Este trabajo fue financiado por el NINDS y la Asociación Americana del Corazón para S.Ge. Este enfoque fue creado y desarrollado originalmente por el autor en el laboratorio del Dr. Hongjun canción en la Universidad Johns Hopkins. Nos gustaría dar las gracias al Dr. Song Hongjun por su orientación y apoyo.

1. Virus de la inyección Alto título de retrovirus modificados (1 x 10 9 unidades / ml) es producida por la co-transfección de vectores retrovirales y VSVG en las células HEK293T, seguido por ultracentrifugación de sobrenadante viral. Para las fuentes y los métodos de producción, de ver la demostración JoVe excelente (3). Nota: adultos jóvenes (4-6 semanas de edad) mujeres ratones C57BL / 6 (Charles River) se encuentran en condiciones normales. Todos los procedimientos siguen la guía del Consejo Nacional de Investigación para el cuidado y uso de animales de laboratorio bajo un protocolo aprobado por la Universidad de Stony Brook IACUC. <ol><li> 2UL deshielo de los retrovirus por animal congelado en el hielo. </li><li> Anestesiar (100 mg de ketamina + 10 xilazina microgramos por gramo de peso corporal) y montar el animal en un marco estereotáxico (Steolting). Quitar el pelo en la cabeza y limpiar la piel con etanol al 70%. </li><li> Exponer el cráneo y perforar cuatro pozos someros con un taladro dental (broca de 0,6 mm) en las siguientes coordenadas: <ul><li> anterioposterior = -2 mm de bregma, lateral = ± 1,6 mm; ventral = 2,5 mm; anterioposterior = 3 mm de bregma, lateral = ± 2,6 mm; ventral = 3,2 mm. </li><li> anterioposterior = -2 mm de bregma, lateral = ± 1,6 mm; ventral = 2,5 mm; anterioposterior = 3 mm de bregma, lateral = ± 2,6 mm; ventral = 3,2 mm. </li></ul></li><li> Montar un Hamilton 1μl, plana punta de la jeringa e inyectar 0,5 l por retrovirus lugar a una tasa de 0,25 l / min en el giro dentado. (Ver las coordenadas por encima de la profundidad ventral.) Pausa 2 minutos después de cada inyección antes de retirar la jeringa lentamente para evitar el reflujo de líquido. Entre las inyecciones, una breve aclaración de la superficie externa de la punta de la jeringa con PBS estéril y un aplicador para eliminar los rastros de sangre. </li><li> Cerrar la herida con material estéril sutura quirúrgica (tipo P-1; Tamaño 6-0) y devolver el animal a las condiciones de vivienda estándar hasta las etapas de tiempo deseado después de la inyección. </li></ol> 2. Preparación Cortar Para obtener cortes de alta calidad aguda de ratones adultos, se utiliza una solución de corte modificado para la preparación de corte (ver más abajo). <ol><li> Pre-corte chill solución a 0-4 º C en el hielo y la burbuja con un 95% de O 2 -5% de CO 2 por lo menos durante 30 minutos. </li><li> Anestesiar y transcardially perfundir un animal con helado de oxígeno de una solución saturada de corte. </li><li> Quite rápidamente el cerebro en un petri plato con papel de filtro pre-humedecido con una solución fría, oxigenada de corte. Cortar el cerebelo y la porción de una superficie de montaje de al menos 1 mm anterior a las coordenadas de la inyección (visible). Pegue el cerebro en una etapa vibrotome y montarlo en la cámara de corte llena con