Los autores desean dar las gracias a la oficina del programa de Post bachillerato académicos e institutos de Novartis para la investigación biomédica en su conjunto para la financiación de este estudio.

1. preparación de las células
Nota: Este protocolo, incluyendo la generación de datos, utiliza las células HEK293 transduced con un baculovirus codificación NR1 y NR2A las células.
<ol><li>Semillas el número apropiado de células HEK293 y añada el virus NR1 y NR2A las concentraciones finales apropiadas (1.00 μl cada una). Para una placa de 384 pozos, utilice 10.000 células/pozo en un volumen final de 30 μl.</li>
	<li>Como alternativa, las células HEK293-NR1-NR2A en el número de células adecuado y el volumen (para una placa de 384 pozos, uso 10.000 células/pozo en un volumen final de 30 μL) de la semilla. Añadir tetraciclina en una concentración de 2 μg/mL inducen la expresión de NR2A y agregar la protección compuesta (100 μm MDL105, 519 o 5 μm CGP07066724).</li>
	<li>Nota: El baculovirus codificación NR1 y N2 deben tener un título aproximado entre 5 x 109 - 10 x 109 infecciosas unidad por mililitro24.</li>
</ol>2. medición de la actividad del receptor de NMDA
<ol><li>Preparar una placa de 384 pocillos con células HEK293 transduced con células HEK293-NR1-NR2A NR1/N2 en presencia de cualquier compuesto de protección. Utilice un fondo claro, marco negro, placa revestida poly-D-lisina. Nota: Protección con 100 μm MDL105, 519 se utiliza para conferir sensibilidad a glicina, mientras que 5 μm CGP070667 se utiliza para conferir sensibilidad al glutamato.</li>
	<li>Incubar la placa a 37 ° C y 5% CO2 para 16 horas (noche).</li>
	<li>Retire la placa de la incubadora y deseche suavemente los medios de comunicación celular, retardando la placa de los bancos sobre un contenedor de residuos biológicos compatibles. Golpear suavemente la placa sobre papel absorbente para limpiar los medios de comunicación fuera de los bordes de la placa y drenar cualquier medio restante.</li>
	<li>Para preparar el tinte calcium6 solubilización de un frasco de tintura de calcium6 (1 tamaño de la placa) en 10 mL de tampón de incubación (HBSS pH 7.5, 20 mM HEPES, 1 mM MgCl2, probenecid 1 mM). Incube la placa durante 2 horas a 37 ° C y 5% CO2.</li>
	<li>Retire la placa de la incubadora y dejar que las células a ajustar a la temperatura ambiente durante 10 minutos.</li>
	<li>Verter suavemente el tinte de calcium6 como se describe en el paso 2.3 y añadir 30 μl de tampón de ensayo (HBSS pH 7.5, 20 mM HEPES, 1,8 mM CaCl2, probenecid 1 mM).</li>
	<li>Repita el paso 2.6 dos veces para un total de 3 lavados. Dejar 30 μl de tampón de ensayo en las células después del último lavado.</li>
	<li>Que las células reposar durante 5 minutos a temperatura ambiente.</li>
	<li>Preparar la placa de ligando, haciendo un caldo 4 veces de 400 μm glicina y el glutamato 400 μm (concentración final de 100 μm, concentración óptima de ligandos que se determinará como se describe a continuación). Transferencia de un mínimo de 25 μl del stock de ligando en una placa de 384 pocillos V-inferior.</li>
	<li>Cargue la placa celular y placa de ligando en la FDSS (sistema de detección de drogas funcional).</li>
	<li>Medir la fluorescencia basal (Fo) de la placa celular durante 30 segundos. Transferir 10 μl de la acción del ligando en la placa de la célula usando la función dispensadora FDSS y mida el flujo de calcio mediante el registro de fluorescencia calcium6-colorante durante 5 minutos.</li>
