Diese Arbeit wurde durch das National Institute of Neurological Disorders and Stroke Interne Research Program (NS002986) unterstützt. AS schrieb das Manuskript und umgesetzt Dekonvolutionsanalyse. JSD entwickelte die erste Version des Dekonvolutionsanalyse und kommentiert den Text.

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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=Neuronal+activity+regulates+glutamate+transporter+dynamics+in+developing+astrocytes.">Neuronal activity regulates glutamate transporter dynamics in developing astrocytes.</a> Glia. 60, 175-188 (2012).</li><li>Hires, S. A., Zhu, Y., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+measurement+of+synaptic+glutamate+spillover+and+reuptake+by+linker+optimized+glutamate-sensitive+fluorescent+reporters.">Optical measurement of synaptic glutamate spillover and reuptake by linker optimized glutamate-sensitive fluorescent reporters.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 4411-4416 (2008).</li><li>Scimemi, A., Meabon, J., Woltjer, R. L., Sullivan, J. M., Diamond, J. S., Cook, D. 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Die Autoren erklären, keinen Interessenkonflikt.

Hier haben wir einen experimentellen Ansatz zur elektrophysiologischen Ableitungen von Astrozyten erhalten beschreiben, leiten ein analytisches Protokoll Glutamattransporter Ströme in Astrozyten isolieren und eine mathematische Methode, um den zeitlichen Verlauf der Glutamat-Clearance von Astrozyten-Transporter Ströme. Der Erfolg der Analyse beruht auf der Fähigkeit, hochwertige Patch-Clamp-Aufnahmen von Astrozyten zu erhalten und auf die Genauigkeit der Anpassungsalgorithmen verwendet, u...

Astrozyten sind eine der am häufigsten vorkommenden Zelltypen im Gehirn mit sternförmigen Morphologie und feine Membran Vorsprünge, die in der gesamten Neuropils erweitern und erreichen benachbarten synaptische Kontakte 1,2. Die Astrozyten &#39;Zellmembran ist dicht mit Glutamat-Transporter-Moleküle 3 verpackt. Unter physiologischen Bedingungen Glutamattransporter schnell binden Glutamat an der extrazellulären Seite der Membran und überträgt es auf Zytoplasma der Zelle. Auf diese Weise erhalten die Transporter günstig die basale Konzentration von Glutamat in den extrazellulären Raum 4. Glutamat-Transporter in feine Astrozytenfortsätze benachbarten Synapsen erregenden sind ideal positioniert, um Glutamat während der synaptischen Ereignisse veröffentlicht, sobald sie weg aus dem synaptischen Spalt diffundiert binden. Damit die Transporter auch begrenzen Glutamat Spillover auf peri-und extra-synaptischen Regionen und auf benachbarten Synapsen, die Verringerung der räumlichen Ausbreitung der erregenden Signals im Gehirn 5-7. Glutamate Transport ist ein Prozess elektrogenen stöchiometrisch auf die Bewegung von 3 gekoppelt Na + und H + 1 entlang ihrer elektrochemischen Gradienten und dem Zähler-Transport von 1 K + 8. (Thiocyanat)&gt; NO - 3 - (Nitrat) ≈ ClO 4 - (Perchlorat)&gt; I -&gt; Br -&gt; Cl -&gt; F -, nicht Glutamate Transport mit (aber nicht stöchiometrisch gekoppelt) ein anionisches Leitwert durchlässig SCN verbunden um CH 3 SO 3 - (Methansulfonat) und C 6 H 11 O 7 - (Gluconat) 9-11. Beide Ströme (stöchiometrischen und nicht-stöchiometrischen) durch Erhalten Whole-Cell-Patch-Clamp-Aufnahmen von Astrozyten, visuell unter Dodt Beleuchtungs-oder Infrarot-Differential-Interferenz-Kontrast (IR-DIC) in acut identifiziert aufgezeichnet werdene Gehirnscheiben 12. Das stöchiometrische Komponente des Stroms mit Glutamat Transport über die Membran verbunden sind, können durch Verwendung von CH 3 SO 3 isoliert werden - oder C 6 H 11 O 7 - basierten intrazellulären Lösungen und kann durch Flash-Uncaging Glutamat auf Astrozyten 13,14 hervorgerufen werden, oder durch die Aktivierung Glutamat-Freisetzung aus benachbarten Synapsen, entweder elektrisch 12 oder mit einer gezielten optogenetische Kontrolle. Der zeitliche Verlauf der stöchiometrischen Komponente des Transporters Strom durch die Lebensdauer des Glutamat-Konzentration in Astrozyten Profil Membranen (zB Glutamat Clearance) geformt, die Kinetik der Glutamat-Transporter, die passive Membran Eigenschaften von Astrozyten und während der synaptischen Reize, durch die Synchronität der Freisetzung von Glutamat in den aktivierten Synapsen 13. Hier beschreiben wir ausführlich: (1) eine experimentelle ca.oach die stöchiometrische Komponente von Glutamat-Transporter Ströme von whole-cell Patch-Clamp-Aufnahmen von Astrozyten mit akuten Maus Hippocampus als Beispiel experimentelle Vorbereitung isolieren, (2) ein analytischer Ansatz zur Ableitung des zeitlichen Verlaufs von Glutamat-Clearance von diesen Aufnahmen 13, 14. Diese Methoden können verwendet werden, um aufzuzeichnen und zu analysieren Glutamattransporter Ströme von Astrozyten in einer beliebigen Region des zentralen Nervensystems werden.

