Name: levator auris longus preparation for examination of mammalian neuromuscular transmission under voltage clamp conditions
Uploaded: 2018-05-05T14:00:00.000+00:00
Duration: 10 min 45 s
Description: Watch this Scientific Journal Video about Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions at JoVE.com

Wir danken Dr. Mark M. Rich und Daniel Miranda für redaktionelle Kommentare, Ahmad Khedraki für die Unterstützung dieser Technik und Wright State University für die finanzielle Unterstützung (Gründerfonds, A.A.V.) zu etablieren.

<ol>
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R., 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=Progressive+Cl-+channel+defects+reveal+disrupted+skeletal+muscle+maturation+in+R6/2+Huntington's+mice.">Progressive Cl- channel defects reveal disrupted skeletal muscle maturation in R6/2 Huntington's mice.</a> J Gen Physiol. 149 (1), 55-74 (2017).</li><li>Waters, C. W., Varuzhanyan, G., Talmadge, R. J., Voss, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Huntington+disease+skeletal+muscle+is+hyperexcitable+owing+to+chloride+and+potassium+channel+dysfunction.">Huntington disease skeletal muscle is hyperexcitable owing to chloride and potassium channel dysfunction.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (22), 9160-9165 (2013).</li><li>Khedraki, A., 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=Depressed+Synaptic+Transmission+and+Reduced+Vesicle+Release+Sites+in+Huntington's+Disease+Neuromuscular+Junctions.">Depressed Synaptic Transmission and Reduced Vesicle Release Sites in Huntington's Disease Neuromuscular Junctions.</a> Journal of Neuroscience. 37 (34), 8077-8091 (2017).</li><li>Greene, E. 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Hier beschrieben ist die Vorbereitung und Verwendung der Maus LAL Muskel für die Messung der neuromuskulären Übertragung unter Strom oder Spannung Klemme Bedingungen. Es gibt mehrere wichtige Punkte zu beachten für Sezieren, die LAL. Reinigung überschüssiges Bindegewebe an der Muskel-Aids in Elektrode Impalement, als die Elektroden kann das Bindegewebe Haken, wenn sie für Impalement Positionierung. Jedoch nur entfernen, Bindegewebe, das weggenommen werden kann ganz einfach, die Wahrscheinlichkeit, dass des Muskels...