	<li>Calcular el cociente de la fluorescencia máxima dividiendo la fluorescencia máxima obtenida por la fluorescencia basal (Fmáx/fo).</li>
</ol>3. optimización de los niveles de expresión de NMDAR
<ol><li>Para determinar la cantidad óptima de baculovirus para utilizar, realizar una titulación de virus NR1 y NR2A. Prepare una placa de 384 pocillos con células HEK293 (recomendación de 10.000 células/pozo, 30 μl de medios de comunicación) e incluyen los compuestos de protección tal como se describe en el paso 2.1.</li>
	<li>Añadir cantidades variables del virus NR1 en filas diferentes de la placa (por ejemplo: agregar 0 μL 2 μl, 1 μl, 0,5 μl, 0.25 μL en filas A-E, respectivamente). Agregar duplicados para la exactitud de los datos.</li>
	<li>Añadir cantidades variables del virus NR2A en diferentes columnas de la placa (por ejemplo: agregar 0 μL 2 μl, 1 μl, 0,5 μl, 0.25 μL en filas A-E, respectivamente). Agregar duplicados para la exactitud de los datos.</li>
	<li>Incubar la placa a 37 ° C y 5% CO2 para 16 horas (noche).</li>
	<li>Determinar la relación óptima y la cantidad de los virus de NR1/NR2A realizando una medición del flujo de calcio en la FDSS (véase arriba).</li>
</ol>4. ligando titulación
<ol><li>Preparar las células como se describe en el paso 2.3.</li>
	<li>Preparar diluciones de cada ligando en presencia de concentraciones (100 μm) se satura de otro ligando como una solución madre 4 veces. Por ejemplo: para una concentración de prueba final de 10 μm de glicina, prepare una solución stock de 40 μm de glicina como 400 μm glutamato.</li>
	<li>Proceder con la medición de la FDSS como se describe en el paso 2.11.</li>
</ol>5. compuesto de prueba
<ol><li>Preparar las células como se describe en el paso 1.</li>
	<li>Preparar la placa de ligando con una 5-fold acción de la concentración de ligando final en el tampón de ensayo.</li>
	<li>Preparar la placa de compuestos con el caldo 4 veces de la concentración final deseada de compuestos en el tampón de ensayo. Para una concentración final compuesta de 10 μm en el tampón de ensayo, preparar una solución stock de 40 μm.<ol>
<li>Mantener la concentración de dimetilsulfóxido (DMSO) constante a través de todas las muestras mediante la preparación de una dilución previa del compuesto en DMSO antes de la mayor dilución en el tampón de ensayo.</li>
		<li>Para una respuesta a la dosis del compuesto en el ensayo final que oscila entre 3 y 30 μm, preparar una dilución previa del compuesto en DMSO que van desde 1 mm a 10 mM. Diluir en el tampón de ensayo a la concentración de acción 4 veces. La concentración final del DMSO no debe exceder 0.5%. Nota: Es aconsejable probar cada muestra en triplicado.</li>
	</ol>
</li>
	<li>Cargue la placa celular, ligando y compuesto en FDSS.</li>
	<li>Medir la fluorescencia basal durante 30 segundos.</li>
	<li>Añadir 10 μl de caldo compuesto y medir la fluorescencia durante 5 minutos.</li>
	<li>Añadir 10 μl del stock de ligando y medir la fluorescencia durante 5 minutos.</li>
	<li>Determinar la integral de la relación de fluorescencia calculando el área bajo la curva de los cocientes fluorescentes durante los últimos 5 minutos de la medida. Nota: Alternativamente, el cociente máximo de fluorescencia después de la adición de ligandos puede utilizarse para el análisis.</li>
</ol>