una solución de corte en frío y oxigenado. </li><li> Prepare cortes coronales u horizontal (300-350μm) y la transferencia con una pipeta de mayor diámetro para una cámara de incubación a temperatura ambiente que contiene ACSF burbujas con un 95% O 2 / 5% CO 2. </li><li> Incubar los cortes, burbujas de forma continua, a 32 ° C durante 30-60 minutos. Devolver la cámara a temperatura ambiente durante la duración de la grabación. </li></ol> 3. Los experimentos electrofisiológicos <ol><li> Los sectores se transfieren a la cámara de grabación que está continuamente perfundidos con ACSF saturada de oxígeno a 32 ° C - 34 ° C. </li><li> Un electrodo de estimulación bipolar de tungsteno se coloca en la capa molecular del giro dentado con un objetivo de microscopio óptico bajo (10 veces). </li><li> PED recién nacido en la zona sub-granular / capa de células granulares se identifican por la expresión de GFP o dTomato. De células enteras de grabación patchclamp se realiza en las neuronas recién nacidas bajo aumento (40X). </li><li> Propiedades de disparo de las células registradas se obtienen mediante la inyección de una serie de corrientes en el modo actual-clamp. </li><li> Estímulos eléctricos son entregados por el electrodo de estimulación a través de un aislador de la estimulación controlada por el software de grabación, y evocó las corrientes postsinápticas en el PED recién nacidos se registran. </li></ol> 4. Análisis de Datos Amplitudes de las respuestas evocadas postsináptica se analizan en las distintas fases de los adultos nacidos en las neuronas. 5. Resultados representante Utilizando el protocolo anterior, la expresión de GFP en el recién nacido neuronas giro dentado similar a la Figura 1 es común. Tenga en cuenta que las neuronas recién nacidas que es fácil ver y tanto dendritas y los axones están marcados con firmeza. Expresión en los axones delgados DGC y pequeñas espinas depende de la calidad y el título del virus, el promotor utilizado y la longitud de la expresión in vivo. En nuestras manos, las células del recién nacido y orgánulos subcelulares son generalmente visibles a los pocos días después de la inyección, y la expresión de la columna vertebral puede ser rastreado desde las primeras etapas de desarrollo. Wcélula-agujero grabaciones de las neuronas recién nacidas en las etapas de tiempo diferentes permitir el estudio de propiedades únicas células del recién nacido, por ejemplo, los potenciales de acción (figura 2a), así como el proceso de integración sináptica en los actuales circuitos neuronales durante la maduración (Figura 2b). <img src="/files/ftp_upload/2548/2548fig1.jpg" alt="Figura 1" /> Figura 1 de 2 fotones imagen confocal de neuronas recién nacidas en una sección horizontal de giro dentado de un ratón adulto. Las células verdes son 21dpi (días después de la inyección) que expresan GFP PED recién etiquetados con PUX-GFP retrovirus. <img src="/files/ftp_upload/2548/2548fig2.jpg" alt="Figura 2" /> Figura 2 a, b) Potencial de acción disparando propiedades de un recién nacido DGC a los 21 dpi.) Representante evocó excitatorios postsinápticos corrientes (EPSCs) grabado en un recién nacido DGC a los 21 dpi.

