Diese Arbeit wurde durch Zuschüsse aus dem NIH (F32 NS059120 zu RLM und R01NS25217 und P30NS048154 zu ABR) unterstützt.

Die Autoren erklären, keine konkurrierenden finanziellen Interessen.

Die hier beschriebenen Methoden ermöglichen die elektrische und morphologische Charakterisierung von sensorischen und motorischen Neuronen von Zebrabärbling Embryonen nach minimaler Zerlegung des Rückenmarks. Neurone bleibt für mindestens 1 h gesund, die Frist auf diesen Aufnahmen auferlegt. Neurone wurden unter Verwendung der Standard Gesamtzellkonfiguration aufgezeichnet, sowie von nukleierten Patches; Das letztere Verfahren minimiert Raum-clamp - Probleme , die eine detaillierte Untersuchung der biophysikalischen...

<html> George Streisinger Pionier bei der Verwendung von Danio rerio, das gemeinhin als Zebrabärbling bekannt, als Modellsystem für die genetische Analyse von Wirbel Entwicklung 10. Das Modell bietet mehrere Vorteile, einschließlich: (1) relativ einfach und kostengünstig Tierhaltung; (2) externe Befruchtung, die einen leichten Zugang zu Embryonen von den frühesten Entwicklungsstadien; und (3) ein transparentes Embryo ermöglicht direkte und wiederholte Beobachtungen von Zellen, Geweben und Organen, wie sie bilden. In den folgenden Jahrzehnten stiegen einige Fortschritte weiter die Leistung des Zebrabärbling-Modells. Insbesondere Vorwärts genetischen Screens und Sequenzierung des gesamten Genoms Bemühungen kritischen Schlüsselrollen bei der Identifizierung von Mutationen und Gene gespielt zu vielen Entwicklungsprozesse 11, 12, 13, 14,„&gt; 15, 16. Gateway - Klonierungsverfahren die Routineanwendung von transgenen erlaubt haben Ansätze 17, 18. Die jüngsten Fortschritte in der Genombearbeitung, beispielhaft dargestellt durch Transkriptionsaktivator-like (TALENS) und gruppierten regelmäßig voneinander beabstandeten kurzen palindromischen Repeats (CRISPR) -Cas9 Nukleasen, ermöglicht die gezielte Einführung von Mutationen sowie Knock-out und knock-in Ansätzen 19, 20, 21, 22. Combined, diese Methoden Zebrabärbling machen ein leistungsfähiges Modell für die Untersuchung der genetischen Mechanismen , bestimmte Verhaltensweisen und mehrere menschliche Krankheiten zugrunde liegen 23, 24, 25, 26, 27. Diese Arbeit konzentriert sich auf die Entwicklung vonpsychische Regulierung und die Rolle der elektrischen Aktivität in neuronalen Entwicklung. Der Schwerpunkt liegt auf dem Rückenmark, für die das Modell Zebrabärbling mehr Vorteile bietet. Erstens ist es relativ einfach, Zebrabärbling an embryonalen und Larvenstadien zuzugreifen; Daher kann eine Rückenmarksfunktion während der Entwicklungsstadien untersuchen , die weniger Neuronen und einfachere Schaltungsanordnung 28, 29 haben. Darüber hinaus hat die Zebrabärbling Rückenmark eine vielfältige Gruppe von Neuronen, ähnlich wie bei anderen Wirbeltieren, als Faktoren , 30, 31, 32, 33, 34, 35 und durch charakteristische Unterscheidungsmuster der Transkription nachgewiesen. Die meisten Studien in Zebrabärbling, das die Mechanismen aufzudecken darauf abzielen, die die Funktion von Rückenmarksschaltungen zugrunde liegen, vor allemdiejenigen , die Fortbewegung, sind verständlicherweise konzentrieren sich auf Larvenstadien 36, 37, 38, 39, 40, 41, 42, 43 unterstützen. Allerdings sind viele der Neuronen, die das Rücken Lokomotive Netzwerken initiieren ihre Differenzierung in frühen Embryonalstadium, ~ 9-10 h nach der Befruchtung (hpf) 44, 