Este estudio fue financiado por la División de Programa de Investigación Intramural (DIRP) del Instituto Nacional de Salud Mental, los Institutos Nacionales de Salud.

<ol>
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No hay conflictos de interés declarado.

1. Cuestiones técnicas: <ol><li> Una técnica estéril. La preparación de los acuerdos ambientales multilaterales y la preparación de la cultura son todas realizadas dentro de una campana de flujo laminar bajo condiciones estériles. Los antibióticos, que afectan a la actividad neuronal, no se utilizan en cualquier momento durante el proceso de preparación y la cultura. </li><li> Plasma / trombina y la coagulación de la adhesión del tejido en la supervivencia de los AMUMA. Tejido en el MEA requiere un cuidadoso equilibrio entre el tiempo requerido para el plasma / trombina coagulación y el tiempo de exposición del tejido a la atmósfera. Un tiempo de coagulación riesgos a corto desprendimiento prematuro de las secciones de la MEA, mientras que la exposición prolongada a la atmósfera provoca degeneración de los tejidos. Porque la fuerza de la solución de trombina determina la velocidad del proceso de coagulación, es un parámetro muy importante para el éxito de unir las culturas saludables a la superficie de los AMUMA. Se obtienen mejores resultados con 1000 unidades (1KU; una unidad de NIH = 0.324 ± 0.073 mg). Es importante destacar que mezcla incompleta de los resultados de solución de plasma / trombina en la coagulación de la promoción de plasma espacial heterogénea-se rompe durante el cultivo. &quot;Agujeros&quot; en estos afectan gravemente a la salud plasma la cultura y separar la cultura de microelectrodos, comprometiendo así la calidad de los registros electrofisiológicos. Trabajar con placas de enfriamiento durante el MEA / tejido montaje disminuye la coagulación y permite la mezcla homogénea de la disolución de plasma / la trombina y el correcto posicionamiento de los cortes de tejido. Del mismo modo, mediante la adición de medio de las gotitas individuales de la cámara de cultivo para sumergir la cultura después de tan sólo 5 minutos de la coagulación, reduce enormemente el riesgo de desprendimiento de los tejidos debido a las tensiones de la superficie. Una cultura de éxito se aplanan en la MEA y un poco ampliar mostrando un crecimiento saludable durante semanas, sin ningún signo importante de la incompleta del tejido y los AMUMA de contacto o la degeneración del tejido (por ejemplo, la figura. 1D). </li><li> Disección de los tejidos. Cuchillos Micro han mejorado mucho nuestro proceso de disección. Utilizamos dos hojas de afeitar borde (Herramientas de Bellas Ciencia - frágiles hojas de bisturí - 100050-00), de la cual nos separamos ~ 2 mm de ancho &quot;hojas&quot; con unos alicates, y los mantienen con el titular de bisturí. Las muestras de tejido disecado de la corte coronal con un movimiento suave, firme vertical de la hoja lo que reduce considerablemente el estrés mecánico debido a la tracción de los tejidos en general mejorar la salud de la cultura. </li><li> Tejido de refrigeración. La correcta refrigeración de los cortes y secciones de tejido durante la preparación es esencial para el éxito de la cultura. Utilizamos a medida platos fríos construida de elementos Peltier adjunta debajo de un disco de metal. El calor producido por el elemento Peltier es eliminado a través de la perfusión de agua fría. Esto reduce considerablemente el tiempo de preparación y estandariza enfriamiento durante cada etapa de la preparación (Laboratorios Dold e Ingeniería 131 Plantación Dr. Seguin, TX 78155;. (830) 560 hasta 1471)). </li><li> Condición de la incubadora. Una incubadora de medida con precisión las condiciones de oscilación interna ha sido crucial para nuestro éxito en el cultivo de segmentos en los acuerdos ambientales multilaterales. Basado en un original en la casa de diseño por Multichannelsystems (actualmente no disponible comercialmente), el dispositivo