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Los autores tienen ningún conflicto de intereses y nada que revelar.

El éxito de este ensayo depende en gran medida la salud de las células HEK. Las células que experimentan un crecimiento exponencial y con número de paso bajo se debe utilizar. Este análisis implica a muchos traslados y adiciones de soluciones, así que con cuidado asegurarán una mayor precisión en sus resultados. Concentraciones de compuestos y otros reactivos deben ser también revisado en cruz para minimizar los errores. Al sustituir los medios de comunicación de la célula con el tampón de ensayo para el aná...

Con los avances actuales en la medicina, la esperanza de vida ha aumentado significativamente; sin embargo, tiene tan la prevalencia de enfermedades relacionadas con la edad. Enfermedades del sistema nervioso central (SNC) tales como esquizofrenia, esclerosis lateral amiotrófica (ELA), enfermedad de Alzheimer y de Parkinson, entre otros, no son la excepción y se han proyectado para aumentar durante el próximo decenio1, 2 , 3. el mal funcionamiento de receptores de glutamato ionotrópicos conocido como receptores N-metil-D-Aspartato (NMDAR) se ha relacionado con la enfermedad de Alzheimer, esquizofrenia, lesión cerebral traumática, accidente cerebrovascular, diabetes y glaucoma entre otros, que garantiza la necesidad de estudiar su biología para el desarrollo de terapias eficaces, modificadores de la enfermedad4,5,6,7.
NMDARs se componen de cuatro monómeros o subunidades4,8,9. La composición estructural de lo NMDAR muestra variabilidad regional y de desarrollo en el cerebro de7,10. NMDARs están implicados en la plasticidad sináptica, la cognición y la generación de los ritmos de respiración y locomoción11,12,13. Como un canal voltaje-bloqueado, es en gran parte no conductoras en el potencial de membrana de reposo (-70 mV) y es bloqueado por magnesio para evitar la penetración de los iones. El canal es activado por la Unión de dos ligandos, glutamato y glicina/D-serina y una simultánea despolarización en la membrana sináptica mediada por receptores AMPA, otra subclase de receptores de glutamato ionotrópicos. La despolarización elimina el bloqueo de magnesio de NMDAR, permitiendo la afluencia de cationes, particularmente calcio14,15,16. A pesar de que la activación de NMDAR es esencial para la supervivencia de la célula, activación excesiva puede conducir a la célula muerte17,18,19 por excitotoxicidad. Esto, además de la compleja naturaleza de los receptores, hace difícil realizar estudios necesarios para el desarrollo de terapias efectivas.
Se han desarrollado diferentes métodos para estudiar el NMDAR. Sin embargo, cada uno tiene acompañamiento de advertencias. Por ejemplo, una técnica ampliamente utilizada es un ensayo basado en la fluorescencia que mide los cambios de NMDAR mediada por calcio intracelular en una línea celular estable bajo el control de un promotor inducible por tetraciclina (Tet-en)20. Sin embargo, en este sistema, las concentraciones de supramaximal de ligandos que se necesitan y el requisito de que los inhibidores de NMDAR están presentes durante la medición es casi imposible detectar la actividad del antagonista competitivo. En otros sistemas similares, la expresión del receptor funcional de causa toxicidad, requiriendo bloqueadores de los canales tales como ketamina21,22 para preservar los cultivos celulares. Estos bloqueadores de los canales se sientan en el corazón del receptor y son difíciles de lavar, sobre todo en un formato basado en la placa, para que interfieran con los estudios funcionales del receptor. Finalmente, en medidas electrofisiológicas como fijación con abrazadera del remiendo, hay limitado rendimiento, y estudios de gran escala son muy caro23. No obstante, los sistemas anteriores son insensibles a la estimulación de la glicina; por lo tanto, estudiando la actividad dependiente de la glicina de la NMDAR se convierte en un reto.
Aquí, describimos un nuevo enfoque para el estudio de NMDAR que supera las limitaciones discutidas. Nuestra técnica aprovecha el sistema de expresión de baculovirus para expresar el receptor a nivel funcional con una relación óptima de las subunidades en tan poco como 16 horas. Además, el uso de baculovirus permite un enfoque simple y combinatorio, que proporciona una caracterización amplia de los distintos subtipos NMDAR recombinantes. A diferencia de otros ensayos, este Protocolo no requiere bloqueadores de los canales por el uso de antagonistas débiles. Ventaja más fuerte del método es que lavado después de la débil antagonista, el receptor es sensible a la modulación de los sitios individuales de glicina y glutamato enlace además de doble modulación de D/glicina-serina y glutamato-ligando sitios. El ensayo recapitula conocido farmacología del receptor NMDAR y los efectos de sus conocidos moduladores positivos y negativos. Finalmente, la generación de este ensayo celular en vitro supera la toxicidad celular causada por la afluencia excesiva de calcio y permite estudios funcionales del receptor de la manera un alto rendimiento, que puede acelerar descubrimientos de moduladores NMDAR en Estados de enfermedad.

