Name: a high-throughput calcium-flux assay to study nmda-receptors with sensitivity to glycine/d-serine and glutamate
Uploaded: 2018-07-10T15:00:00.000+00:00
Duration: 4 min 48 s
Description: 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

Die Autoren möchten den Post Baccalaureate Scholars Program Office und Novartis Institutes for BioMedical Research als Ganzes für die Finanzierung dieser Studie danken.

1. Vorbereitung der Zellen
Hinweis: Dieses Protokoll, einschließlich der Datengenerierung, verwendet HEK293 Zellen mit einem Baculovirus Codierung NR1 und NR2A Zellen ausgestrahlt.
<ol><li>Die entsprechende Anzahl an HEK293 Zellen Saatgut und NR1 und/oder NR2A Virus am entsprechenden Endkonzentrationen (1,00 µL) hinzufügen. Verwenden Sie für ein 384-Well-Platte 10.000 Zellen/Brunnen in einem Endvolumen von 30 µL.</li>
	<li>Alternativ HEK293-NR1-NR2A Samenzellen über die entsprechende Zellenzahl und Volumen (für ein 384-Well-Platte, Verwendung 10.000 Zellen/Brunnen in einem Endvolumen von 30 µL). Tetracyclin in einer Konzentration von 2 µg/mL zu induzieren NR2A Ausdruck und fügen Sie den zusammengesetzten Schutz hinzufügen (100 µM MDL105, 519 oder 5 µM CGP07066724).</li>
	<li>Hinweis: Das Baculovirus Codierung NR1 und NR2 sollte eine ungefähre Titer zwischen 5 x 10-9 - 10 x 109 infektiösen Einheiten pro Milliliter24haben.</li>
</ol>2. Messung der NMDA-Rezeptor-Aktivität
<ol><li>Bereiten Sie eine 384-Well-Platte mit HEK293 Zellen mit NR1/NR2 oder HEK293-NR1-NR2A Zellen in Anwesenheit von entweder schützende Verbindung ausgestrahlt. Verwenden Sie eine klare Boden, schwarzer Rahmen, Poly-D-Lysin beschichtete Platte. Hinweis: Schutz mit 100 µM MDL105, 519 wird verwendet, um die Empfindlichkeit zu Glycin, während 5 µM verleihen CGP070667 wird verwendet, um die Empfindlichkeit gegenüber Glutamat zu verleihen.</li>
	<li>16 Stunden (über Nacht) inkubieren Sie die Platte bei 37 ° C und 5 % CO2 .</li>
	<li>Entfernen Sie die Platte aus dem Inkubator und entsorgen Sie sanft die Zelle Medien durch die Verlangsamung, spiegeln die Platte über eine kompatible Bioabfälle Container. Tippen Sie die Platte auf ein Papiertuch zu reinigen die Medien Kanten der Platte und entwässern alle übrigen Medien vorsichtig an.</li>
	<li>Bereiten Sie den calcium6-Farbstoff durch Gefäss-eine Phiole mit calcium6 Farbstoff (1 Plattengröße) in 10 mL Inkubation Puffer (HBSS pH 7.5, 20 mM HEPES, 1 mM MgCl2, 1 mM Probenecid). 2 Stunden bei 37 ° C und 5 % CO2inkubieren Sie Platte.</li>
	<li>Entfernen Sie die Platte aus dem Inkubator und lassen Sie die Zellen, die für 10 Minuten auf Raumtemperatur anpassen.</li>
	<li>Gießen Sie vorsichtig, des calcium6-Farbstoffes, wie unter Punkt 2.3 beschrieben und 30 µL testpuffer (HBSS pH 7.5, 20 mM HEPES, 1,8 mM CaCl2, 1 mM Probenecid).</li>
	<li>Wiederholen Sie Schritt 2.6 zweimal für eine Gesamtmenge von 3 Wäschen. Lassen Sie 30 µL testpuffer auf die Zellen nach dem letzten waschen.</li>
	<li>Lassen Sie die Zellen für 5 Minuten bei Raumtemperatur ruhen.</li>
	<li>Bereiten Sie die Liganden-Platte machen ein 4-fold Lager von 400 µM Glycin und 400 µM Glutamat (Endkonzentration von 100 µM; optimale Konzentration des Liganden ermittelt werden, wie unten beschrieben). Übertragen Sie ein Minimum von 25 µL des Liganden bestand in einem V-Boden-384-Well-Platte.</li>
	<li>Laden Sie die Liganden Platte und Zellplatte in FDSS (funktionelle Drug Screening-System).</li>
