Los autores desean agradecer a las siguientes organizaciones: Fundación de Ciencias naturales nacionales de China (31171018, 31171355), la ciencia y la tecnología de división de Guangdong (2013KJCX0054), la Fundación de Ciencias naturales de la provincia de Guangdong ( 2014A030313418, 2014A030313440) y Guangzhou ciencia y Technology Bureau (201607010320).

Alojamiento de los animales y todos los animales se realizaron experimentos de acuerdo con procedimientos aprobados por el Comité de ética para la investigación animal en South China Normal University, según las directrices para el cuidado del Animal establecido por el Instituto Nacional de Salud.
 1. preparación de soluciones <ol><li>preparar 500 mL de estándar artificial líquido cerebroespinal (ACSF) que contiene los componentes se describe en nuestro trabajo publicado 7 como se indica en la tabla 1.
	<ol><li>Uso limpia vidrio vasos de precipitados con agua desionizada para preparar una solución fresca de la ACSF. Limpiar espátulas con agua desionizada y seque antes de usar Añadir NaCl, KCl, NaH 2 PO 4, NaHCO 3, MgSO 4, D-glucosa y añadir CaCl 2 después de haber burbujas la solución con 95% O 2 / 5% CO 2 mezcla de ~ 20 minutos (como indica en la tabla 1) a 500 mL de agua desionizada. Revolver hasta que esté disuelto completamente.</li>
		<li>Asegurar la ACSF es completamente clara sin ninguna precipitación. Luego, ajustar el pH a 7.2-7.4. Burbuja con una mezcla de gases compuesta de 95% O 2 y 5% CO 2 ~ 20 min</li>
	</ol></li> <li>Preparar 250 mL de solución de corte de rebanada que contienen los productos químicos 7 descritos en la tabla 2.
	<ol><li>Añadir KCl, MgCl 2, NaH 2 PO 4, NaHCO 3, choline·Cl, D-glucosa y añadir CaCl 2 últimos como se indica en 1.1.1, a 250 mL de agua desionizada y disolver como se describe en 1.1.1 y 1.1.2.</li>
		<li>Después de burbujeo con 95% O 2 / 5% CO 2, coloque la solución en el congelador durante 15-20 min para que se convierte en la solución de corte parcialmente congelados y helados. 
		Nota: Utilizamos esta solución de corte al hacer rodajas de 1-2 mes-ratones del viejo; la fórmula puede variar para los ratones mayores de 2 meses.</li>
		<li>Solución de LPS preparar disolviendo en solución salina libre de endotoxina isotónica estéril (0.9% NaCl). LPS es administrado por vía intraperitoneal (0, 5 mg/kg) y comenzar a grabar 2 h más tarde.</li>
	</ol></li></ol> 2. Preparación de Corteza Prefrontal <ol><li>tomar la solución de corte fuera del congelador y homogeneizar para obtener un granizado. Coloque la solución en hielo y burbujas constantemente con 95% O 2 / 5% CO 2.</li>
	<li>Con unas tijeras grandes y afiladas, rápidamente decapitar un ratón como descrito 9 e inmediatamente puso la cabeza en un vaso llenado de frías (0-4 ° C) solución de corte.</li>
	<li>Cortar la parte superior del cráneo a lo largo de la línea media en dirección caudal nasal con tijeras finas y retirar las porciones lateral del cráneo. Quitar el cerebro con una espátula fina y colocar en solución de corte helada. 
	Nota: Pasos 2.2-2.3 deben realizarse rápidamente (idealmente &#60; 1 min).</li>
	<li>Poner el cerebro en la tapa de un plato de Petri de 9 cm con hielo. Lavar el cerebro con la solución de corte helado. Corta el cerebelo y el bulbo olfatorio con una cuchilla de afeitar.</li>
	Titular de <li>Aplicar pegamento del cianocrilato a la muestra de un vibratome. Coloque cuidadosamente el bloque del cerebro sobre la gota de pegamento, lado rostral y sumerja inmediatamente en la solución helada corte.</li>