<table><tbody><tr><td>HEK-293</td><td>ATCC</td><td>CRL-1573</td><td></td></tr><tr><td>Human NMDA (NR1/NR2A) Receptor Cell Line</td><td>ChanTest Corporation</td><td>CT6120</td><td></td></tr><tr><td>pFastBac Dual Expression Vector</td><td>ThermoFisher Scientific</td><td>10712-024</td><td></td></tr><tr><td>Corning 384-well Clear Flat Bottom Microplate</td><td>Corning Life Sciences</td><td>3844</td><td></td></tr><tr><td>FLIPR Calcium 6-QF Assay Kit</td><td>Molecular Devices</td><td>R8192</td><td></td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td>G7126</td><td></td></tr><tr><td>Glutamate</td><td>Sigma-Aldrich</td><td>49621</td><td></td></tr><tr><td>D-serine</td><td>Sigma-Aldrich</td><td>S4250</td><td></td></tr><tr><td>L701,324</td><td>Tocris</td><td>907</td><td></td></tr><tr><td>HEPES Buffer</td><td>Boston Bio Product</td><td>BB-103</td><td></td></tr><tr><td>Magnesium Chloride Solution</td><td>Sigma-Aldrich</td><td>63069</td><td></td></tr><tr><td>Calcium Chloride</td><td>VWR</td><td>E506</td><td></td></tr><tr><td>HBSS</td><td>ThermoFisher Scientific</td><td>14025-092</td><td></td></tr><tr><td>Probenecid</td><td>ThermoFisher Scientific</td><td>P36400</td><td></td></tr><tr><td>DMEM/F-12, GlutaMAX media</td><td>ThermoFisher Scientific</td><td>10565018</td><td></td></tr><tr><td>MDL105,519</td><td>NIBR</td><td>Synthesized in house</td><td></td></tr><tr><td>NVP-AAM077</td><td>NIBR</td><td>Synthesized in house</td><td></td></tr><tr><td>CGP070667</td><td>NIBR</td><td>Synthesized in house</td><td></td></tr></tbody></table>

a high-throughput calcium-flux assay to study nmda-receptors with sensitivity to glycine/d-serine and glutamate

Los receptores N-metil-D-Aspartato (NMDA) (NMDAR) se clasifican como receptores de glutamato ionotrópicos y tienen un papel crítico en el aprendizaje y la memoria. Mal funcionamiento NMDAR, expresado como sea sobre - o debajo - activity causada por mutaciones, expresión alterada, tráfico y localización, puede contribuir a numerosas enfermedades, especialmente en el sistema nervioso central. Por lo tanto, entender la biología del receptor, así como facilitar el descubrimiento de compuestos y moléculas pequeñas es crucial en los esfuerzos para combatir enfermedades neurológicas. Enfoques actuales del estudio de los receptores tienen limitaciones como bajo rendimiento, alto costo y la incapacidad para el estudio de sus capacidades funcionales debido a la necesaria presencia de bloqueadores de los canales para evitar la excitotoxicidad mediada de NMDAR. Además, los sistemas de análisis existentes son sensibles a la estimulación por el glutamato solo y carecen de sensibilidad a la estimulación por glicina, otro ligando co del NMDAR. Presentamos el primer ensayo de placa de base con la energía de alto rendimiento para el estudio de un receptor NMDA con sensibilidad a ligandos Co, glutamato y D-serina/glicina. Este enfoque permite el estudio de las diferentes composiciones de subunidad NMDAR y estudios funcionales del receptor en modos sensibles a glutamato y glicina. Además, el método no requiere la presencia de inhibidores durante las mediciones. Los efectos de los moduladores alostéricos positivos y negativos se pueden detectar con este ensayo y la farmacología conocida de NMDAR ha sido replicada en nuestro sistema. Esta técnica supera las limitaciones de los métodos existentes y es económica. Creemos que esta nueva técnica acelerará el descubrimiento de terapias para patologías de NMDAR-mediada.