45, 46, 47, 48, 49, 50, 51 bilden. Im Hinblick darauf, das Verständnis, wie die morphologischen und elektrischen Eigenschaften von spinalen Neuronen entstehen und Wechsel zwischen den embryonalen und Larvenstadien ist wichtig für eine Marktpreisefürll Verständnis der lokomotorischen Schaltungsbildung und -funktion. Die Dissektion hier beschriebenen Methoden erlauben Patch-Clamp-Aufnahmen von spinalen Neuronen und haben an embryonalen Stadien (~ 17-48 HPF) und Larvenstadien (~ 3-7 Tage nach der Befruchtung [dpf]) erfolgreich angewandt worden. Dieser Ansatz begrenzt die Menge der Dissektion erforderlich, um Zugang zu den Neuronen von Interesse bereitzustellen. Das Protokoll unterscheidet mich von der Mehrzahl der anderen veröffentlichten Verfahren für in Tierarzt dieses Naht Leim aus Zebrabärbling spinalen Neuronen Aufzeichnung verwendet wird, sondern als ein feiner Wolframstift, den Embryo oder Larve auf die Aufzeichnungskammer zu befestigen. Die Verfügbarkeit von zwei unterschiedlichen Ansätzen (dh Naht Klebstoff gegenüber dem Wolframstift) für die Zebrabärblingembryonen oder Larven für elektrophysiologische Analyse Montage bietet Forscher mit alternativen Optionen ihre spezifischen experimentellen Ziele zu erreichen. Als erstes Verfahren für den Zugriff auf und die Aufnahme von einem Pop<html> ulation von primären sensorischen Neuronen, Rohon Beard-Zellen, beschrieben. Die Zellkörper dieser Neuronen liegen im dorsalen Rückenmark. Rohon-Beard - Zellen existieren in zahlreichen Wirbeltierarten unterscheiden früh in der Entwicklung, und liegen unter der embryonalen Touch - Reaktion 6, 44, 47, 48. Zweitens Verfahren für den Zugriff auf und die Aufnahme von spinalen motorischen Neuronen sind detailliert. Zebrafisch-spinale motorische Neuronen entstehen während zwei Wellen des Neurogenese. Die früheren geborenen primären motorischen Neuronen entstehen am Ende der Gastrulation (~ 9-16 HPF), mit nur 3-4 primären motorischen Neuronen , die pro hemisegment 45, 46, 49. Im Gegensatz dazu ist die später geborene Bevölkerung von sekundären motorischen Neuronen zahlreicher und stellt sich während eines längeren Zeitraums, beginnend bei ~ 14 HPFef &quot;&gt; 45, 50. Secondary Motoneuron Genese Mitte trunk Segmente meistens von 51 HPF 50. Secondary motorischen Neuronen sind als abgeschlossen ist das Gegenstück von Motoneuronen in amniotes 46 sein. Interessanterweise supraspinalen Neuronen über Dopamin, regulieren Lokomotion in der Larve und sekundären Motoneuron Genese im Embryo und junge 50 Larve, 51. primäre und sekundäre Motorneuronen umfassen , die jeweils mehrere verschiedene Subtypen. jeder primäre Motoneuron - Subtyps projiziert einen peripheren Axonen , die eine charakteristische Muskelgruppe innerviert, in einer stereotypen führt, axonalen Trajektorie identifiziert. im Allgemeinen sekundäre Motorneuronen die axonale Wege zuvor eingesetzten primären motorischen Neuronen folgen. Somit wird mit Bezug auf die axonale Trajektorien, primäre und sekundäre Motorneuronen sind ähnlich, mit der Ausnahme, dass axonale Dicke und Größe a somatawieder größer für die primären Motorneuronen 45. Drittens Methoden für die Aufzeichnung von einigen Arten von Inter diskutiert. In diesen Fällen ist jedoch eine begrenzte Menge an Entfernung anderer Rückenmarkszellen erforderlich ist, und damit das Rückenmark ist weniger intakt als bei Aufnahmen von Rohon Beard-Zellen oder motorischen Neuronen.