	<li>Ajustar el sujetador de la cuchilla de vibratome a un ángulo de 15° en relación con el plano horizontal y preparar 350 μm rebanadas de cerebro coronales (frecuencia de la vibración de la lámina = 85 Hz, velocidad = 0,1 mm/s).</li>
	<li>Con una pipeta, transfiera las rebanadas en la cámara de recuperación llenan con ACSF, incubar durante 1 h (a RT) con burbujeo constante de 95% O 2 / 5% CO 2.</li>
</ol> 3. Grabación de parche de celulares <ol><li>hacer vidrio Micropipetas de borosilicato para utilizar como electrodos de grabación o puff Micropipetas según las directrices específicas en el Manual de operación del tirador. Para la grabación de los electrodos, las resistencias de punta están en el rango 4-6 M &#8486;, mientras que el soplo Micropipetas tienen diámetros de punta en el rango de 2-5 μm.</li>
	<li>Agregar bloqueador de canales de Na + (TTX, 1 μm), bloque rápido Na + de canales y así los potenciales de acción, a la ACSF y mantener un flujo constante de esta solución de 2-3 mL/min en la sala de grabación, por bomba peristáltica en la cámara de y aspiraciones de ella. Burbuja la ACSF con 95% O 2 / 5% CO 2 constantemente para garantizar la viabilidad de las rodajas de.</li>
	<li>Transferir un trozo de cerebro en la sala de grabación con una pipeta Pasteur modificada cuya punta fina fue cortada para ajustarse al tamaño de rebanada. Cubre la rebanada con un ancla de platino rebanada con hilos de nylon paralelo espaciados &#8805; 2 mm aparte hacer la rebanada en la plataforma de.</li>
	<li>Llenar la micropipeta de grabación con solución interna 7 (tabla 3) y se llenan de las Micropipetas puff GABA en 10 μm, 50 μm y 100 μm (disuelto en ACSF) o ACSF como control del vehículo.</li>
	<li>Para evitar la obstrucción con residuos, aplique ligera presión positiva utilizando una jeringa de 1 mL antes de sumergir el electrodo de la grabación en la ACSF. Utilizando un micromanipulador bajo un microscopio (objetivo del x 4), colocar la micropipeta de electrodo y puff de grabación para que aparezcan las puntas en el centro de la imagen en el monitor ( figura 1 A).</li>
	<li>Ajustar el enfoque del microscopio (objetivo x 40) reduciendo gradualmente la micropipeta de hojaldre y colocar por encima de la neurona de la grabación en un ángulo de 45°. Mantener la distancia entre la punta de la micropipeta de hojaldre y la neurona registrada entre 20-40 μm ( figura 1 B). Lenta y cuidadosamente abordar la neurona con el electrodo de la grabación y luego suelte la presión positiva. Aplicar una succión débil y breve a través del tubo conectado a la pinza portaelectrodos para crear un sello de giga ohmios.</li>
	<li>Mantener la tensión a 0 mV. Después de la formación del sello giga ohmios, compensar rápido y lento capacitancia manualmente o automáticamente. Luego aplicar una succión breve y fuerte a través del tubo mencionado arriba para entrar en la célula entera.</li>
	Modo de <li>eIPSC de registro en la abrazadera de tensión. Presión baja de gas (nitrógeno, 4-6 psi) se aplican a la micropipeta de hojaldre utilizando un Picospritzer controlado por un generador de paso de voltaje Master 8 para entregar GABA unitarios o emparejado soplos ( figura 2).</li>
	<li>Cambiar la duración del soplo para obtener los mejores resultados de la eIPSC ( figura 3) y medir el eIPSC en el modelo de la depresión inducida por LPS ( figura 4).</li>
</ol>