Studium der synaptischen Übertragung an der neuromuskulären Synapse bietet Einblicke in die dynamische Beziehung zwischen dem Nerven- und Skelett-Muskelsystem und ist ein hervorragendes Modell für die Prüfung von synaptischen Physiologie. Die Levator Auris Longus (LAL) ist eine dünne Muskel, so dass für die neuromuskulären Verbindungen leicht visualisiert werden. Frühere Berichten haben die Bequemlichkeit der Nutzung der LAL, synaptische Drogen und Giftstoffe zu untersuchen und charakterisiert die skelettartigen Muskel Faser Art Eigenschaften des LAL1,2beschrieben. Zahlreiche Studien haben die LAL verwendet, um neuromuskuläre Physiologie3,4,5,6,7,8zu untersuchen. Für Elektrophysiologie die Fähigkeit, LAL neuromuskuläre Kreuzungen leicht beobachten ermöglicht die genaue Platzierung von Mikroelektroden an der motorischen Endplatte und Klemme Platzprobleme in der synaptischen Übertragung Aufnahme reduziert. Strom-Clamp Aufnahmen der Muskel Membran Eigenschaften, wie die Membran Zeitkonstante (τm) und Eingangswiderstand (Rin) werden leicht erreicht. Darüber hinaus können diese Eigenschaften von den gleichen Muskelfasern zur Aufzeichnung der neuromuskulären Übertragung, so dass ein direkter Vergleich der synaptischen Funktion an den Muskel-Membran-Eigenschaften verwendet gemessen werden. Analyse dieser Daten liefern wichtige Erkenntnisse über die physikalischen Mechanismen der vielen neuromuskulären Erkrankungen und Zustände der veränderte Aktivität.
Ein wichtiger Aspekt der hier beschriebenen Technik ist die Verwendung der Spannung-Klemme für synaptische Aufnahmen, die nicht unterliegen die Nichtlinearitäten im Strom-Clamp begegnet und sind unabhängig von der Muskel-Membran-Eigenschaften. Vorteile der Verwendung von Voltage-Clamp im Gegensatz zu Strom-Clamp, um neuromuskulären Übertragung zu untersuchen wurden von bahnbrechenden Bemühungen in den 1950er Jahren9eingerichtet. Unter Strom-Clamp sind EPPs, die 10-15 mV Amplitude überschreiten keine lineare Produkt der Nahostfriedensprozess Amplitude9. Zum Beispiel, wenn die durchschnittliche Nahostfriedensprozess 1 mV, einem EVP von 5 mV kann davon ausgegangen werden, das Produkt von 5 mEPPs (QC 5); in der Erwägung, eine EVP von 40 mV wird das Produkt von mehr als 40 mEPPs sein. Diese nicht-Linearität bei größeren EPPs tritt auf, weil die treibende Kraft für die EVP, was ist der Unterschied zwischen der Membran und Gleichgewicht-Potenzial für den Acetylcholin-Rezeptor (~-10 mV), erheblich sinkt, während große EPPs. Dieses Problem wird in Voltage-Clamp-Experimenten vermieden, weil der Muskel Membran Potenzial während Voltage-Clamp-Experimenten nicht ändert. Ein Nachteil ist, dass Spannung-Clamp Experimente sind technisch schwieriger als Strom-Clamp Aufnahme. Mit diesem im Verstand entwickelt McLachlan und Martin eine einfache mathematische Korrektur, die Nichtlinearitäten im Strom-Clamp Aufnahmen von EPPs10ausmacht. Die Korrekturen funktionieren gut11,12,13, aber wichtiger ist, davon ausgehen, dass die Muskel-Membran-Eigenschaften wurden nicht gestört.
Die Muskel-Membran-Eigenschaften sind besonders wichtig zu prüfen, ob Studium der Bedingungen oder Krankheitszuständen, die den Muskel zu stören. Skelettartiger Muskel aus dem R6/2 transgenen Modell der Huntington-Krankheit ist beispielsweise übererregbar durch eine schrittweise Verringerung der ruhenden Chlorid und Kalium Ströme14,15. Infolgedessen sind mEPPs und EPPs in der Skelettmuskulatur R6/2 verstärkt. Natürlich können zusätzliche Faktoren mEPPs und EPPs ändern. Arbeiten Sie mit einem anderen Modell von Chorea Huntington Mäusen (R6/1) Änderungen in EPPs, die im Zusammenhang mit SNARE-Proteine8schien gefunden. Um die Mechanismen, die veränderten neuromuskulären Übertragung zu beurteilen, wäre es vorteilhaft, die Auswirkungen der veränderten Muskel Membran Eigenschaften mithilfe einer Spannung-Klemme zu beseitigen. In einer aktuellen Studie wurde die R6/2 neuromuskuläre Übertragung Bedingungen sowohl Strom und Spannung-Klemme mit der beschriebenen Technik hierin untersucht. Die Gesamtheit der motorischen endplatten wurden Spannung eingespannten mit weniger als 1 % Fehler, indem man zwei Mikroelektroden in die Konstante Länge der Endplatte16. Es zeigte sich, dass Spannung-Klemme und Strom-Clamp Aufzeichnungen ergab kontrastierende Messungen der neuromuskulären Übertragung in R6/2 Muskel korrigiert. Dies macht deutlich, dass es schwierig sein kann, EPPs Nichtlinearitäten zu korrigieren, wenn der Muskel-Membran-Eigenschaften geändert wurden und zeigt die Vorteile der Voltage-Clamp-Datensätze, die unabhängig von der Muskel-Membran-Eigenschaften sind zu erhalten. Das Protokoll enthaltenen eignet sich für die Prüfung von Bedingungen oder Krankheitszustände, die synaptische Übertragung und der postsynaptischen Membran Eigenschaften beeinflussen.