de oscilación interna consiste en bandejas que se unen a las dos ruedas laterales. Motores paso a paso y los controles computarizados permiten una trayectoria oscilante precisa (balanceo punto de vista, balanceándose velocidad y pausas intermitentes). En última instancia, las culturas corte deben ser expuestos al medio ambiente y la cultura en alternancia lento. El método tradicional consiste en colocar las culturas cortar en tubos estrechos que gire lentamente a lo largo de no más largo del eje. Aquí, la rotación lenta no produce estrés mecánico debido a la propia rotación y la velocidad de rotación es lo suficientemente alta para obtener una óptima &quot;de alimentación / respiración&quot; del ciclo de alrededor de 5 a 10 min de duración. El interior más compacto de la cámara de MEA, su volumen total de aproximadamente 2 cc, y el volumen medio de cultivo pequeña necesaria para el acondicionamiento medio por el propio tejido, plantea un reto importante. Por la oscilación entre la MEA ± ~ 70 º de ángulo (tiempo de ciclo: ~ 200 s), lo que frena la velocidad de balanceo, como las transiciones de la cultura entre la fase líquida y la atmósfera, y detener la oscilación en los ángulos extremos de una exposición prolongada a la atmósfera sido esencial para la supervivencia de la cultura. </li></ol> 2. Edad de desarrollo de las culturas corteza para estudiar las avalanchas neuronales Rodajas aguda de la corteza de la rata se toma comúnmente a PND 0-1 y se cultivan durante varias semanas en el MEA. Los primeros estudios han demostrado claramente que una sola rebanada de culturas corteza, después de varias semanas in vitro, mantener una estructura en capas con los tipos de células de identificación que puede ser fácilmente comparado con las clases de células en vivo 18,18-21. La organización en capas en el sistema in vitro ha sido convenientemente utilizado para el estudio del tálamoinervación de la corteza durante el desarrollo de 22 a 24, así como para la conducción de las estructuras subcorticales como el estriado 25,26. De hecho, la especificidad en la formación de conexiones neuronales dentro de las regiones del cerebro permite la construcción de complejos sistemas in vitro de que la recuperación de numerosos sistemas de proyección detallada, por ejemplo, la de los ganglios basales, la corteza de circuitos 27-30. Después de 4 - 6 semanas in vitro, de un solo rodajas de corteza 31 y rodajas de corteza co-cultivadas con estriado 26 o 32 tálamo muestran espontánea hacia arriba y hacia los estados se encuentran típicamente en vivo en ratas anestesiadas con uretano 33. La buena organización temporal de los estados arriba lleva el sello de anidado θ y γ-oscilaciones indicativo de una red electrofisiológica de las neuronas piramidales maduro y rápido remate interneuronas GABAérgicas 31. Es importante destacar que, en ausencia de dopamina D2-estimulación de los receptores, la maduración de parvalbúmina positivo interneuronas corticales se demora alrededor de 2 semanas en las culturas corte corteza 34. En línea con estos resultados, la evolución en el tiempo de desarrollo de anidado θ, β-y γ-se corresponde con las oscilaciones que en vivo cuando rebanadas corteza son co-cultivadas con el área tegmental ventral (VTA), que contiene las neuronas dopaminérgicas que se proyectan hacia el corteza 6. Estos estudios indican que, al estudiar las avalanchas neuronales, que dependen fundamentalmente de la inhibición GABAérgica madura rápido y se encuentran en las capas superficiales de la corteza 2,4, el gran cuidado debe tenerse para asegurar la correcta maduración del tejido cortical. Mientras que las avalanchas neuronales surgen en las culturas corteza sola en el transcurso de tiempo de 2 - 5 semanas 4, cuando se requiere un curso de tiempo de desarrollo que se corresponde con el desarrollo en vivo, rodajas de corteza necesita un receptor adecuado de dopamina estimulación, por ejemplo, rodajas de corteza de cultivo co- con el VTA 6.