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Los autores no tienen nada que revelar.

Aplicación de hojaldre es ampliamente utilizado para evaluar la función de receptores postsinápticos3,4,7, pero requiere un control preciso en cada experimento. Aquí describimos un procedimiento que implica sujeción parche de células enteras, que demuestra que GABA-puff inducida IPSCs (es decir, eIPSCs) en rebanadas de cerebro cortical prefrontal. La resistencia del electrodo de registro es MΩ cerca de 5, mientras que el ...

La técnica de patch-clamp, una herramienta primaria para la investigación de las señales eléctricas en las neuronas, se desarrolló en la década de 19701,2. Una gran ventaja de esta técnica es que proporciona conocimiento sobre tratamientos específicos cómo (por ejemplo, farmacológicas) pueden alterar las funciones neuronales o canales en tiempo real3. Evaluación farmacológica de la función neuronal durante la grabación en una rebanada del cerebro de células completas requiere la aplicación de fármacos, como agonistas o antagonistas de receptores específicos, las neuronas se registran. Este método permite la identificación de alteraciones neuronales que ocurren después de la aplicación de un medicamento específico, lo que conduce a una mejor comprensión de las propiedades fisiológicas y patológicas de las neuronas4. Aunque la administración farmacológica puede llevarse a cabo mediante perfusión5 o soplo6, este último es la técnica superior. En particular: () aplicación de hojaldre rápido aumenta la droga concentración alrededor de la neurona grabada a un nivel tal que se evita la desensibilización de los receptores de membrana; (ii) el volumen de droga inflado es extremadamente bajo, por lo que hay poco efecto en la solución del baño, que por lo tanto reduce los efectos indeseables de las sustancias químicas administradas en rebanadas de cerebro; (iii) el protocolo de hojaldre puede ser establecido y guardado, haciendo el experimento precisamente reproducible; (iv) puff aplicación representa uso económico de agonistas/antagonistas, especialmente donde tales reactivos son caros o difíciles de conseguir.
Aquí, nos centraremos en la grabación de celulares corrientes inducidas por GABA el soplar en rebanadas de cerebro agudo preparado, una preparación que tiene la ventaja de circuitos cerebrales relativamente bien conservado. Cómo llevamos a cabo corrientes inhibitorios inducidos por el soplo7 se describen en este artículo. Utilizando cesio (Cs+)-soluciones internas y el mantenimiento de las neuronas a 0 mV, presentamos GABA-puff evocado inhibitorias postsynaptic corrientes (eIPSCs) con las características técnicas adecuadas. Utilizando un modelo de ratón de la depresión inducida por lipopolisacárido (LPS) inyección8, se muestra que el eIPSC amplitud evocada por un soplo de GABA se reduce significativamente en rebanadas de ratones inyectados por LPS comparados con el control del vehículo. Nuestra intención es que este artículo debería mostrar cómo la técnica de soplo es ampliamente aplicable a estudios dirigidos a evaluar el efecto de productos químicos, compuestos o drogas sobre actividades neuronales en rebanadas de cerebro.

<table><tbody><tr><td>Glass Borosilicate micropipettes</td><td>Shutter Instruments</td><td>BF150-86-10</td><td>1.50 mm outer diameter; 0.86 mm inner diameter</td></tr><tr><td>Micropipette Puller</td><td>Shutter Instruments</td><td>MODEL P-97&amp;nbsp;</td><td>Flaming/Brow Micropipette Puller</td></tr><tr><td>Micromanipulators</td><td>Shutter Instruments</td><td>MP-285</td><td></td></tr><tr><td>Computer controlled Amplifier&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Molecular Devices</td><td>Multiclamp 700B</td><td></td></tr><tr><td>Digital Acquisition system&amp;nbsp;</td><td>Molecular Devices</td><td>Digidata 1440A</td><td></td></tr><tr><td>Imaging Camera</td><td>Nikon&amp;nbsp;</td><td>2115001</td><td>Inspection equipment</td></tr><tr><td>Microscopy&amp;nbsp;</td><td>Nikon&amp;nbsp;</td><td>Eclipse FN1&amp;nbsp;</td><td></td></tr><tr><td>Master 8</td><td>A.M.P.I.</td><td>Master-8 Pulse stimulator</td><td></td></tr><tr><td>Vibratome Slicer</td><td>Leica</td><td>VT 1000S</td><td></td></tr><tr><td>Razor blade</td><td>Gillette</td><td>74-S</td><td>FLYING EAGLE</td></tr><tr><td>Video monitor</td><td>Panasonic</td><td>WV-BM 1410</td><td></td></tr><tr><td>502 Glue</td><td>Deli</td><td>7146</td><td>Cyanoacrylate Glue</td></tr><tr><td>Peristaltic pump</td><td>Shanghai JIA PENG Corporation</td><td>BT100-1F</td><td></td></tr><tr><td>Video Camera</td><td>Olympus America Medical</td><td>OLY-150</td><td></td></tr><tr><td>Transfer Pipets</td><td>Biologix</td><td>30-0138A1</td><td></td></tr></tbody></table>

whole-cell currents induced by puff application of gaba in brain slices

Administración farmacológica es de uso general cuando se realizan células completas-abrazadera del remiendo en rebanadas de cerebro. Uno de los mejores métodos de aplicación de la droga durante la grabación electrofisiológica es la técnica de soplo, que puede utilizarse para estudiar el efecto de reactivos farmacológicos en actividades neuronales en rebanadas de cerebro. La mayor ventaja del uso del hojaldre es que la concentración del fármaco en el sitio de grabación aumenta rápidamente, evitando así la desensibilización de receptores de membrana. Exitosa aplicación del soplo implica especial atención a los siguientes elementos: la concentración del fármaco, los parámetros de la micropipeta de hojaldre, la distancia entre la punta de una micropipeta de hojaldre y la neurona registrada y la duración y presión conduce el soplo (libras por pulgada cuadrada, psi). Este artículo describe un procedimiento paso a paso para registrar celulares corrientes inducidas por ácido gamma - aminobutírico (GABA) en una neurona de un segmento cortical prefrontal de soplar. En particular, el mismo procedimiento se puede aplicar con pequeñas modificaciones a otras áreas del cerebro como el hipocampo y el striatum y a diferentes preparaciones, tales como los cultivos celulares.