<ol>
	<li>Zhao, C., Deng, W., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+functional+implications+of+adult+neurogenesis.">Mechanisms and functional implications of adult neurogenesis.</a> Cell. 132, 645-660 (2008).</li><li>Ge, S., Goh, E. L., Sailor, K. A., Kitabatake, Y., Ming, G. L., Song, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+regulates+synaptic+integration+of+newly+generated+neurons+in+the+adult+brain.">GABA regulates synaptic integration of newly generated neurons in the adult brain.</a> Nature. 439, 589-593 (2006).</li><li>Gavrilescu, L. C., Van Etten, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+replication-defective+retrovirus+by+transient+transfection+of+293T+cells.">Production of replication-defective retrovirus by transient transfection of 293T cells.</a> J Vis Exp. , (2007).</li></ol>

Neurogénesis continua en el hipocampo de cerebros de los mamíferos adultos proporciona un sistema único modelo experimental para estudiar el desarrollo y la integración neuronal en el cerebro maduro. El protocolo se presenta aquí combina etiquetado retrovirales y métodos electrofisiológicos para estudiar la integración de los recién nacidos las neuronas granulares dentadas en los cerebros de ratones adultos. Para asegurarse de que sus experimentos tienen éxito, se recomienda lo siguiente durante las etapas críticas del procedimiento: Para evitar la infección y la inflamación no deseados, todos los instrumentos deben ser esterilizados (por autoclave, cualquier tipo de esterilización, o el 70% de etanol) antes de la cirugía. Use PBS estéril para lavar la punta de la aguja de la jeringa antes de la inyección. Para la inyección de virus, los animales deben estar bien montado en el dispositivo estereotáxico y fuentes de los movimientos innecesarios deben ser minimizadas durante la inyección. Asegúrese de que la cabeza está bien fijo y firme, y ajustar la posición de la nariz del ratón a nivel de bregma y lambda antes de la inyección de cálculo de coordenadas. Extracto de virus en la jeringa rápidamente para minimizar la exposición a temperatura ambiente - Los retrovirus son particularmente sensibles a la temperatura. Inyectar lentamente virus en la dosis recomendada para minimizar el daño de presión. Uso de la recomendada de punta plana jeringa (vs. punta biselada común) se asegurará una distribuida uniformemente zona dentada infección giro. Antes de preparar las rebanadas, corte de la solución y la incubación de ACSF se burbujea con un 95% de CO 2 O 2 al 5% por lo menos 30 minutos para permitir que el oxígeno para saturar las soluciones. Los animales deben ser perfundidos con rapidez y mantener el tejido en frío, saturada de oxígeno solución para la mejor calidad de corte. Para la grabación, aumento de la intensidad del estímulo gradualmente hasta que haya una respuesta postsináptica. Cambiar la ubicación de los electrodos de estimulación en la capa molecular del giro dentado según sea necesario. En resumen, el enfoque recomendado ofrece una manera de explorar distintas propiedades y posible papel funcional de los adultos nacidos en las neuronas durante las primeras etapas de integración en los circuitos activos, maduros.

<ul><li> Solución de corte (en mm): 110 cloruro de colina, 2,5 KCl, 1,3 KH 2 PO 4, 25,0 NaHCO3, 0,5 CaCl 2, 7 MgCl 2, 10 de dextrosa, 1.3 ascorbato de sodio, 0,6 piruvato de sodio, ácido 5,0 quinurénico. </li><li> Artificial líquido cefalorraquídeo (ACSF, en mM): NaCl 125,0, 25,0 NaHCO3, 2,5 KCl, 2 CaCl 2, 1,3 KH 2 PO 4, 1,3 MgCl 2, 1,3 ascorbato de sodio, 0,6 piruvato de sodio, 10 de dextrosa. </li><li> Solución interna de células enteras patchclamp (en mM): 120 gluconato de potasio, KCl 15, 4 MgCl 2, 0,1 EGTA, HEPES 10,0, 4 MgATP, 0,3 Na 3 GTP, 7 fosfocreatina (pH 7,4, 300 mOsm). </li></ul>