<table><tbody><tr><td>Vacuum filter/Storage bottle, 0.22 mm pore</td><td>Corning</td><td>431096</td><td></td></tr><tr><td>Syringe filter 0.2 mm</td><td>Whatman</td><td>6780-2502</td><td></td></tr><tr><td>Tricaine</td><td>Sigma</td><td>A-5040</td><td>Ethyl 3-aminobenzoate methanesulfonate salt&amp;nbsp;</td></tr><tr><td>&amp;alpha;-bugarotoxin</td><td>Tocris</td><td>11032-79-4</td><td></td></tr><tr><td>Tetrodotoxin</td><td>Tocris</td><td>4368-28-9</td><td></td></tr><tr><td>Alexa-549 hydrazine salt</td><td>Molecular Probes</td><td>A-10438</td><td>fluorescent dye</td></tr><tr><td>Spin-X centrifuge tube filter</td><td>Corning</td><td>8161</td><td></td></tr><tr><td>Glass microscope slide</td><td>Fisher</td><td>12-550C</td><td></td></tr><tr><td>Sylgard silicone elastomer kit</td><td>Dow Corning</td><td>184</td><td>silicone elastomer</td></tr><tr><td>Petri dishes</td><td>Falcon</td><td>351029</td><td></td></tr><tr><td>Borosilicate glass capillaries</td><td>Harvard Apparatus</td><td>30-0038</td><td>inner and outer diameters of 0.78 and 1.0 mm (thin walled glass capillaries)</td></tr><tr><td>Borosilicate glass capillaries</td><td>Drummond Scientific</td><td>1-000-1000-100</td><td>inner and outer diameters of 1.13 and 1.55 mm (thick walled glass capillaries)</td></tr><tr><td>Miniature barbed polypropylene fitting&amp;nbsp;</td><td>Cole-Palmer</td><td>6365-90</td><td></td></tr><tr><td>Vetbond tissue adhesive&amp;nbsp;</td><td>3M</td><td>1469SB</td><td></td></tr><tr><td>Collagenase XI</td><td>Sigma</td><td>C7657</td><td></td></tr><tr><td>Microelectrode puller</td><td>Sutter Instruments</td><td>&amp;nbsp;Model P-97&amp;nbsp;</td><td></td></tr><tr><td>Amplifier</td><td>Molecular Devices</td><td>Axopatch 200B</td><td></td></tr><tr><td>Head stage</td><td>Molecular Devices</td><td>CV203BU</td><td></td></tr><tr><td>Motorized micromanipulator&amp;nbsp;&amp;nbsp;</td><td>Sutter Instruments</td><td>MP-285</td><td></td></tr><tr><td>Tygon tubing</td><td>Fisher</td><td>14-169-1B</td><td>ID 1/16 IN, OD 1/8 IN and WALL 1/32 IN (flexible laboratory tubing)</td></tr><tr><td>Electrode holder</td><td>Molecular Devices</td><td>1-HC-U</td><td></td></tr><tr><td>Pharmaseal Three-Way Stopcocks&amp;nbsp;</td><td>Baxter</td><td>K75</td><td></td></tr><tr><td>Digitizer</td><td>Axon Instruments</td><td>Digidata 1440A</td><td></td></tr><tr><td>Inverted microscope&amp;nbsp;</td><td>Zeiss</td><td>Axioskop2 FS plus</td><td></td></tr><tr><td>40X/0.80W Achroplan objective</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Data acquisition and analysis software&amp;nbsp;</td><td>Axon Instruments</td><td></td><td>PClamp 10 - Clampex and Clampfit&amp;nbsp;</td></tr><tr><td>Micropipette puller</td><td>Sutter Instruments</td><td>Model P-97</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;Dissection and Recording Solutions (in mM)&lt;/strong&gt;</td></tr><tr><td>All solutions, except the intracellular, are stable for ~2-3 months when filtered (0.22 mm filter cups) and stored at room temperature (RT).</td><td></td><td></td><td></td></tr><tr><td>The intracellular solution is filtered (0.2 mm syringe filters) and stored frozen (-20 &amp;deg;C) in small aliquots that are individually thawed on the day of use.&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Dissection/Ringer&amp;rsquo;s solution</td><td></td><td></td><td>145 NaCl, 3 KCl, 1.8 CaCl&lt;sub&gt;2&lt;/sub&gt;.2H&lt;sub&gt;2&lt;/sub&gt;O, 10 HEPES; pH 7.4 (with NaOH)</td></tr><tr><td>Pipette (intracellular) recording solution</td><td></td><td></td><td>135 KCl, 10 EGTA-acid, 10 HEPES; pH 7.4 (with KOH).</td></tr><tr><td>Bath (extracellular) recording solution/voltage and current-clamp</td><td></td><td></td><td>125 NaCl, 2 KCl, 10 CaCl&lt;sub&gt;2&lt;/sub&gt;.2H&lt;sub&gt;2&lt;/sub&gt;O, 5 HEPES; pH 7.4 (with NaOH).</td></tr><tr><td>Alexa-594 hydrazine salt stock solution.&amp;nbsp;</td><td></td><td></td><td>Prepare a 13.2 mM stock in ddH&lt;sub&gt;2&lt;/sub&gt;O, aliquot (~100 &amp;micro;l) and store at -20 &amp;deg;C. For use, dilute the stock solutiond 132 fold with pipette solution to a final concentration of 100 mM. After dilution, filter the Alexa-594 containing pipette solution&amp;nbsp; with a centrifuge tube filter.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Immobilizing agents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.4% ethyl 3-aminobenzoate methanesulfonate salt (Tricaine)</td><td></td><td></td><td>Prepare a 0.4% stock solution in 0.2 M Tris, pH 9 (0.4 g Tricaine/100 mL 0.2 M Tris</td></tr><tr><td colspan="4">Adjust pH to 7 with NaOH and store at -20 &amp;deg;C.</td></tr><tr><td colspan="4">For use, dilute the stock solution ~25 fold in embryo media</td></tr><tr><td>250 mM &amp;alpha;-bungarotoxin</td><td></td><td></td><td>Prepare a 250 mM stock in ddH&lt;sub&gt;2&lt;/sub&gt;O (1 mg/500 mL), prepare 100 &amp;micro;L aliquots, and sotre at -20 &amp;deg;C.</td></tr><tr><td colspan="4">For use, dilute 2,500-fold with extracellular solution to a final concentration of 100 nM.</td></tr><tr><td>1 mM Tetrodotoxin</td><td></td><td></td><td>Prepare a 1 mM stock in ddH2O (1 mg/3 mL), prepare 100 &amp;micro;L aliquots, and sotre at -20 &amp;deg;C.</td></tr><tr><td colspan="4">For use, dilute 2,000-fold with extracellular solution to a final concentration of 500 nM.</td></tr></tbody></table>