La técnica de soplo permite a los investigadores a estudiar el efecto de las drogas en particular receptores en la superficie de una neurona determinada. En este artículo, nos centramos en corrientes mediadas por receptores de GABA. La figura 1 muestra las Micropipetas puff y grabación. Para eliminar cualquier efecto en las neuronas registradas de la presión física generado por el soplo, ACSF y soplos de sacarosa (figura 2<s...

Watch this Scientific Journal Video about Whole-cell Currents Induced by Puff Application of GABA in Brain Slices at JoVE.com

Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

Celulares corrientes inducidas por Puff aplicación de GABA en rebanadas de cerebro

Describimos la técnica de soplo, que reactivos farmacológicos pueden ser administrados durante la grabación de la abrazadera del remiendo de célula entera y resaltar diversos aspectos de las características que son cruciales para su éxito.

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug ...

whole-cell-currents-induced-puff-application-gaba-brain

Research

JoVE Journal

Neuroscience

9.3K vistas.  South China Normal University. Describimos la técnica de soplo, que reactivos farmacológicos pueden ser administrados durante la grabación de la abrazadera del remiendo de célula entera y resaltar diversos aspectos de las características que son cruciales para su éxito.

Video: Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

GABA-activated Single-channel and Tonic Currents in Rat Brain Slices

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, respectively 4,5,6,7 The GABAA channel is a pentameric GABA-gated chloride channel. The channel subunits are grouped into 8 families (&alpha;1-6, &beta;1-3, &gamma;1-3, &delta;, &#949;, &theta;, &pi; and &rho;). Two alphas, two betas and one 3rd subunit form the functional channel 8. By combining studies of sub-type specific GABA-activated single-channel molecules with studies including all populations of GABAA channels in the neuron it becomes possible to understand the basic mechanism of neuronal inhibition and how it is modulated by pharmacological agents.
We use the patch-clamp technique 9,10 to study the functional properties of the GABAA channels in alive neurons in hippocampal brain slices and record the single-channel and whole-cell currents. We further examine how the channels are affected by different GABA concentrations, other drugs and intra and extracellular factors. For detailed theoretical and practical description of the patch-clamp method please see The Single-Channel Recordings edited by B Sakman and E Neher 10.

We use the patch-clamp technique to measure GABA-activated single-channel currents (GABAA channels, GABAA receptors) and the synaptic and tonic currents they generate in neurons. Activation of the channels decreases neuronal excitability in health and disease 1,2,3,4.

GABA corrientes activadas de un solo canal y Tonic en rodajas de cerebro de rata

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, ...

Investigación

Neurociencias

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Plenario de células patch-clamp grabaciones de Morphologically- e identificados-neuroquímicamente interneuronas del hipocampo

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments that largely determines the quality of results. It has been shown that omitting the cooling step during cutting procedure is beneficial in obtaining healthy slices and cells, especially when dealing with highly myelinated brain structures from mature animals. Even though the precise mechanism whereby elevated temperature supports neural health can only be speculated upon, it stands to reason that, whenever possible, the temperature in which the slicing is performed should be close to physiological conditions to prevent temperature related artifacts. Another important advantage of this method is the simplicity of the procedure and therefore the short preparation time. In the demonstrated method adult mice are used but the same procedure can be applied with younger mice as well as rats. Also, the following patch clamp experiment is performed on horizontal cerebellar slices, but the same procedure can also be used in other planes as well as other posterior areas of the brain.

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

Slice It Hot: Agudo cerebrales en adultos rebanar en la temperatura fisiológica

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

Las grabaciones de células enteras Patch-clamp en rodajas de cerebro

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Proyección de imagen de calcio de dos fotones en las dendritas neuronales en rebanadas de cerebro

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Methods for the Discovery of Novel Compounds Modulating a Gamma-Aminobutyric Acid Receptor Type A Neurotransmission

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the identification of novel molecules active in preclinical assays from an in vitro recombinant receptor to their pharmacological effects at native receptors in rodent brain slices. For compounds binding at the benzodiazepine site of the receptor, the first step is to set up a primary screen that consists of developing radioligand binding assays on cell membranes expressing the major GABAA subtypes. Then, taking advantage of the heterologous expression of rodent and human GABAA receptors in Xenopus oocytes or HEK 293 cells, it is possible to explore, in electrophysiological assays, the physiological properties of the different receptor subtypes and the pharmacological properties of the identified compounds. The Xenopus oocyte system will be presented here, starting with the isolation of the oocytes and their microinjection with different mRNAs, up to the pharmacological characterization using two-electrode voltage clamps. Finally, recordings conducted in rodent brain slices will be described that are used as a secondary physiological test to assess the activity of molecules at their native receptors in a well-defined neuronal circuit. Extracellular recordings using population responses of multiple neurons are demonstrated together with the drug application.

