Name: whole-cell currents induced by puff application of gaba in brain slices
Uploaded: 2017-10-12T14:00:00
Description: Watch this Scientific Journal Video about Whole-cell Currents Induced by Puff Application of GABA in Brain Slices at JoVE.com

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...

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D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+determining+the+recruitment+of+inositol+trisphosphate+receptor+channels+during+calcium+puffs.">Factors determining the recruitment of inositol trisphosphate receptor channels during calcium puffs.</a> Biophys J. 105 (11), 2474-2484 (2013).</li><li>Lee, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Columnar+distribution+of+activity+dependent+gabaergic+depolarization+in+sensorimotor+cortical+neurons.">Columnar distribution of activity dependent gabaergic depolarization in sensorimotor cortical neurons.</a> Mol Brain. 5, 33 (2012).</li><li>Glykys, J., Mody, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+main+source+of+ambient+GABA+responsible+for+tonic+inhibition+in+the+mouse+hippocampus.">The main source of ambient GABA responsible for tonic inhibition in the mouse hippocampus.</a> J Physiol. 582, 1163-1178 (2007).</li><li>Chen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=APP+modulates+KCC2+expression+and+function+in+hippocampal+GABAergic+inhibition.">APP modulates KCC2 expression and function in hippocampal GABAergic inhibition.</a> Elife. 6, (2017).</li><li>Dunn, A. J., Swiergiel, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+interleukin-1+and+endotoxin+in+the+forced+swim+and+tail+suspension+tests+in+mice.">Effects of interleukin-1 and endotoxin in the forced swim and tail suspension tests in mice.</a> Pharmacol Biochem Behav. 81 (3), 688-693 (2005).</li><li>Engel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+Patch-clamp+Recordings+from+the+Somatodendritic+Domain+of+Nigral+Dopamine+Neurons.">Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons.</a> J Vis Exp. (117), (2016).</li><li>Wondolowski, J., Frerking, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subunit-dependent+postsynaptic+expression+of+kainate+receptors+on+hippocampal+interneurons+in+area+CA1.">Subunit-dependent postsynaptic expression of kainate receptors on hippocampal interneurons in area CA1.</a> J Neurosci. 29 (2), 563-574 (2009).</li><li>Yang, L., Wang, Z., Wang, B., Justice, N. J., Zheng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+precursor+protein+regulates+Cav1.2+L-type+calcium+channel+levels+and+function+to+influence+GABAergic+short-term+plasticity.">Amyloid precursor protein regulates Cav1.2 L-type calcium channel levels and function to influence GABAergic short-term plasticity.</a> J Neurosci. 29 (50), 15660-15668 (2009).</li><li>Huo, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prefrontal+Cortical+GABAergic+Dysfunction+Contributes+to+Aberrant+UP-State+Duration+in+APP+Knockout+Mice.">Prefrontal Cortical GABAergic Dysfunction Contributes to Aberrant UP-State Duration in APP Knockout Mice.</a> Cereb Cortex. , (2016).</li><li>Zhou, W. L., Gao, X. B., Picciotto, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylcholine+Acts+through+Nicotinic+Receptors+to+Enhance+the+Firing+Rate+of+a+Subset+of+Hypocretin+Neurons+in+the+Mouse+Hypothalamus+through+Distinct+Presynaptic+and+Postsynaptic+Mechanisms.">Acetylcholine Acts through Nicotinic Receptors to Enhance the Firing Rate of a Subset of Hypocretin Neurons in the Mouse Hypothalamus through Distinct Presynaptic and Postsynaptic Mechanisms.</a> eNeuro. 2 (1), (2015).</li></ol>

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.

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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 ...