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			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments </td>
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			<td>CGP52432</td>
			<td>Tocris</td>
			<td>1246</td>
			<td></td>
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			<td>(R,S)-CPP</td>
			<td>Tocris</td>
			<td>173</td>
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			<td>DPCPX</td>
			<td>Tocris</td>
			<td>439</td>
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			<td>LY341495 disodium salt</td>
			<td>Tocris</td>
			<td>4062</td>
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			<td>MSOP</td>
			<td>Tocris</td>
			<td>803</td>
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			<td>Tocris</td>
			<td>1044</td>
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			<td>D,L-TBOA</td>
			<td>Tocis</td>
			<td>1223</td>
			<td></td>
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			<td>Picrotoxin</td>
			<td>Sigma</td>
			<td>P1675</td>
			<td></td>
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			<td>MNI-L-glutamate</td>
			<td>Tocris</td>
			<td>1490</td>
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			<td>Alexa 594</td>
			<td>Life Technologies</td>
			<td>A10438</td>
			<td>Optional</td>
		 </tr>
		 <tr>
			<td>Matrix electrodes</td>
			<td>Frederick Haer Company</td>
			<td>MX21AES(JD3)</td>
			<td></td>
		 </tr>
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			<td>Borosilicate glass capillaries</td>
			<td>World Precision Instruments</td>
			<td>PG10165-4</td>
			<td></td>
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		 <tr>
			<td>Dual-stage glass micro-pipette puller</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Loctite 404 instant adhesive</td>
			<td>Ted Pella</td>
			<td>46551</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Xe lamp</td>
			<td>Rapp OptoElectronic</td>
			<td>FlashMic</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Igor Pro 6</td>
			<td>Wavemetrics</td>
			<td></td>
			<td></td>
		 </tr>
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 </table>

deriving the time course of glutamate clearance with a deconvolution analysis of astrocytic transporter currents

Die höchste Dichte von Glutamat-Transporter im Gehirn in Astrozyten gefunden. Glutamattransporter Paar die Bewegung von Glutamat durch die Membran mit dem Co-Transport von 3 Na + und H + 1 und der Gegenelektrode Transport von 1 K +. Das stöchiometrische Strom, der durch den Transport Prozess erzeugt wird, kann mit whole-cell Patch-Clamp-Aufnahmen von Astrozyten überwacht werden. Der zeitliche Verlauf der aufgezeichneten Strom wird durch den zeitlichen Verlauf der Glutamatkonzentration Profil auf die Astrozyten ausgesetzt sind, die Kinetik der Glutamat-Transporter, und die passive elektrotonischen Eigenschaften von Astrozyten Membranen geformt. Hier beschreiben wir die experimentellen und analytischen Methoden, die verwendet werden, um Glutamattransporter Ströme in Astrozyten erfassen und isolieren den zeitlichen Verlauf der Glutamat-Clearance von allen anderen Faktoren, die die Wellenform der Astrozyten-Transporter Ströme formen kann. Die hier beschriebenen Methoden können verwendet werden, um die Lebensdauer o abzuschätzenf Flash-uncaged und Glutamat in Astrozyten Membranen in jeder Region des zentralen Nervensystems bei Gesundheit und Krankheit synaptisch-released.