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> Conjunto integrado multielectrodo plana </td><td> Sistemas multicanal ( <a href="www.multichannelsystems.com">www.multichannelsystems.com</a> ) ALA Scientific Instruments (Www.alascience.com) </td><td> <a href="http://www.multichannelsystems.com/products-mea/product-details/products/158/20030ir-ito-wo.html">200/30iR-ITO-w/o</a> </td><td> El nitrato de titanio (TiN) Electrodos (30 mm de diámetro) tienen gran superficie que resulta en baja impedancia (alrededor de 1,5 kW a 1 kHz) y una excelente banda ancha grabaciones (W / O - sin anillo) </td></tr><tr><td> Cámara de vidrio </td><td> www.aceglass.com </td><td> 7620-32 </td><td> Cilindro de vidrio roscado </td></tr><tr><td> Cámara de tapa </td><td> www.aceglass.com </td><td> 7622-114 </td><td> Tapa de plástico con teflón inserción </td></tr><tr><td> Sylgard 184 </td><td> www.wpiinc.com </td><td> SYLG184 </td><td> Dos partes de elastómero de silicona </td></tr><tr><td> Poli-D-lisina </td><td> Sigma-Aldrich </td><td> P6407-5mg </td><td> γ-irradiados, polvo liofilizado de células, cultivadas a prueba. Reconstituye con 5 ml de agua desionizada antes de su uso. </td></tr><tr><td> Solución salina equilibrada de Gey </td><td> Sigma-Aldrich </td><td> G9779-500 ml </td><td> filtra de manera estéril y se cultivaron a prueba </td></tr><tr><td> pollo plasma </td><td> Sigma-Aldrich </td><td> P3266-5mL </td><td> Reconstituir liofilizados, con 5 ml de agua desionizada antes de su uso. </td></tr><tr><td> trombina </td><td> Sigma-Aldrich </td><td> T6634-1KU </td><td> a partir de plasma de bovino, en forma de polvo liofilizado. </td></tr><tr><td> suero de caballo </td><td> Sigma-Aldrich </td><td> H1138-100 ml </td><td> manada de los donantes, el calor inactiva la cultura, la celda de prueba </td></tr><tr><td> Basal medio de Eagle </td><td> Invitrogen </td><td> 21010-046 </td><td> 1x, 500 ml - (+) Las sales de Earle, (-) L-glutamina), </td></tr><tr><td> Solución salina tamponada de Hank </td><td> Invitrogen </td><td> 24020-117 </td><td> 500 ml - (+) de magnesio, (+) de calcio, w / rojo fenol) </td></tr><tr><td> Cámara de diapositivas </td><td> Lab-Tek-Cámara de diapositivas w / tapa (Nunc), estéril </td><td> 177429 </td><td></td></tr><tr><td> Uridina </td><td> Sigma-Aldrich </td><td> U3003 </td><td></td></tr><tr><td> ARA-C de citosina-β-D-arabinofuranoside </td><td> Sigma-Aldrich </td><td> C6645 </td><td></td></tr><tr><td> 5-fluoro-2&#39;-desoxiuridina </td><td> Sigma-Aldrich </td><td> F0503 </td><td></td></tr></tbody></table>