The overall goal of this puff technique is to conduct the pharmacological administration in whole-cell patch-clamp recording. This method can help answer key questions in the electrophysiology field, such as pharmacological administration. The main advantage of this technique is that the drug concentration around the recording site increases rapidly, thus preventing the membrane receptors from desensitization.Generally, individual new to this method will struggle because the preparation of acute prefrontal cortex slice and the position of puff micropipette are relatively difficult. To begin this procedure, crush the frozen cutting solution to obtain a slush. Keep the slushy solution on ice, and bubble it with 95%oxygen, 5%carbon dioxide.Next, place the dissected head into a beaker filled with ice-cold cutting solution, and take it out after a few seconds. Cut the skull along the midline in a caudal-rostral direction with fine scissors, and remove the lateral skull portions. Then, remove the brain with a fine spatula, and drop it into the ice-cold cutting solution.Afterward, place the brain on the lid of a nine-centimeter Petri dish filled with ice. Rinse the brain with ice-cold cutting solution. Then, cut off the cerebellum and olfactory bulb with a razor blade.Subsequently, apply cyanoacrylate glue to the Vibratome specimen holder. Carefully place the brain block on the drop of glue, rostral side up, and immediately immerse it in the ice-cold cutting solution. Adjust the blade holder to an angle of 15 degrees with reference to the horizontal plane.Section the brain tissues into 350 micron coronal slices. Then, transfer the slides into the recovery chamber filled with ACSF, and incubate them for one hour with constant bubbling of 95%oxygen, 5%carbon dioxide. In our experiment, the preparation of acute prefrontal cortex slice is also critical in the procedure.A healthy brain slice means active neurons that can be patched tightly. In this procedure, make glass borosilicate micropipettes for use as recording electrodes or puff micropipettes. For recording electrodes, tip resistances are in the range of four to six megaohm, while puff micropipettes have tip diameters in the range of two to five micrometers.Add sodion channel blocker to ACSF to block fast sodion channel and thus action potentials, and maintain a constant flow rate of this solution of two to three milliliter per minute in the recording chamber. Bubble the ACSF with 95%oxygen, 5%carbon dioxide constantly to ensure the viability of the slices. Transfer a brain slice to the recording chamber, using a Pasteur pipette with its fine tip cut to fit the size of the slice.Next, place a platinum slice anchor above the slice to hold it on the platform. After that, fill the recording micropipettes with internal solution, and fill the puff micropipettes with GABA at 10 micromolar, 50 micromolar, and 100 micromolar, or ACSF as vehicle control. To prevent blockage with debris, apply slight positive pressure using a one-milliliter syringe before immersing the recording electrode in the ACSF.Under a microscope, position the recording electrode and puff micropipette so that the tips appear in the center of the monitor. Then, identify a target neuron. Adjust the microscope focus while gradually lowering the puff micropipette, and place it above the recording neuron at an angle of 45 degrees.Keep the distance between the tip of the puff micropipette and the target neuron in the range of 20 to 40 micrometers. Slowly and carefully approach the neuron with the recording electrode, and then release the positive pressure. Apply a weak and brief suction through the tubing connected to the electrode holder to create a gigaohm seal.Maintain the voltage at zero millivolts. After the formation of the gigaohm seal, compensate the fast and slow capacitance manually or automatically. Then, apply a brief and strong suction through the tubing to break into the whole-cell mode.Subsequently, record evoked IPSC currents in voltage clamp mode. Deliver single or paired GABA puffs through the puff micropipette controlled by a Master-8 voltage-step generator. Change the puff duration to obtain the best evoked IPSC currents results, and measure the evoked IPSC currents in the LPS-induced depression model.Shown here, are the sample IPSCs induced by 10, 50, and 100 micromolar GABA. And here are the puff duration response curves. The repetitive currents evoked by GABA at 50 micromolar puffs at interpuff intervals of one, two, or three seconds are shown here.One mastered, this technique can be done in two hours if it is performed properly. While attempting this procedure, it's important to remember to obtain healthy brain slices. After its development, this technique paved the way for researchers in the field of pharmacological administration to explore the effect of drug in electrophysiology.After watching this video, you should have a good understanding of how to conduct the pharmacological administration in whole-cell patch-clamp recording. Don't forget that working with TTX can be extremely hazardous and precautions, such as wearing gloves, should always be taken while performing this procedure.

Research

JoVE Journal

Neuroscience

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

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

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

La aplicación local de medicamentos para estudiar la función del receptor nicotínico de acetilcolina en rodajas de cerebro de ratón

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent ...

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

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 ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

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 ...

Whole-cell Patch-clamp Recordings in Brain Slices

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 ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Aplicación de la abrazadera de parche guiada por imágenes automatizada para el estudio de neuronas en cortes cerebrales

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

Evaluación de la densidad de sinapsis en rebanadas de hipocampo cerebral de roedor

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Grabación y modulación de la actividad epileptiforme en rebanadas de cerebro de roedor juntada a arreglos de microelectrodos

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Preparación de láminas agudas de la médula espinal para celulares Patch-clamp de grabación en las neuronas de la sustancia Gelatinosa

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Evaluación de la inducción a largo plazo de la depresión en rodajas de cerebelosores adultos

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...