Der Erfolg der analytischen hier beschriebene Ansatz hängt entscheidend von hoher Qualität zu erhalten elektrophysiologischen Ableitungen von Transporter Ströme von Astrozyten in jeder Region des zentralen Nervensystems. In akuten Maus Hippocampus kann Astrozyten leicht unter Dodt Beleuchtung oder IR-DIC werden wegen ihrer kleinen Zellkörper (Ø = 10 um) und prominente Kern (Abbildung 1) identifiziert. Ihre markante sternförmige Morphologie mit Epifluoreszenz, konfokale oder Zwei-Photonen-Laser-Sca...

Watch this Scientific Journal Video about Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents at JoVE.com

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

Wir beschreiben eine analytische Methode, um die Lebensdauer von Glutamat an astrozytären Membranen schätzen von elektrophysiologischen Ableitungen von Glutamat-Transporter Ströme in Astrozyten.

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 ...

deriving-time-course-glutamate-clearance-with-deconvolution-analysis

Research

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Neuroscience

10.2K Aufrufe.  National Institutes of Health. Wir beschreiben eine analytische Methode, um die Lebensdauer von Glutamat an astrozytären Membranen schätzen von elektrophysiologischen Ableitungen von Glutamat-Transporter Ströme in Astrozyten.

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

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

Isolation von sensorischen Neuronen von<em&gt; Aplysia californica</em&gt; Für Patch-Clamp-Recordings von Glutamaterge Currents

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Forschung

Neurowissenschaften

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

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Schnelle Micro-Iontophorese von Glutamat und GABA: Ein nützliches Tool, um Synaptic Integration Untersuchen

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Induktion von Plastizität Astrozytäre Rezeptoren durch Manipulation der neuronalen Feuerraten

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

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 ...

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.

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer&#039;s Mouse Models

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 Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

Ein Protokoll für die Verwaltung von Real-Time-fMRT Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

Live-Bilder von GLUT4 Protein Menschenhandel Maus primäre hypothalamische Neurone mit Dekonvolution Mikroskopie

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

Fokale Macropatch Aufnahmen der synaptischen Ströme aus der Drosophila larvalen neuromuskulären Synapse

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

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

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR malfunction, expressed as either over- or under-activity caused by mutations, altered expression, trafficking, or localization, can contribute to numerous diseases, especially in the central nervous system. Therefore, understanding the receptor&#39;s biology as well as facilitating the discovery of compounds and small molecules is crucial in ongoing efforts to combat neurological diseases. Current approaches to studying the receptor have limitations including low throughput, high cost, and the inability to study its functional abilities due to the necessary presence of channel blockers to prevent NMDAR-mediated excitotoxicity. Additionally, the existing assay systems are sensitive to stimulation by glutamate only and lack sensitivity to stimulation by glycine, the other co-ligand of the NMDAR. Here, we present the first plate-based assay with high-throughput power to study an NMDA receptor with sensitivity to both co-ligands, glutamate and D-serine/glycine. This approach allows the study of different NMDAR subunit compositions and allows functional studies of the receptor in glycine- and/or glutamate-sensitive modes. Additionally, the method does not require the presence of inhibitors during measurements. The effects of positive and negative allosteric modulators can be detected with this assay and the known pharmacology of NMDAR has been replicated in our system. This technique overcomes the limitations of existing methods and is cost-effective. We believe that this novel technique will accelerate the discovery of therapies for NMDAR-mediated pathologies.

The goal of this protocol is to facilitate the study of NMDA-receptors (NMDAR) at a larger scale and allow the examination of modulatory effects of small molecules and their therapeutic applications.

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

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

Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing neuronal dysfunction which eventually leads to cognitive decline. Hyperphosphorylated tau aggregates in the neuron are believed to cause most of the pathology associated with AD. These aggregates are assumed to be released into the extracellular compartment and taken up by adjacent healthy neurons where they induce further tau aggregation. This &#34;prion-like&#34; spreading can be interrupted by antibodies capable of binding and &#34;neutralizing&#34; extracellular tau aggregates as shown in preclinical mouse models of AD. One of the proposed mechanisms by which therapeutic antibodies reduce pathology is antibody-mediated uptake and clearance of pathological aggregated forms of tau by microglia. Here, we describe a quantitative cell-based assay to assess tau uptake by microglia. This assay uses the mouse microglial cell line BV-2, allows for high specificity, low variability and medium throughput. Data generated with this assay can contribute to a better characterization of anti-tau antibody effector functions.

Here we describe a cell-based assay to quantitatively assess tau uptake by microglia with the aim of creating an investigational tool to better characterize the mechanisms of action of anti-tau antibodies.

Zellbasierte Assays, Antikörper-vermittelten Tau Clearance von Mikroglia zu studieren

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing ...