendrites, are achieved during development, it is important to be able to accurately assay microtubule organization. Here we describe a robust immunohistological labeling method to examine microtubule organization of dendritic arborization neuron sensory dendrites, trachea, muscle, and other Drosophila larva body wall tissues.

Etiquetado inmunohistológica de microtúbulos en las dendritas neurona sensorial, tráqueas y los músculos en el Drosophila Larva pared del cuerpo

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

Difusión lateral y exocitosis de proteínas de membrana en neuronas cultivadas evaluó a través de la recuperación de fluorescencia y la pérdida de fluorescencia photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.
We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.

The Proboscis Extension Response (PER) conditioning protocol, developed for the honey bee (Apis mellifera), provides an ecologically-relevant and easily quantifiable means for studying several different mechanisms of learning in many insect species.

Un Protocolo de Respuesta de probóscide Extensión para la Investigación de la plasticidad de comportamiento en Insectos: Aplicación a Basic, Biomédica y de Investigación Agrícola

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

La inducción de la plasticidad de astrocítico Receptores por manipulación de Neuronales cocción Precios

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Developing HiPSC Derived Serum Free Embryoid Bodies for the Interrogation of 3-D Stem Cell Cultures Using Physiologically Relevant Assays

Although a number of in vitro disease models have been developed using hiPSCs, one limitation is that these two-dimensional (2-D) systems may not represent the underlying cytoarchitectural and functional complexity of the affected individuals carrying suspected disease variants. Conventional 2-D models remain incomplete representations of in vivo-like structures and do not adequately capture the complexity of the brain. Thus, there is an emerging need for more 3-D hiPSC-based models that can better recapitulate the cellular interactions and functions seen in an in vivo system.
Here we report a protocol to develop a 3-D system from undifferentiated hiPSCs based on the serum free embryoid body (SFEB). This 3-D model mirrors aspects of a developing ventralized neocortex and allows for studies into functions integral to living neural cells and intact tissue such as migration, connectivity, communication, and maturation. Specifically, we demonstrate that the SFEBs using our protocol can be interrogated using physiologically relevant and high-content cell based assays such as calcium imaging, and multi-electrode array (MEA) recordings without cryosectioning. In the case of MEA recordings, we demonstrate that SFEBs increase both spike activity and network-level bursting activity during long-term culturing. This SFEB protocol provides a robust and scalable system for the study of developing network formation in a 3-D model that captures aspects of early cortical development.

Here we report a protocol to develop a three-dimensional (3-D) system from human induced-pluripotent stem cells (hiPSCs) called the serum free embryoid body (SFEB). This 3-D model can be used like an organotypic slice culture to model human cortical development and for the physiological interrogation of developing neural circuits.

Desarrollo de cuerpos embrionarios libres de suero derivados de HiPSC para la interrogación de cultivos de células madre tridimensionales usando ensayos fisiológicamente relevantes

Although a number of in vitro disease models have been developed using hiPSCs, one limitation is that these two-dimensional (2-D) systems may not ...

Screening of Axonal Degeneration in Carpal Tunnel Syndrome Using Ultrasonography and Nerve Conduction Studies

Axonal degeneration, indicative of surgical decompression, may coexist in carpal tunnel syndrome (CTS) as the disease progresses. However, the current diagnostic and severity gradation system cannot clearly indicate its coexistence, resulting in&#160;confusion of appropriate treatment prescription. There are also constraints in conventional methods for&#160;differentiation as well. This study aims at introducing an innovative, efficient, and quick screening protocol to differentiate axonal degeneration associated with CTS, using ultrasound and nerve conduction studies (NCS). It starts by using NCS to perform orthodromic stimulation at the wrist, to obtain the sensory conduction of the median and the ulnar nerves respectively. Meanwhile, the motor conduction&#160;of the median nerve is collected by stimulating the palm, wrist, and elbow, followed by the stimulation of the ulnar nerve at the wrist,&#160;below and above the elbow. Then, an ultrasound assessment is performed, using a linear array transducer, with cross-sectional area (CSA) and perimeter (P) at the wrist and at the one-third distal forearm calipered. Ratios (R-CSA, R-P) and changes from wrist to one-third distal forearm (&#916;CSA and &#916;P) are calculated according to a standard format. Potential axonal degeneration coexisting in CTS will be screened according to the criteria of NCS and cut-off values of ultrasound measurements established in a previous study. In terms of its noninvasiveness, low cost, convenience, and efficiency, it is easy to apply ultrasound complimentarily in clinical practice to prescreen patients with potential coexisting axonal degeneration. Nevertheless, the ultrasonographic imaging cannot directly reflect axonal degeneration. It still relies on conventional but invasive methods&#160;such as electromyography (EMG) and biopsy&#160;for confirmation if needed.