studying the integration of adult-born neurons

Neurogénesis ocurre en adultos cerebro de los mamíferos en la zona sub-ventricular (SVZ) del ventrículo lateral y en la zona sub-granular (SGZ) de la circunvolución dentada del hipocampo durante toda la vida. Estudios previos han mostrado que la neurogénesis del hipocampo adulto se asocia con trastornos cerebrales diversos, como la epilepsia, la esquizofrenia, la depresión y la ansiedad (1). Descifrar el proceso de integración de las neuronas normales y aberrantes de adultos nacidos pueden arrojar luz sobre la etiología de estas enfermedades e informar el desarrollo de nuevas terapias. Neurogénesis adulta SGZ espejos desarrollo neuronal embrionario y postnatal, incluyendo las etapas de especificación de destino, la migración, la integración sináptica, y la maduración. Sin embargo, la plena integración se produce durante un prolongado período de 6 semanas. Entrada inicial sináptica de adultos nacidos en las células granulares SGZ dentado (PED) es GABAérgicas, seguido por la entrada glutamatérgica a los 14 días (2). Los factores específicos que regulan la formación de circuitos de adultos nacidos en las neuronas del giro dentado se desconoce en la actualidad. Nuestro laboratorio utiliza una replicación deficiente vector retroviral basado en el virus de la leucemia murina Moloney para entregar las proteínas fluorescentes y la hipótesis de los genes reguladores de estas células en proliferación. Esta técnica ofrece una alta especificidad viral y la resolución para el análisis de la fecha de nacimiento de células, el linaje, la morfología y sinaptogénesis. Un experimento típico emplea a menudo dos o tres tipos de virus que contiene la etiqueta única, los transgenes, y los elementos promotores de una sola célula de análisis de un proceso de desarrollo deseado en vivo. El siguiente protocolo describe un método para el análisis de la integración funcional de las neuronas recién nacido con un único color verde (GFP) o rojo (dTomato) retrovirus proteína fluorescente y patch clamp-electrofisiología.

Watch this Scientific Journal Video about Studying the Integration of Adult-born Neurons at JoVE.com

Studying the Integration of Adult-born Neurons

Una manera de estudiar la integración de los recién nacidos las células granulares dentadas en los animales adultos se describe. Esta técnica utiliza un retrovirus modificados para etiquetar las neuronas recién nacidas, seguido por los registros electrofisiológicos para determinar la integración funcional en vivo.

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 ...

studying-the-integration-of-adult-born-neurons

Research

JoVE Journal

Neuroscience

13.8K vistas.  State University of New York at Stony Brook. Una manera de estudiar la integración de los recién nacidos las células granulares dentadas en los animales adultos se describe. Esta técnica utiliza un retrovirus modificados para etiquetar las neuronas recién nacidas, seguido por los registros electrofisiológicos para determinar la integración funcional en vivo.

Video: Studying the Integration of Adult-born Neurons

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

En el útero de electroporación seguido por cultivos neuronales primarias para estudiar la función génica en el subconjunto de neuronas corticales

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Investigación

Neurociencias

Studying the Neural Basis of Adaptive Locomotor Behavior in Insects

Studying the neural basis of walking behavior, one often faces the problem that it is hard to separate the neuronally produced stepping output from those leg movements that result from passive forces and interactions with other legs through the common contact with the substrate. If we want to understand, which part of a given movement is produced by nervous system motor output, kinematic analysis of stepping movements, therefore, needs to be complemented with electrophysiological recordings of motor activity. The recording of neuronal or muscular activity in a behaving animal is often limited by the electrophysiological equipment which can constrain the animal in its ability to move with as many degrees of freedom as possible. This can either be avoided by using implantable electrodes and then having the animal move on a long tether (i.e. Clarac et al., 1987; Duch &amp; Pfl&uuml;ger, 1995; B&ouml;hm et al., 1997; Gruhn &amp; Rathmayer, 2002) or by transmitting the data using telemetric devices (Kutsch et al, 1993; Fischer et al., 1996; Tsuchida et al. 2004; Hama et al., 2007; Wang et al., 2008). Both of these elegant methods, which are successfully used in larger arthropods, often prove difficult to apply in smaller walking insects which either easily get entangled in the long tether or are hindered by the weight of the telemetric device and its batteries. In addition, in all these cases, it is still impossible to distinguish between the purely neuronal basis of locomotion and the effects exerted by mechanical coupling between the walking legs through the substrate. One solution for this problem is to conduct the experiments in a tethered animal that is free to walk in place and that is locally suspended, for example over a slippery surface, which effectively removes most ground contact mechanics. This has been used to study escape responses (Camhi and Nolen, 1981; Camhi and Levy, 1988), turning (Tryba and Ritzman, 2000a,b; Gruhn et al., 2009a), backward walking (Graham and Epstein, 1985) or changes in velocity (Gruhn et al., 2009b) and it allows the experimenter easily to combine intra- and extracellular physiology with kinematic analyses (Gruhn et al., 2006).
We use a slippery surface setup to investigate the timing of leg muscles in the behaving stick insect with respect to touch-down and lift-off under different behavioral paradigms such as straight forward and curved walking in intact and reduced preparations.