	<li>Die Baseline-Fluoreszenz (Fo) der Zellplatte für 30 Sekunden zu messen. Übertragen Sie 10 µL der Liganden bestand in der Zellplatte mit FDSS Abgabe Funktion und Maßnahme der Kalzium-Fluss durch die Aufnahme calcium6-Farbstoff Fluoreszenz für 5 Minuten.</li>
	<li>Berechnen Sie die maximale Fluoreszenz-Verhältnis dividiert die maximale Fluoreszenz durch die Basislinie Fluoreszenz (Fmax/fo) gewonnen.</li>
</ol>(3) Optimierung der NMDAR Ausdruck Niveaus
<ol><li>Um die optimale Menge an Baculovirus nutzen zu ermitteln, führen Sie eine Titrierung NR1 und NR2A Viren. Bereiten Sie eine 384-Well-Platte mit HEK293 Zellen (Empfehlung von 10.000 Zellen/gut, 30 µL der Medien) und umfassen Sie die Schutz-Verbindungen, wie unter Punkt 2.1 beschrieben.</li>
	<li>Fügen Sie unterschiedliche Mengen des Virus NR1 in verschiedenen Reihen der Platte (zum Beispiel: Fügen Sie 0 µL, 2 µL, 1 µL, 0,5 µL 0.25 µL bzw. in den Reihen A-E). Fügen Sie Duplikate für Korrektheit der Daten.</li>
	<li>Fügen Sie unterschiedliche Mengen an das NR2A-Virus in verschiedenen Spalten der Platte (zum Beispiel: Fügen Sie 0 µL, 2 µL, 1 µL, 0,5 µL 0.25 µL bzw. in den Reihen A-E). Fügen Sie Duplikate für Korrektheit der Daten.</li>
	<li>16 Stunden (über Nacht) inkubieren Sie die Platte bei 37 ° C und 5 % CO2 .</li>
	<li>Bestimmen Sie die optimale Verhältnis und die Höhe der NR1 und NR2A Viren durch Ausführen einer Kalzium-flussmessung FDSS (siehe oben).</li>
</ol>(4) Liganden Titration
<ol><li>Bereiten Sie die Zellen, wie unter Punkt 2.3 beschrieben.</li>
	<li>Bereiten Sie Verdünnungen von jeder Ligand in Anwesenheit von Sättigung Konzentrationen (100 µM vor) des anderen Liganden als eine 4-fold Stammlösung. Zum Beispiel: für eine 10 µM Abschlusstest Konzentration von Glycin, bereiten Sie eine 40 µM-Stammlösung von Glycin einschließlich 400 µM Glutamat.</li>
	<li>Fahren Sie mit der Messung von FDSS wie in Schritt 2.11 beschrieben.</li>
</ol>5. Verbindung testen
<ol><li>Bereiten Sie die Zellen, wie in Schritt 1 beschrieben.</li>
	<li>Bereiten Sie die Liganden-Platte mit einem 5-fold bestand der endgültigen Liganden-Konzentration in der Assay-Puffer.</li>
	<li>Bereiten Sie die Verbindungen-Platte mit dem 4-fold bestand der gewünschten Endkonzentration von Verbindungen in der Assay-Puffer. Bereiten Sie eine zusammengesetzte Endkonzentration von 10 µM in der Assay-Puffer eine Stammlösung 40 µM.<ol>
<li>Halten Sie die Konzentration von Dimethyl Sulfoxid (DMSO) konstant über alle Proben durch die Vorbereitung einer Vorverdünnung der Verbindung in DMSO vor weiteren Verdünnung in den Assay-Puffer.</li>
		<li>Bereiten Sie für eine Dosis-Antwort der Verbindung in der letzten Probe zwischen 3 und 30 µM eine Vorverdünnung der Verbindung im Bereich von 1 bis 10 mM DMSO. In der Assay-Puffer, die 4-fold Lager Konzentration zu verdünnen. Die Endkonzentration der DMSO sollte 0,5 % nicht überschreiten. Hinweis: Es wird empfohlen, jede Probe in Triplicates zu testen.</li>
	</ol>
</li>
	<li>Laden Sie die Liganden, die Verbindung und die Zellplatte in FDSS.</li>
	<li>Messen Sie die Grundlinie Fluoreszenz für 30 Sekunden.</li>
	<li>Fügen Sie 10 µL des zusammengesetzten Materials und Messung der Fluoreszenz für 5 Minuten.</li>
	<li>Fügen Sie 10 µL des Liganden Lager und Messung der Fluoreszenz für 5 Minuten.</li>
	<li>Das Integral der Fluoreszenz-Verhältnis durch die Berechnung der Fläche unter der Kurve der fluoreszierenden Verhältnisse während der letzten 5 Minuten der Messung zu bestimmen. Hinweis: Alternativ kann das maximale Verhältnis der Fluoreszenz nach Ligand Zugabe für die Analyse verwendet werden.</li>
</ol>