zebrafish in situ spinal cord preparation for electrophysiological recordings from spinal sensory and motor neurons

Zebrafisch, zunächst als Entwicklungsmodell eingeführt hat an Popularität gewinnt in vielen anderen Bereichen. Die Leichtigkeit der Aufzucht einer großen Anzahl von sich rasch entwickelnden Organismen, mit der embryonalen optische Klarheit kombiniert, diente als Ausgangs zwingenden Attribute dieses Modells. In den letzten zwei Jahrzehnten hat sich der Erfolg dieses Modells weiter durch seine Ansprechbarkeit auf groß angelegte Mutagenese-Bildschirme und durch die Leichtigkeit der Transgene angetrieben worden. In jüngster Zeit haben Gen-Editing Ansätze, um die Leistung des Modells erweitert. Für Studien der neurologischen Entwicklung der Larve Zebrafischembryo und liefern ein Modell, an dem mehrere Methoden angewendet werden können. Hier konzentrieren wir uns auf Methoden, die die Studie einer wesentlichen Eigenschaft von Neuronen, elektrische Erregbarkeit ermöglichen. Unsere Vorbereitung für die elektrophysiologische Untersuchung von Zebrabärbling spinalen Neuronen beinhaltet die Verwendung von Tierarzt Naht Kleber, um die Vorbereitung auf eine Aufzeichnungskammer zu sichern. Alternative Methoden zur Aufzeichnungvon Zebrabärbling - Embryonen und Larven betreffen die Befestigung des Präparates an die Kammer eine feine Wolframstift 1, 2, 3, 4, 5 verwendet wird . Ein Wolframstift wird am häufigsten verwendet , um die Zubereitung in einer seitlichen Orientierung zu montieren, obwohl sie verwendet wurde Larve dorsale Seite nach oben 4 zu montieren. Der Naht Leim verwendet worden Embryonen und Larven in beiden Orientierungen zu montieren. Unter Verwendung des Klebers kann eine minimale Dissektion durchgeführt werden, die den Zugang zu spinalen Neuronen ohne die Verwendung einer enzymatischen Behandlung, um dadurch eventuelle Schäden zu vermeiden. Doch für die Larven, ist es notwendig, eine kurze Enzymbehandlung anzuwenden, um das Muskelgewebe rund um das Rückenmark zu entfernen. Die hier beschriebenen Methoden sind verwendet worden, die intrinsischen elektrischen Eigenschaften von Motorneuronen, Inter und sensorischen Neuronen an mehreren developmenta zu studierenl Stufen 6, 7, 8, 9.