Antes de probar los efectos de moléculas pequeñas, uno debe determinar los niveles de expresión óptima de NMDARs, así como las concentraciones de ligando óptima. Como se describe, las células fueron sembradas a 10.000 células por pocillo en una placa de 384 pozos, HEK293 en presencia de 5 μm CGP060667, luego transduced con cantidades variables de NR1 y NR2A virus. Después de incubación durante la noche, inducida por el ligando calcio se midió flujo (figura...

Watch this Scientific Journal Video about A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate at JoVE.com

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

Un análisis del flujo de calcio de alto rendimiento para el estudio de los receptores NMDA con sensibilidad D/glicina-serina y glutamato

El objetivo de este protocolo es facilitar el estudio de receptores de NMDA (NMDAR) a una escala mayor y permitir el examen de los efectos moduladores de pequeñas moléculas y sus aplicaciones terapéuticas.

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR ...

a-high-throughput-calcium-flux-assay-to-study-nmda-receptors-with

Research

JoVE Journal

Neuroscience

9.2K vistas.  Novartis Institutes for BioMedical Research. El objetivo de este protocolo es facilitar el estudio de receptores de NMDA (NMDAR) a una escala mayor y permitir el examen de los efectos moduladores de pequeñas moléculas y sus aplicaciones terapéuticas.

Video: A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

Glutamine Flux Imaging Using Genetically Encoded Sensors

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for biological molecules became available in the past 15 years, some of which became indispensable tools that are used routinely in many laboratories.
One of the exciting applications of genetically encoded sensors is the use of these sensors in investigating cellular transport processes. Properties of transporters such as kinetics and substrate specificities can be investigated at a cellular level, providing possibilities for cell-type specific analyses of transport activities. In this article, we will demonstrate how transporter dynamics can be observed using genetically encoded glutamine sensor as an example. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed.

This article will demonstrate how to monitor glutamine dynamics in live cells using FRET. Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed for genetically encoded glutamine sensors.

Glutamina Flux Imaging utilizando sensores codificados genéticamente

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for ...

Investigación

Bioingeniería

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high spatial and temporal resolution. Combined with electrophysiology, it is widely used to study activity-related calcium signals in small subcellular compartments such as dendrites and dendritic spines. In addition to calcium transients, synaptic activity also induces postsynaptic sodium signals, the properties of which are only marginally understood. Here, we describe a method for combined whole-cell patch-clamp and multi-photon sodium imaging in cellular micro domains of central neurons. Furthermore, we introduce a modified procedure for ultra-violet (UV)-light-induced uncaging of glutamate, which allows reliable and focal activation of glutamate receptors in the tissue. To this end, whole-cell recordings were performed on Cornu Ammonis subdivision 1 (CA1) pyramidal neurons in acute tissue slices of the mouse hippocampus. Neurons were filled with the sodium-sensitive fluorescent dye SBFI through the patch-pipette, and multi-photon excitation of SBFI enabled the visualization of dendrites and adjacent spines. To establish UV-induced focal uncaging, several parameters including light intensity, volume affected by the UV uncaging beam, positioning of the beam as well as concentration of the caged compound were tested and optimized. Our results show that local perfusion with caged glutamate (MNI-Glutamate) and its focal UV-uncaging result in inward currents and sodium transients in dendrites and spines. Time course and amplitude of both inward currents and sodium signals correlate with the duration of the uncaging pulse. Furthermore, our results show that intracellular sodium signals are blocked in the presence of blockers for ionotropic glutamate receptors, demonstrating that they are mediated by sodium influx though this pathway. In summary, our method provides a reliable tool for the investigation of intracellular sodium signals induced by focal receptor activation in intact brain tissue.

We describe the combination of focal UV-induced photo-activation of neuro-active compounds with whole-cell patch-clamp and multi-photon imaging of intracellular sodium transients in dendrites and spines of hippocampal neurons in acute tissue slices of the mouse brain.