We describe a method to record motor activity, timed to the electrically recorded tarsal contact signal in a tethered insect, walking on a slippery surface. This is used to study the neural basis of adaptive behavior under reduced influence of mechanical interaction between legs through the substrate.

Estudiar la base neural de la conducta adaptativa Locomotor en insectos

Studying the neural basis of walking behavior, one often faces the problem that it is hard to separate the neuronally produced stepping output from those ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

Transducción viral selectiva de adultos nacidos en las neuronas olfativas de las enfermedades crónicas En vivo Estimulación Optogenetic

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

La especificación de telencefálicas neuronas glutamatérgicas a partir de células madre pluripotentes

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Fast Micro-iontoforesis de glutamato y GABA: una herramienta útil para investigar la integración sináptica

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

Métodos para el estudio de los mecanismos de acción de los fármacos antipsicóticos en<em&gt; Caenorhabditis elegans</em

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

Etiquetado diferencial de la superficie celular e internalizado Proteínas después Antibody alimentación de vivo cultivadas neuronas

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with neurodegenerative diseases or injuries, but also in the normal brain. Here, we describe a newly-established micromechanical system to rapidly, reliably, and reversibly induce axonal varicosities, allowing us to understand the mechanisms governing varicosity formation and heterogeneous protein composition. This system represents a novel means to evaluate the effects of compression and shear stress on different subcellular compartments of neurons, different from other in vitro systems that mainly focus on the effect of stretching. Importantly, owing to the unique features of our system, we recently made a novel discovery showing that the application of pressurized fluid can rapidly and reversibly induce axonal varicosities through the activation of a transient receptor potential channel. Our biomechanical system can be utilized conveniently in combination with drug perfusion, live cell imaging, calcium imaging, and patch clamp recording. Therefore, this method can be adopted for studying mechanosensitive ion channels, axonal transport regulation, axonal cytoskeleton dynamics, calcium signaling, and morphological changes related to traumatic brain injury.

This protocol describes a physiologically relevant, pressurized fluid approach for rapid and reversible induction of varicosities in neurons.

Un sistema de Microbiomechanical para estudiar la formación de várices y recuperación en axones de la neurona Central

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with ...

An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). Although mouse models of tauopathy have provided a valuable resource for investigating the neurotoxic mechanisms of aggregated tau, it is becoming increasingly apparent that, due to interspecies differences in neurophysiology, the mouse brain is unsuitable for modeling the human condition. Advances in cell culture methods have made human neuronal cultures accessible for experimental use in vitro and have aided in the development of neurotherapeutics. However, despite the adaptation of human neuronal cell cultures, in vitro models of human tauopathy are not yet widely available. This protocol describes a cellular model of tau aggregation in which human neurons are transduced with lentiviral-derived vectors that code for pathogenically mutated tau fused to a yellow fluorescent protein (YFP) reporter. Transduced cultures produce tau aggregates that stain positively for thioflavin and display markers of neurotoxicity, such as decreased axonal length and increased lysosomal volume. This procedure may be a useful and cost-effective model for studying human tauopathies.

This protocol details a procedure in which human neuronal cultures are transduced with lentiviral constructs coding for mutant human tau. Transduced cultures display tau aggregates and associated pathologies.

Un modelo in vitro para estudiar la agregación de Tau utilizando la transducción de neuronas humanas mediada por lentivirales

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

Grabación electrofisiológica inalámbrica de las neuronas por tetrodes móviles en la natación libre de peces

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...