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Der Erfolg dieser Assay hängt die Gesundheit der HEK Zellen verwendet. Zellen, exponentielles Wachstum erlebt und mit niedrigen Passagenanzahl sollte verwendet werden. Dieser Test beinhaltet viele Transfers und Ergänzungen von Lösungen, also mit Vorsicht sorgt für höhere Genauigkeit des Ergebnisses. Konzentrationen von Verbindungen und alle anderen Reagenzien sollten auch abgeglichen, Fehler zu minimieren. Beim Austausch der Zelle Medien mit dem Assay-Puffer für die Kalzium-Flux-Assay sollte die Platte vorsichtig g...

Mit den aktuellen Fortschritten in der Medizin hat die Lebenserwartung deutlich zugenommen; Allerdings hat also die Prävalenz der altersbedingten Erkrankungen. Erkrankungen des zentralen Nervensystems (ZNS) wie Amyotrophe Lateralsklerose (ALS), Schizophrenie, Alzheimer-Krankheit und Parkinson-Krankheit, unter anderem sind da keine Ausnahme und haben voraussichtlich in den nächsten zehn Jahren1, zu erhöhen 2 , 3. die Fehlfunktion von Ionotropic Glutamat-Rezeptoren bekannt als N-Methyl-D-Aspartat-Rezeptoren (NMDAR), Alzheimer-Krankheit, Schizophrenie, Schädel-Hirn-Verletzungen, Schlaganfall, Diabetes und Glaukom u.a. die Optionsscheine verknüpft wurde die Ihre Biologie für die Entwicklung von wirksamen, krankheitsmodifizierende Therapien4,5,6,7studieren müssen.
NMDARs bestehen aus vier Monomeren oder Untereinheiten4,8,9. Das Strukturbild der NMDAR zeigt Entwicklungs- und regionale Streuung innerhalb der Gehirn-7,-10. NMDARs engagieren sich in Synaptische Plastizität, Kognition und die Generation der Rhythmen für Atmung und Fortbewegung11,12,13. Als Voltage-gated-Kanal, es ist weitgehend nicht leitend bei ruhenden Membranpotential (-70 mV) und wird durch Magnesium zur Verhinderung weiterer Permeation von Ionen blockiert. Der Kanal wird durch die Bindung von zwei Liganden, Glutamat und Glycin/D-Serin aktiviert, und eine gleichzeitige Depolarisation an der synaptischen Membran durch AMPA-Rezeptoren, eine andere Unterklasse von Ionotropic Glutamat-Rezeptoren vermittelt. Die Depolarisation entfernt die Magnesium-Blockade der NMDAR, so dass des Zustroms von Kationen, insbesondere Kalzium14,15,16. Obwohl die Aktivierung von NMDAR essentiell für das Überleben der Zelle ist, kann übermäßige Aktivierung zu Tod17,18,19 bis Excitotoxizität Zelle führen. Dies macht neben der komplexen Natur des Rezeptors, es schwierig durchzuführenden Studien notwendig für die Entwicklung wirksamer Therapien.