Here we present a protocol using nerve conduction studies and ultrasound to screen potential axonal degeneration associated with carpal tunnel syndrome. The criteria for differentiation are established. Compared to conventional approaches, this method is noninvasive, convenient, and efficient, with an overall satisfactory accuracy, sensitivity, and specificity.

Detección de la degeneración Axonal en el síndrome del túnel carpiano mediante ecografía y estudios de conducción nerviosa

Axonal degeneration, indicative of surgical decompression, may coexist in carpal tunnel syndrome (CTS) as the disease progresses. However, the current ...

Comprehensive Autopsy Program for Individuals with Multiple Sclerosis

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 days a year. Participants consent to donate their brain and spinal cord. Most patients were followed by neurologists at the Cleveland Clinic Mellen Center for MS Treatment and Research. Their clinical courses and neurological disabilities are well-characterized. Soon after death, the body is transported to the MS Imaging Center, where the brain is scanned in situ by 3 T magnetic resonance imaging (MRI). The body is then transferred to the autopsy room, where the brain and spinal cord are removed. The brain is divided into two hemispheres. One hemisphere is immediately placed in a slicing box and alternate 1 cm-thick slices are either fixed in 4% paraformaldehyde for two days or rapidly frozen in dry ice and 2-methylbutane. The short-fixed brain slices are stored in a cryopreservation solution and used for histological analyses and immunocytochemical detection of sensitive antigens. Frozen slices are stored at -80 &#176;C and used for molecular, immunocytochemical, and in situ hybridization/RNA scope studies. The other hemisphere is placed in 4% paraformaldehyde for several months, placed in the slicing box, re-scanned in the 3 T magnetic resonance (MR) scanner and sliced into centimeter-thick slices. Postmortem in situ MR images (MRIs) are co-registered with 1 cm-thick brain slices to facilitate MRI-pathology correlations. All brain slices are photographed and brain white-matter lesions are identified. The spinal cord is cut into 2 cm segments. Alternate segments are fixed in 4% paraformaldehyde or rapidly frozen. The rapid procurement of postmortem MS tissues allows pathological and molecular analyses of MS brains and spinal cords and pathological correlations of brain MRI abnormalities. The quality of these rapidly-processed postmortem tissues (usually within 6 h of death) is of great value to MS research and has resulted in many high-impact discoveries.

Multiple sclerosis is an inflammatory demyelinating disease with no cure. Analysis of brain tissue provides important clues to understanding the pathogenesis of the disease. Here we discuss the methodology and downstream analysis of MS brain tissue collected through a unique rapid autopsy program in operation at the Cleveland Clinic.

Programa integral de autopsias para individuos con esclerosis múltiple

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 ...

3D Modeling of Dendritic Spines with Synaptic Plasticity

Computational modeling of diffusion and reaction of chemical species in a three-dimensional (3D) geometry is a fundamental method to understand the mechanisms of synaptic plasticity in dendritic spines. In this protocol, the detailed 3D structure of the dendrites and dendritic spines is modeled with meshes on the software Blender with CellBlender. The synaptic and extrasynaptic regions are defined on the mesh. Next, the synaptic receptor and synaptic anchor molecules are defined with their diffusion constants. Finally, the chemical reactions between synaptic receptors and synaptic anchors are included and the computational model is solved numerically with the software MCell. This method describes the spatiotemporal path of every single molecule in a 3D geometrical structure. Thus, it is very useful to study the trafficking of synaptic receptors in and out of the dendritic spines during the occurrence of synaptic plasticity. A limitation of this method is that the high number of molecules slows the speed of the simulations. Modeling of dendritic spines with this method allows the study of homosynaptic potentiation and depression within single spines and heterosynaptic plasticity between neighbor dendritic spines.

The protocol develops a three-dimensional (3D) model of a dendritic segment with dendritic spines for modeling synaptic plasticity. The constructed mesh can be used for computational modeling of AMPA receptor trafficking in the long-term synaptic plasticity using the software program Blender with CellBlender and MCell.

Modelado 3D de espinas dendríticas con plasticidad sináptica

Computational modeling of diffusion and reaction of chemical species in a three-dimensional (3D) geometry is a fundamental method to understand the ...