Multi-fotón intracelular de sodio Imaging Combinado con Desembalado focal UV-mediada de glutamato en las neuronas CA1 Piramidal

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high ...

Neurociencias

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

Un ensayo de fluorescencia de calcio de "doble adición" para el cribado de alto rendimiento de receptores acoplados a proteínas G recombinantes

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

Bioquímica

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

El uso de biosensores basados ​​en enzimas para medir tónica y fásica glutamato en modelos de ratones con Alzheimer

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate (NMDA) Receptor in Blood

The presence of anti-NMDA receptor autoantibody can cause various neuropsychiatric symptoms in the affected patients, termed anti-NMDA receptor autoimmune encephalitis. Detection of the specific autoantibody against the NMDA receptor in the blood or cerebrospinal fluid (CSF) is essential for the accurate diagnosis of this condition. The NMDA receptor is an ion channel protein complex that contains four subunits, including two mandatory NMDA receptor subunit 1 (NR1) and one or two NMDA receptor subunit 2A (NR2A), NMDA receptor subunit 2B (NR2B), NMDA receptor subunit 2C (NR2C), or NMDA receptor subunit 2D (NR2D). The epitope of anti-NMDA receptor autoantibody was reported to be present at the extracellular N-terminal domain of the NR1 subunit of the NMDA receptor. The goal of this study is to develop a simple cell-based immunofluorescence assay that can be used as a screening test to detect the presence of autoantibodies against NR1 subunit of the NMDA receptor in the blood to facilitate the clinical and basic research of anti-NMDA receptor autoimmune encephalitis.

A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate NMDA Receptor in Blood

We ectopically expressed NR1 subunit of NMDA receptor tagged with green fluorescent protein in human embryonic cells (HEK293) as antigen to detect autoantibodies against NMDA receptor in the blood of patients suspected with autoimmune encephalitis. This simple method may be suitable for screening purposes in clinical settings.

Un análisis de la inmunofluorescencia celular Simple para detectar autoanticuerpos contra el Receptor N-metil-D-Aspartato (NMDA) en sangre

The presence of anti-NMDA receptor autoantibody can cause various neuropsychiatric symptoms in the affected patients, termed anti-NMDA receptor autoimmune ...

A High-content Assay for Monitoring AMPA Receptor Trafficking

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different stimuli. The &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are responsible for fast excitatory synaptic transmission in neurons, are trafficked to and from the postsynaptic surface to dynamically alter neuronal excitability. AMPA receptor trafficking is essential for synaptic plasticity and can be disrupted in neurological disease. However, prevalent approaches for quantifying receptor trafficking ignore entire receptor pools, are overly time- and labor-intensive, or potentially disrupt normal trafficking mechanisms and therefore complicate the interpretation of resulting data. We present a high-content assay for the quantification of both surface and internal AMPA receptor populations in cultured primary hippocampal neurons using dual fluorescent immunolabeling and a near-infrared fluorescent 96-well microplate scanner. This approach facilitates the rapid screening of bulk internalized and surface receptor densities while minimizing sample material. However, our method has limitations in obtaining single-cell resolution or conducting live cell imaging. Finally, this protocol may be amenable to other receptors and different cell types, provided proper adjustments and optimization.

Neuronal excitability can be modulated through a dynamic process of endo- and exocytosis of excitatory ionotropic glutamate receptors. Described here is an accessible, high-content assay for quantifying surface and internal receptor population pools.

Un análisis de contenido de alta para seguimiento AMPA del Receptor de tráfico

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different ...