Verschiedene Methoden wurden entwickelt, um die NMDAR zu studieren. Allerdings hat jeweils begleitenden Einschränkungen. Beispielsweise ist eine weit verbreitete Technik eine Fluoreszenz basierende Assays, die NMDAR-vermittelte Änderungen in intrazellulären Calcium in eine stabile Zelllinie unter der Kontrolle eines Tetracyclin-induzierbaren Promotors (Tet-On)20misst. Jedoch in diesem System macht supramaximalen Konzentrationen von Liganden, die benötigt werden und die Anforderung, dass NMDAR Inhibitoren vorhanden während der Messung sind es nahezu unmöglich, Aktivität der kompetitiver Antagonist zu erkennen. In anderen ähnlichen Systemen verursacht der Ausdruck des funktionalen Rezeptors Toxizität, erfordern-Antagonisten wie Ketamin21,22 , die Zellkulturen zu bewahren. Diese Kanalblocker das Herzstück des Rezeptors sitzen und sind schwierig zu waschen, vor allem in einem Teller-basierte Format, damit sie funktionellen Studien des Rezeptors beeinträchtigt. Zu guter Letzt in elektrophysiologische Messungen wie Patch spannen, gibt es begrenzter Durchsatz, und groß angelegte Studien sind sehr teuer23. Dennoch sind die oben genannten Systeme unempfindlich gegen Glycin Stimulation; Daher wird das Studium Glycin-abhängige Aktivität von der NMDAR eine Herausforderung.
Hier beschreiben wir einen neuartigen Ansatz für das Studium NMDAR, die die besprochenen Grenzen überwindet. Unsere Technik nutzt Baculovirus Expressionssystems Rezeptor auf Funktionsebenen mit optimalem Verhältnis der Untereinheiten in weniger als 16 Stunden zum Ausdruck zu bringen. Darüber hinaus ermöglicht die Verwendung von Baculovirus für einen einfachen und kombinatorische Ansatz, wonach eine breite Charakterisierung der verschiedenen rekombinanten NMDAR Untertypen. Im Gegensatz zu anderen Assays erfordert dieses Protokoll nicht-Kanal-Blocker wegen des Einsatzes von schwachen Antagonisten. Stärkste Vorteil der Methode ist, dass nach dem Auswaschen des schwachen Antagonisten, der Rezeptor reagiert empfindlich auf Modulation der einzelnen Glycin - und Glutamat-Bindungsstellen neben duale Modulation von Glycin/D-Serin und Glutamat Ligand-Bindung Seiten. Der Assay rekapituliert bekannten Pharmakologie des NMDAR-Rezeptors und die Auswirkungen seiner bekannten positiven und negativen Modulatoren. Zu guter Letzt Generation von in-vitro- zelluläre Assays überwindet die zelluläre Toxizität verursacht durch übermäßigen Kalzium Zustrom und ermöglicht funktionelle Studien des Rezeptors in einer Hochdurchsatz-Mode, die Entdeckungen der NMDAR Modulatoren beschleunigen können bei Krankheitszuständen.