Here, we present protocols to discover compounds active at GABAA receptors, from the binding to the physiology and pharmacology.

Métodos para el descubrimiento de nuevos compuestos modular un Receptor del ácido Gamma - Aminobutírico tipo una neurotransmisión

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the ...

Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics and investigate its underlying mechanisms. Acute seizure models are convenient, offer the ability to perform electrophysiological recordings, and can generate a large volume of electrographic seizure-like (ictal) events. The promising findings from acute seizure models can then be advanced to chronic epilepsy models and clinical trials. Thus, studying seizures in acute models that faithfully replicate the electrographic and dynamical signatures of a clinical seizure will be essential for making clinically relevant findings. Studying ictal events in acute seizure models prepared from human tissue is also important for making findings that are clinically relevant. The key focus in this paper is on the cortical 4-AP model due to its versatility in generating ictal events in both in vivo and in vitro studies, as well as in both mouse and human tissue. The methods in this paper will also describe an alternative method of seizure induction using the Zero-Mg2+ model and provide a detailed overview of the advantages and limitations of the epileptiform-like activity generated in the different acute seizure models. Moreover, by taking advantage of commercially available optogenetic mouse strains, a brief (30 ms) light pulse can be used to trigger an ictal event identical to those occurring spontaneously. Similarly, 30 - 100 ms puffs of neurotransmitters (Gamma-Amino Butyric Acid or glutamate) can be applied to the human tissue to trigger ictal events that are identical to those occurring spontaneously. The ability to trigger ictal events on-demand in acute seizure models offers the newfound ability to observe the exact sequence of events that underlie seizure initiation dynamics and efficiently evaluate potential anti-seizure therapies.

Acute seizure models are important for studying the mechanisms underlying epileptiform events. Furthermore, the ability to generate epileptiform events on-demand provides a highly efficient method to study the exact sequence of events underlying their initiation. Here, we describe the acute 4-aminopyridine cortical seizure models established in mouse and human tissue.

Generación y demanda inicio de la actividad Ictal aguda en roedores y tejido humano

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics ...

Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect distinct cellular and network components of these circuits remains unclear. Ex vivo brain slices provide a means by which investigators may probe discrete components of complex networks and disentangle potential mechanisms underlying the effects of volatile anesthetics on evoked responses. To isolate potential cell type- and pathway-specific drug effects in brain slices, investigators must be able to independently activate afferent fiber pathways, identify non-overlapping populations of cells, and apply volatile anesthetics to the tissue in aqueous solution. In this protocol, methods to measure optogenetically-evoked responses to two independent afferent pathways to neocortex in ex vivo brain slices are described. Extracellular responses are recorded to assay network activity and targeted whole-cell patch clamp recordings are conducted in somatostatin- and parvalbumin-positive interneurons. Delivery of physiologically relevant concentrations of isoflurane via artificial cerebral spinal fluid to modulate cellular and network responses is described.

Ex vivo brain slices can be used to study the effects of volatile anesthetics on evoked responses to afferent inputs. Optogenetics are employed to independently activate thalamocortical and corticocortical afferents to non-primary neocortex, and synaptic and network responses are modulated with isoflurane.

Activación optogenética de vías aferentes en cortes cerebrales y modulación de respuestas por anestésicos volátiles

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

Rebanadas hipocampales horizontales del cerebro del ratón

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

Recording Whole-Cell Currents with GABA Puffing in a Mouse Brain Slice

Source: Feng, Y. et al., <a href="https://app.jove.com/v/56387">Whole-cell Currents Induced by Puff Application of GABA in Brain Slices.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates the recording of whole-cell currents induced by gamma-aminobutyric acid, or GABA, in a mouse prefrontal cortical brain slice. Controlled delivery of GABA opens chloride channels in a postsynaptic neuron, generating inhibitory postsynaptic currents, or IPSCs. These IPSCs increase with higher GABA concentrations, reflecting GABA&#39;s inhibitory activity on synaptic transmission.

Registro de corrientes de células enteras con resoplido de GABA en un corte de cerebro de ratón

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Preparation of Solutions

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