An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population of surface-expressed receptors is constantly adapting and subject to strict mechanisms of regulation. The paradigmatic example and one of the most studied trafficking events in biology is the regulated control of the synaptic expression of glutamate receptors (GluRs). GluRs mediate the vast majority of excitatory neurotransmission in the central nervous system and control physiological activity-dependent functional and structural changes at the synaptic and neuronal levels (e.g., synaptic plasticity). Modifications in the number, location, and subunit composition of surface expressed GluRs deeply affect neuronal function and, in fact, alterations in these factors are associated with different neuropathies. Presented here is a method to study GluR trafficking in dissociated hippocampal primary neurons. An &#34;antibody-feeding&#34; approach is used to differentially visualize GluR populations expressed at the surface and internal membranes. By labeling surface receptors on live cells and fixing them at different times to allow for receptors endocytosis and/or recycling, these trafficking processes can be evaluated and selectively studied. This is a versatile protocol that can be used in combination with pharmacological approaches or overexpression of altered receptors to gain valuable information about stimuli and molecular mechanisms affecting GluR trafficking. Similarly, it can be easily adapted to study other receptors or surface expressed proteins.

This article presents a method to study glutamate receptor (GluR) trafficking in dissociated primary hippocampal cultures. Using an antibody-feeding approach to label endogenous or overexpressed receptors in combination with pharmacological approaches, this method allows for the identification of molecular mechanisms regulating GluR surface expression by modulating internalization or recycling processes.

Un enfoque de alimentación de anticuerpos para estudiar el tráfico de receptores de glutamato en cultivos hipocampales primarios disociados

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

Indicadores de sinapsis únicas de liberación y aceptación de glutamato en rebanadas cerebrales agudas de ratones normales y Huntington

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

Cuantificación de los flujos espontáneos de Ca2+ y sus efectos aguas abajo en las neuronas primarias de cerebro medio de ratón

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Un canal celular conectado a Patch-clamp grabación

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Un análisis del flujo de calcio de alto rendimiento para el estudio de los receptores NMDA con sensibilidad D/glicina-serina y glutamato

Resumen

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Un ensayo de fluorescencia de calcio de "doble adición" para el cribado de alto rendimiento de receptores acoplados a proteínas G recombinantes

El uso de biosensores basados en enzimas para medir tónica y fásica glutamato en modelos de ratones con Alzheimer

Un análisis de la inmunofluorescencia celular Simple para detectar autoanticuerpos contra el Receptor N-metil-D-Aspartato (NMDA) en sangre

Un análisis de contenido de alta para seguimiento AMPA del Receptor de tráfico

Un enfoque de alimentación de anticuerpos para estudiar el tráfico de receptores de glutamato en cultivos hipocampales primarios disociados

Indicadores de sinapsis únicas de liberación y aceptación de glutamato en rebanadas cerebrales agudas de ratones normales y Huntington

Cuantificación de los flujos espontáneos de Ca²⁺ y sus efectos aguas abajo en las neuronas primarias de cerebro medio de ratón

Un canal celular conectado a Patch-clamp grabación

Un análisis del flujo de calcio de alto rendimiento para el estudio de los receptores NMDA con sensibilidad D/glicina-serina y glutamato

Resumen

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Capítulos en este video

Glutamina Flux Imaging utilizando sensores codificados genéticamente

Multi-fotón intracelular de sodio Imaging Combinado con Desembalado focal UV-mediada de glutamato en las neuronas CA1 Piramidal

Un ensayo de fluorescencia de calcio de "doble adición" para el cribado de alto rendimiento de receptores acoplados a proteínas G recombinantes

El uso de biosensores basados ​​en enzimas para medir tónica y fásica glutamato en modelos de ratones con Alzheimer

Un análisis de la inmunofluorescencia celular Simple para detectar autoanticuerpos contra el Receptor N-metil-D-Aspartato (NMDA) en sangre

Un análisis de contenido de alta para seguimiento AMPA del Receptor de tráfico

Un enfoque de alimentación de anticuerpos para estudiar el tráfico de receptores de glutamato en cultivos hipocampales primarios disociados

Indicadores de sinapsis únicas de liberación y aceptación de glutamato en rebanadas cerebrales agudas de ratones normales y Huntington

Cuantificación de los flujos espontáneos de Ca2+ y sus efectos aguas abajo en las neuronas primarias de cerebro medio de ratón

Un canal celular conectado a Patch-clamp grabación

El uso de biosensores basados en enzimas para medir tónica y fásica glutamato en modelos de ratones con Alzheimer

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