<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

N-Methyl-D-Aspartat-(NMDA)-Rezeptoren (NMDAR) gelten als Ionotropic Glutamat-Rezeptoren und haben entscheidende Rollen in lernen und Gedächtnis. NMDAR Störung, ausgedrückt als entweder über - oder unter - activity verursacht durch Mutationen, veränderten Ausdruck, Menschenhandel oder Lokalisierung, kann zahlreiche Krankheiten, insbesondere in das zentrale Nervensystem beitragen. Daher unbedingt Verständnis der Rezeptor Biologie sowie zur Erleichterung der Entdeckung von Verbindungen und kleine Moleküle in laufenden Bemühungen zur Bekämpfung von neurologischer Erkrankungen. Aktuelle Ansätze zur Untersuchung des Rezeptors haben Beschränkungen, einschließlich niedriger Durchsatz, hohen Kosten und der Unfähigkeit, seine funktionellen Fähigkeiten durch die notwendige Anwesenheit von Kalziumkanalblocker NMDAR-vermittelten Excitotoxizität verhindern zu studieren. Darüber hinaus die bestehenden Assay-Systeme sind empfindlich auf die Stimulation durch Glutamat nur und Empfindlichkeit auf Anregung von Glycin, die andere Co Liganden der NMDAR fehlt. Hier präsentieren wir die erste Platte-basierte Assay mit hohem Durchsatz macht einen NMDA-Rezeptor mit Sensibilität für Co-Liganden, Glutamat sowohl D-Serin/Glycin zu studieren. Dieser Ansatz ermöglicht die Untersuchung der verschiedenen NMDAR Untereinheit Kompositionen und funktionelle Studien des Rezeptors in Glycin und/oder Glutamat-sensitiven Modi ermöglicht. Darüber hinaus erfordert die Methode nicht die Anwesenheit von Inhibitoren während der Messung. Die Auswirkungen der positive und negative allosterische Modulatoren können mit diesem Test erkannt werden und die bekannten Pharmakologie des NMDAR in unserem System repliziert wurden. Diese Technik überwindet die Grenzen der bestehenden Methoden und ist kostengünstig. Wir glauben, dass diese neuartige Technik die Entdeckung von Therapien für NMDAR-vermittelten Erkrankungen beschleunigen wird.

Vor der Prüfung der Auswirkungen von kleinen Molekülen, muss die optimalen Ausdruck Ebenen des NMDARs sowie die optimale Liganden-Konzentrationen festgestellt werden. Wie beschrieben, HEK293 Zellen bei 10.000 Zellen pro Bohrloch in einem 384-Well-Platte ausgesät wurden, in Anwesenheit von 5 µM CGP060667, dann ausgestrahlt mit unterschiedlichen Mengen von NR1 und NR2A Viren. Nach Inkubation über Nacht, Liganden-induzierte Kalzium Flussmittel gemessen wurde (Abbild...

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

Eine Hochdurchsatz-Kalzium-Flux-Assay, NMDA-Rezeptoren mit Sensibilität für Glycin/D-Serin und Glutamat zu studieren

Das Ziel dieses Protokolls ist es, das Studium der NMDA-Rezeptoren (NMDAR) in einem größeren Maßstab erleichtern und ermöglichen die Prüfung der modulierende Effekte von kleinen Molekülen und deren therapeutische Anwendungen.

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 Aufrufe.  Novartis Institutes for BioMedical Research. Das Ziel dieses Protokolls ist es, das Studium der NMDA-Rezeptoren (NMDAR) in einem größeren Maßstab erleichtern und ermöglichen die Prüfung der modulierende Effekte von kleinen Molekülen und deren therapeutische Anwendungen.

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

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

Die Ableitung der zeitliche Verlauf der Glutamate Ausverkauf mit Dekonvolutionsanalyse von Astrozytäre Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

Forschung

Neurowissenschaften

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-Photonen-Intrazelluläre Sodium Imaging Kombination mit UV-vermittelten Brenn Uncaging von Glutamat in CA1 Pyramidenzellen

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

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.

Ein "Dual-Addition"-Calciumfluoreszenz-Assay für das Hochdurchsatz-Screening von rekombinanten G-Protein-gekoppelten Rezeptoren

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

Biochemie

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).

Enzym-basierte Biosensoren Mit Tonic und Phasic Glutamate in der Alzheimer-Maus-Modellen zu messen

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

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.

Ein High-Content-Assay für die Überwachung der AMPA Rezeptor Menschenhandel

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.

Ein Antikörper-Fütterungsansatz zur Untersuchung des Schleimatrezeptorhandels in dissoziierten primären Hippocampuskulturen

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.

Einzelne Synapsenindikatoren für Glutamatfreisetzung und Aufnahme in akuten Gehirnscheiben von normalen und Huntington-Mäusen

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.

Quantifizierung von Spontan-Ca2+-Fluxes und ihren nachgelagerten Auswirkungen in primären Maus-Midbrain-Neuronen

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.

Ein-Kanal-Cell-Attached-Patch-Clamp-Aufnahme

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

Biologie

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

Echtzeit-Fluoreszenzmessung synaptischer Funktionen in Modellen der amyotrophen Lateralsklerose

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...