<table><tbody><tr><td>Olympus Compound Microscope</td><td>Olympus</td><td>BX51WI</td><td></td></tr><tr><td>10x Objective</td><td>Olympus</td><td>UMPLFLN10XW</td><td></td></tr><tr><td>40x Objective</td><td>Olympus</td><td>LUMPLFLN40XW</td><td></td></tr><tr><td>Borosilicate Glass</td><td>Sutter Instruments</td><td>BF150-86-7.5</td><td></td></tr><tr><td>CCD Camera</td><td>Santa Barbara Instruments Group</td><td>ST-7XMEI</td><td></td></tr><tr><td>Mater-9 Pulse Generator</td><td>AMPI</td><td></td><td></td></tr><tr><td>Iso-flex Stimulus Isolator</td><td>AMPI</td><td></td><td></td></tr><tr><td>pCLAMP 10 Data Acquisition and Analysis Software</td><td>Molecular Devices</td><td>1-2500-0180</td><td></td></tr><tr><td>Concentric Bipolar Electrode</td><td>FHC</td><td>CBDSH75</td><td></td></tr><tr><td>Ball-joint Manipulator</td><td>Narishige&amp;nbsp;</td><td></td><td></td></tr><tr><td>Non-metalic Syringes 34 Gauge</td><td>World Precision Instruments</td><td>MF34G-5</td><td></td></tr><tr><td>Nikon Stereomicroscope</td><td>Nikon</td><td>SMZ800N</td><td></td></tr><tr><td>No. 5 Forceps</td><td>Fine Science Tools</td><td></td><td></td></tr><tr><td>Spring Scissors</td><td>Fine Science Tools</td><td>15006-09</td><td></td></tr><tr><td>No. 2 Forceps</td><td>Roboz</td><td>RS-5Q41</td><td></td></tr><tr><td>Microdissecting Scissors</td><td>Roboz</td><td>RS-5912SC</td><td></td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning</td><td>2404019862</td><td></td></tr><tr><td>Hair Removal Cream</td><td>Nair</td><td></td><td></td></tr><tr><td>Grass SD9 Stimulator</td><td>Grass Medical</td><td></td><td></td></tr><tr><td>Model P-1000 Micropipette Puller</td><td>Sutter Instruments</td><td>P-1000</td><td></td></tr><tr><td>Axon Digidata 1550 Low-noise Data Acuisition System</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Low Pass Bessell Filter</td><td>Warner Instrument Corp.</td><td>LPF-8</td><td></td></tr><tr><td>Left-handed Micromanipulator</td><td>Siskiyou Corp.</td><td>MX1641/45DL</td><td></td></tr><tr><td>Right-handed Micromanipulator</td><td>Siskiyou Corp.</td><td>MX1641/45DR</td><td></td></tr><tr><td>Single Motion Controler</td><td>Siskiyou Corp.</td><td>MC100e</td><td></td></tr><tr><td>Crossed Roller Micromanipulator</td><td>Siskiyou Corp.</td><td>MX1641R</td><td>This was added to the Z-axis of the Left and Right-handed micromanipulators to allow the z axis to be motorized. This custom set-up is cheaper and less bulky than buying a 4-axis motorized micromanipulator. It also allows us to control both micromanipulators with one controller</td></tr><tr><td>All chemicals were orded from Fisher except,</td><td></td><td></td><td></td></tr><tr><td>BTS</td><td>Toronto Research Chemicals</td><td>B315190</td><td></td></tr><tr><td>CTX</td><td>Alomone Labs</td><td>C-270</td><td></td></tr><tr><td>4-Di-2-Asp</td><td>Molecular Probes</td><td></td><td>Molecular probes is no longer a company. Now ordered through Fisher</td></tr></tbody></table>

levator auris longus preparation for examination of mammalian neuromuscular transmission under voltage clamp conditions