multi-electrode array recordings of neuronal avalanches in organotypic cultures

La corteza es espontáneamente activo, incluso en ausencia de cualquier entrada o salida del motor. Durante el desarrollo, esta actividad es importante para la migración y diferenciación de los tipos de células corteza y la formación de conexiones neuronales 1. En el animal adulto, la actividad en curso refleja el pasado y el actual estado de un animal en la que los estímulos sensoriales están perfectamente integrados para calcular las futuras acciones. Por lo tanto, una clara comprensión de la organización del curso la actividad espontánea es decir, es un requisito previo para comprender la función de la corteza. Numerosas técnicas de grabación reveló que la actividad en curso en la corteza se compone de muchas neuronas, cuyas actividades individuales transitoriamente suma a los grandes eventos que se pueden detectar en el potencial del campo local (LFP) con microelectrodos extracelulares, o en el electroencefalograma (EEG), la magnetoencefalograma (MEG ), y la señal BOLD de resonancia magnética funcional (fMRI). La LFP es actualmente el método de elección para estudiar la actividad neuronal de la población con una alta resolución temporal y espacial en la escala mesoscópica (varios miles de neuronas). En el microelectrodo extracelular, a nivel local sincronizado actividades de resultado espacialmente neighbored neuronas en desviaciones rápido en la LFP hasta varios cientos de microvoltios. Cuando se utiliza una serie de microelectrodos, las organizaciones de tales desviaciones pueden ser convenientemente controlados en el espacio y el tiempo. Avalanchas neuronales describir la organización espacio-temporal invariante en escala de la actividad en curso neuronal en el cerebro 2,3. Que son específicos de las capas superficiales de la corteza a lo establecido en 4,5 in vitro, in vivo en ratas anestesiadas 6, y en el mono despierto 7. Es importante destacar que los estudios teóricos y empíricos sugieren que 2,8-10 avalanchas neuronales indican una dinámica crítica exquisitamente equilibrado estado de la corteza que optimiza la transferencia de información y procesamiento de la información. Con el fin de estudiar los mecanismos de desarrollo avalancha neuronal, el mantenimiento y la regulación, en preparaciones in vitro son muy beneficiosos, ya que permiten grabaciones de actividad estable avalancha en condiciones controladas con precisión. El actual protocolo describe la forma de estudiar las avalanchas neuronales in vitro mediante el aprovechamiento de desarrollo de la capa superficial de la corteza organotípicos culturas, las culturas, es decir corte, crecido en matrices de plano, de microelectrodos integrados (MEA, ver también 11-14).

Watch this Scientific Journal Video about Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures at JoVE.com

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

Una forma sólida para estudiar las avalanchas neuronales, es decir, invariante en escala espacio-temporal de ráfagas de actividad, lo que indica la dinámica de estado crítico en la corteza. Avalanchas surgen espontáneamente en el desarrollo de las capas superficiales de la corteza cultivo que permite mediciones a largo plazo de la actividad con planos las redes distribuidas multi-electrodo (MEA) en condiciones controladas con precisión.

Electrodos múltiples grabaciones matriz de avalanchas neuronal en cultivos organotípicos

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

multi-electrode-array-recordings-neuronal-avalanches-organotypic

Research

JoVE Journal

Neuroscience

26.3K vistas.  National Institute of Mental Health. Una forma sólida para estudiar las avalanchas neuronales, es decir, invariante en escala espacio-temporal de ráfagas de actividad, lo que indica la dinámica de estado crítico en la corteza. Avalanchas surgen espontáneamente en el desarrollo de las capas superficiales de la corteza cultivo que permite mediciones a largo plazo de la actividad con planos las redes distribuidas multi-electrodo (MEA) en condiciones controladas con precisión.

Video: Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

Multielectrode Array Recordings of the Vomeronasal Epithelium

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is planar multielectrode array (MEA) recording. Here we document the use of MEAs to study the mouse vomeronasal organ (VNO), which plays an essential role in the detection of pheromones and social cues via a diverse population of sensory neurons expressing hundreds of types of receptors. Combining MEA recording with a robotic liquid handler to deliver chemical stimuli, the sensory responses of a large and diverse population of neurons can be recorded. The preparation allows us to remove the intact neuroepithelium of the VNO from the mouse and stimulate with a battery of chemicals or potential ligands while monitoring the electrical activity of the neurons for several hours. Therefore, this technique serves as a useful method for assessing ligand activity as well as exploring the properties of receptor neurons. We present the techniques needed to prepare the vomeronasal epithelium, MEA recording, and chemical stimulation.

Multielectrode array (MEA) recordings provide a method for studying the electrical activity of large populations of neurons. Here, we present the details of a MEA preparation to record from the mouse vomeronasal epithelium while simultaneously stimulating the tissue.

Grabaciones multielectrodo matriz del epitelio vomeronasal

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is ...

Investigación

Neurociencias

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

Organotípicos culturas rebanada hipocampo

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

Culturas organotípicos rebanada de mesencéfalo ventral embrionario: Un sistema para estudiar el desarrollo neuronal dopaminérgica In vitro</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

El análisis de la morfología de células dendríticas Purkinje en cultivos de corte organotípicos

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Dos registros electrofisiológicos de sinápticamente respuestas evocadas neuronal y astroglial en agudas rodajas de hipocampo

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic targeting for microcircuit dissection and disease modeling. The introduction of optogenetics, for example, has allowed for bidirectional manipulation of genetically-identified neurons, at an unprecedented temporal resolution. To capitalize on these tools and gain insight into dynamic interactions among brain microcircuits, it is essential that one has the ability to record from ensembles of neurons deep within the brain of this small rodent, in both head-fixed and freely behaving preparations. To record from deep structures and distinct cell layers requires a preparation that allows precise advancement of electrodes towards desired brain regions. To record neural ensembles, it is necessary that each electrode be independently movable, allowing the experimenter to resolve individual cells while leaving neighboring electrodes undisturbed. To do both in a freely behaving mouse requires an electrode drive that is lightweight, resilient, and highly customizable for targeting specific brain structures.
A technique for designing and fabricating miniature, ultralight weight, microdrive electrode arrays that are individually customizable and easily assembled from commercially available parts is presented. These devices are easily scalable and can be customized to the structure being targeted; it has been used successfully to record from thalamic and cortical regions in a freely behaving animal during natural behavior.

Understanding the neural substrates of behavior requires brain circuit ensemble recording. Because of its genetic tractability, the mouse offers a model for circuit dissection and disease mimicry. Here, a method of designing and fabricating miniaturized probes is described that is suitable for targeting deep brain structure in the mouse.

Diseño y fabricación de Ultralight Peso, ajustables Sondas Multi-electrodos para electrofisiológicos grabaciones en ratones

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Pareadas Grabaciones de células enteras en organotípicos de hipocampo rebanadas

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

La investigación de la regeneración funcional en organotípicos médula espinal Co-culturas cultivadas en Arrays Multi-electrodos

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the stimulation parameters (e.g., orientation, field strength, and stimulation duration) need to be examined in mice brain slice preparations. Testing and arranging the orientation of the electrode relative to the position of the mice brain slice are feasible. The present method preserves the thalamocingulate pathway to evaluate the effect of DCS on anterior cingulate cortex seizure-like activities. The results of the multichannel array recordings indicated that cathodal DCS significantly decreased the amplitude of the stimulation-evoked responses and duration of 4-aminopyridine and bicuculline-induced seizure-like activity. This study also found that cathodal DCS applications at 15 min caused long-term depression in the thalamocingulate pathway. The present study investigates the effects of DCS on thalamocingulate synaptic plasticity and acute seizure-like activities. The current procedure can test the optimal stimulation parameters including orientation, field strength, and stimulation duration in an in vitro mouse model. Also, the method can evaluate the effects of DCS on cortical seizure-like activities at both the cellular and network levels.

Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.

La estimulación de corriente directa y multi-electrodo matriz de registro de la actividad convulsiva en ratones cerebro rebanada preparación

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the ...

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early postnatal development, cerebellar Purkinje cells are known to go through a vulnerable period, during which they undergo programmed cell death. Here, we provide a detailed protocol to perform mouse organotypic cerebellar slice culture during this critical time. The slices are further labeled to assess Purkinje cell survival and the efficacy of neuroprotective treatments. This method can be extremely valuable to screen for new neuroactive molecules.

Organotypic slice cultures are a powerful tool to study neurodevelopmental or degenerative/regenerative processes. Here, we describe a protocol that models the neurodevelopmental death of Purkinje cells in mouse cerebellar slice cultures. This method may benefit research in neuroprotective drug discovery.

Supervivencia de células Purkinje en cultivos de rebanadas de cerebeloso organotípico

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early ...