Dieses Protokoll beschreibt eine Technik zur Aufzeichnung synaptische Übertragung aus der neuromuskulären Synapse unter Strom-Klemme und Spannung-Klemme. Eine ex-Vivo -Vorbereitung des Levator Auris Longus (LAL) wird verwendet, weil es eine dünne Muskel ist, der einfache Visualisierung von der neuromuskulären Synapse Mikroelektrode Impalement an der motorischen Endplatte vorsieht. Diese Methode ermöglicht die Aufzeichnung von spontanen Miniatur Endplatte Potentiale und Ströme (mEPPs und mEPCs), Endplatte Nerv-evozierten Potentiale und Ströme (EPPs und EPCs), sowie die Eigenschaften der Membran der motorischen Endplatte. Ergebnisse, die von dieser Methode auch die Quanten Inhalte (QC), Anzahl der Vesikel freisetzungsstandorten (n), Wahrscheinlichkeit des vesikels Release (pRel), synaptische Erleichterung und Depression sowie Muskel-Membran-Zeitkonstante (τ m) und Eingangswiderstand. Anwendung dieser Technik zu Mausmodellen menschlicher Erkrankungen kann wichtige Pathologien bei Krankheitszuständen markieren und identifizieren neue Behandlungsstrategien. Klemmend voll Spannung-eine einzige Synapse, stellt diese Methode eine der ausführlichsten Analysen der synaptischen Übertragung derzeit verfügbar.

Abbildung 8 zeigt ein Beispiel für die Stromimpulse (Abb. 8A) und die Spannung Antworten (Abbildung 8 b) aus einem LAL Faser unter Strom-Klammer aus einem 12 Wochen alten Wildtyp R6/2-Maus. Das Vorhandensein von mEPPs zeigt, dass diese Aufzeichnungen aus der motorischen Endplatte aufgenommen wurden. Die Aufzeichnungen wurden in normaler physiologischer Kochsalzlösung erhalten. Dieser Strom-Clamp-Da...

Watch this Scientific Journal Video about Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions at JoVE.com

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

Levator Auris Longus Prüfungsvorbereitung der Säugetier-neuromuskulären Übertragung unter Spannung Klemme Bedingungen

Das Protokoll in diesem Dokument beschriebenen verwendet die Maus Levator Auris Longus (LAL) Muskeln, um spontan aufnehmen und Nerv-evozierten postsynaptischen Potenziale (Strom-Klemme) und Ströme (Voltage-Clamp) an der neuromuskulären Synapse. Diese Technik können wichtige Einblicke in die Mechanismen der synaptischen Übertragung unter normalen und Krankheit darstellen.

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...

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Neuroscience

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

9.3K Aufrufe.  Wright State University. Das Protokoll in diesem Dokument beschriebenen verwendet die Maus Levator Auris Longus (LAL) Muskeln, um spontan aufnehmen und Nerv-evozierten postsynaptischen Potenziale (Strom-Klemme) und Ströme (Voltage-Clamp) an der neuromuskulären Synapse. Diese Technik können wichtige Einblicke in die Mechanismen der synaptischen Übertragung unter normalen und Krankheit darstellen.

Serie: Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats

Patch clamp studies from dorsal root ganglia (DRGs) neurons have increased our understanding of the peripheral nervous system. Currently, the majority of recordings are conducted on dissociated DRG neurons, which is a standard preparation for most laboratories. Neuronal properties, however, can be altered by axonal injury resulting from enzyme digestion used in acquiring dissociated neurons. Further, dissociated neuron preparations cannot fully represent the microenvironment of the DRG since loss of contact with satellite glial cells that surround the primary sensory neurons is an unavoidable consequence of this method. To overcome the limitations in using conventional dissociated DRG neurons for patch clamp recordings, in this report we describe a method to prepare intact DRGs and conduct patch clamp recordings on individual primary sensory neurons ex vivo. This approach permits the fast and straightforward preparation of intact DRGs, mimicking in vivo conditions by keeping DRG neurons associated with their surrounding satellite glial cells and basement membrane. Furthermore, the method avoids axonal injury from manipulation and enzyme digestion such as when dissociating DRGs. This ex vivo preparation can additionally be used to study the interaction between primary sensory neurons and satellite glial cells.

This manuscript describes how to prepare intact dorsal root ganglia for patch clamp recordings. This preparation maintains the microenvironment for neurons and satellite glial cells, thus avoiding the phenotypic and functional changes seen using dissociated DRG neurons.

Patch-Clamp-Aufnahmen auf Intact Dorsalwurzelganglien von erwachsenen Ratten

Patch clamp studies from dorsal root ganglia (DRGs) neurons have increased our understanding of the peripheral nervous system. Currently, the majority of ...

Forschung

Biochemie

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP&#8217;s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP&#8217;s and minature excitatory junction potentials (mEJP&#8217;s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP&#8217;s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

Elektrophysiologischen Methoden für die Aufzeichnung Synaptic Potentials aus dem NMJ von Drosophila-Larven

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

Biologie

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

Dissecting und Aufnahme von C. elegans neuromuskulären Synapse

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

Elektrophysiologische Aufzeichnung in der Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

Implementieren Dynamische Clamp mit Synaptic und künstliche Leitfähigkeiten in Maus retinalen Ganglienzellen

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

Neurowissenschaften

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons are involved in proprioceptor signaling and also report on metabolic state and injury related events. We describe an adult mouse in vitro muscle-nerve preparation for studies on stretch-activated muscle afferents.

Ein<em&gt; In-vitro-</em&gt; Adult Maus Muskel-Nerven Vorbereitung für das Studium der Brenneigenschaften des Muskel-Afferenzen

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional ...

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and latency detect chronic axon loss and demyelination, respectively. Axonal excitability techniques &#34;by threshold tracking&#34; expand upon these measures by providing information regarding the activity of ion channels, pumps and exchangers that relate to acute function and may precede degenerative events. As such, the use of axonal excitability in animal models of neurological disorders may provide a useful in vivo measure to assess novel therapeutic interventions. Here we describe an experimental setup for multiple measures of motor axonal excitability techniques in the rat ulnar nerve.
The animals are anesthetized with isoflurane and carefully monitored to ensure constant and adequate depth of anesthesia. Body temperature, respiration rate, heart rate and saturation of oxygen in the blood are continuously monitored. Axonal excitability studies are performed using percutaneous stimulation of the ulnar nerve and recording from the hypothenar muscles of the forelimb paw. With correct electrode placement, a clear compound muscle action potential that increases in amplitude with increasing stimulus intensity is recorded. An automated program is then utilized to deliver a series of electrical pulses which generate 5 specific excitability measures in the following sequence: stimulus response behavior, strength duration time constant, threshold electrotonus, current-threshold relationship and the recovery cycle.
Data presented here indicate that these measures are repeatable and show similarity between left and right ulnar nerves when assessed on the same day. A limitation of these techniques in this setting is the effect of dose and time under anesthesia. Careful monitoring and recording of these variables should be undertaken for consideration at the time of analysis.

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Axonal excitability techniques provide a powerful tool to examine pathophysiology and biophysical changes that precede irreversible degenerative events. This manuscript demonstrates the use of these techniques on the ulnar nerve of anesthetized rats.

In Vivo Elektrophysiologische Messungen der Ratte ulnaris mit axonalen Erregbarkeit testen

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

Vorbereitungen und Protokolle für die ganze Zelle Patch Clamp Aufnahme von Xenopus Laevis Tectal Neuronen

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

Registration of Calcium Transients in Mouse Neuromuscular Junction with High Temporal Resolution using Confocal Microscopy

Estimation of the presynaptic calcium level is a key task in studying synaptic transmission since calcium entry into the presynaptic cell triggers a cascade of events leading to neurotransmitter release. Moreover, changes in presynaptic calcium levels mediate the activity of many intracellular proteins and play an important role in synaptic plasticity. Studying calcium signaling is also important for finding ways to treat neurodegenerative diseases. The neuromuscular junction is a suitable model for studying synaptic plasticity, as it has only one type of neurotransmitter. This article describes the method for loading a calcium-sensitive dye through the cut nerve bundle into the mice's motor nerve endings. This method allows the estimation of all parameters related to intracellular calcium changes, such as basal calcium level and calcium transient. Since the influx of calcium from the cell exterior into the nerve terminals and its binding/unbinding to the calcium-sensitive dye occur within the range of a few milliseconds, a speedy imaging system is required to record these events. Indeed, high-speed cameras are commonly used for the registration of fast calcium changes, but they have low image resolution parameters. The protocol presented here for recording calcium transient allows extremely good spatial-temporal resolution provided by confocal microscopy.

The protocol describes the method of loading a fluorescent calcium dye through the cut nerve into mouse motor nerve terminals. In addition, a unique method for recording fast calcium transients in the peripheral nerve endings using confocal microscopy is presented.

Registrierung von Kalziumtransienten in neuromuskulären Verbindungen von Mäusen mit hoher zeitlicher Auflösung mittels konfokaler Mikroskopie

Estimation of the presynaptic calcium level is a key task in studying synaptic transmission since calcium entry into the presynaptic cell triggers a ...

<meta charset="utf8"></head><body>Elektroporation und Nervenverletzung im Levator Auris longus-Muskel der Maus

Combined In Vivo Electroporation and Short&#45;Term Reinnervation of the Cranial Levator Auris Longus Skeletal Muscle

The neuromuscular junction (NMJ) is the peripheral synapse controlling the contraction of skeletal muscle fibers to allow the coordinated movement of many organisms. At the NMJ, a presynaptic motor axon terminal contacts a muscle postsynaptic domain and is covered by terminal Schwann cells. The integrity and function of the NMJ is compromised under several conditions, including aging, neuromuscular diseases, and traumatic injuries. To analyze the potential contribution of muscle-derived proteins to NMJ maintenance and regeneration, an in vivo gene transfer strategy has been combined with the denervation of the cranial levator auris longus (LAL) muscle after mechanical nerve injury. Previous findings showed that the forced expression of control fluorescent proteins does not alter NMJ organization or neurotransmission. This procedure aims to describe a detailed method of in vivo electroporation of the LAL muscle followed by transection or crushing of the specific branch of the facial nerve innervating cranial muscles, leading to NMJ denervation and reinnervation, respectively. The combination of these experimental strategies in the convenient LAL muscle constitutes an efficient method to study the potential contribution of muscle protein overexpression or silencing in the context of short-term NMJ reinnervation.

<meta charset="utf8"></head><body>Autor im Rampenlicht: Regenerative Rollen von Muskelproteinen an der neuromuskulären Verbindung nach Nervenverletzung

Here, we present a protocol that combines in vivo electroporation and denervation of the cranial levator auris longus (LAL) muscle. This procedure enables the study of the potential role of muscle-derived proteins in the regeneration of the neuromuscular synapse.

Kombinierte In vivo Elektroporation und kurzfristige Reinnervation des kranialen Levator Auris Longus Skelettmuskel

The neuromuscular junction (NMJ) is the peripheral synapse controlling the contraction of skeletal muscle fibers to allow the coordinated movement of many ...