Wir haben erfolgreich von Rohon Beard-Neuronen in 17 hpf Embryonen bis 7 dpf Larven (5A und 5B) aufgezeichnet. Wenn Rohon Beard-Zellen aufgenommen wurden, wurde die Zubereitung montiert dorsal-Seite nach oben. Eine solche Montage ermöglicht die eindeutige Identifizierung von Rohon-Beard auf ihrer oberflächlichen dorsalen Positionen und große Größen soma Zellen basieren. Die Identifizierung wird durch das stereotypisch hyperpolarisiertem Ruhemembranp...

Watch this Scientific Journal Video about Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons at JoVE.com

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Dieses Manuskript beschreibt Verfahren zur elektrophysiologischen Aufnahmen von spinalen Neuronen Zebrabärblingembryonen und Larven. Die Herstellung hält Neuronen in situ und beinhaltet oft minimale Dissektion. Diese Verfahren ermöglichen die elektro Untersuchung einer Vielzahl von spinalen Neuronen, von der ersten elektrischen Erregbarkeit Erwerb durch den frühen Larvenstadien.

Zebrafisch<em&gt; In Situ</em&gt; Spinal Cord Vorbereitung für elektrophysiologische Aufzeichnungen von Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

zebrafish-situ-spinal-cord-preparation-for-electrophysiological

Research

JoVE Journal

Neuroscience

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

11.4K Aufrufe.  University of Colorado Anschutz Medical Campus (UCAMC).  Dieses Manuskript beschreibt Verfahren zur elektrophysiologischen Aufnahmen von spinalen Neuronen Zebrabärblingembryonen und Larven. Die Herstellung hält Neuronen in situ und beinhaltet oft minimale Dissektion. Diese Verfahren ermöglichen die elektro Untersuchung einer Vielzahl von spinalen Neuronen, von der ersten elektrischen Erregbarkeit Erwerb durch den frühen Larvenstadien. 

Serie: Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

Gepaart Patch Clamp Messungen von Motor-Neuron-und Target Skelettmuskulatur bei Zebrafisch

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

Forschung

Neurowissenschaften

Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 &mu;l during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds4. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.

A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

Vorderhirn Elektrophysiologische Aufzeichnung im Larven Zebrafisch

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

Patch-Clamp-Aufnahmen aus embryonalen Zebrafisch-Mauthner-Zellen

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

Dissection und seitlicher Anbau von Zebrafisch-Embryonen: Analyse der Rückenmarksentwicklung

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

Elektrophysiologische Aufnahme im Gehirn von erwachsenen Zebrafisch Intact

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

Rückenmarksdurchtrennung in der Zebrafisch-Larven

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

Zebra II: Advanced ECG System for Multichannel Zebrafish Cardiovascular Studies

Zebra II as A Novel System to Record Electrophysiological Signals in Zebrafish

Zebrafish and their mutant lines have been extensively used in biomedical investigations, cardiovascular studies, and drug screening. In the current study, the commercial version of the novel system, Zebra II, is presented. The protocol demonstrates electrocardiogram (ECG) acquisition and analysis from multiple zebrafish within controllable working environments. The device is composed of an external and independent perfusion system, a 4-point electrode, temperature sensors, and an embedded electronic system. In previous studies, the device prototype underwent validation against the established iWORX system through several tests, demonstrating similar data quality and ECG response to drug interventions. Following this, the study delved into examining the impact of anesthetic drugs and temperature fluctuations on zebrafish ECG, necessitating instant data evaluation. Thanks to the apparatus&#39;s capacity for consistent delivery of anesthetics and drugs, it was possible to extend ECG data collection up to 1 h, markedly longer than the 5 min duration supported by current systems. This paper introduces a pioneering, cloud-based, automated analysis utilizing data from four zebrafish, offering an efficient method for conducting combination experiments and significantly reducing time and effort. The system proved effective in capturing and analyzing ECG, especially in detecting drug-induced arrhythmias in wild-type zebrafish. Additionally, the capability to gather data across multiple channels facilitated the execution of randomized controlled trials with zebrafish models. The developed ECG system overcomes existing limitations, showing the potential to greatly expedite drug discovery and cardiovascular research involving zebrafish.

The study introduces Zebra II, an advanced system for prolonged ECG acquisition and analysis in zebrafish. This system features an independent perfusion system and multiple-point electrodes for handling multiple fish in controlled environments. Acute effects of Amiodarone were assessed and analyzed with wild-type zebrafish.

Zebrafish and their mutant lines have been extensively used in biomedical investigations, cardiovascular studies, and drug screening. In the current ...

Biotechnik

Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates reafferent cues that animals must disentangle from relevant sensory cues of the surrounding environment. For example, when a fish swims, flow generated from body undulations is detected by the mechanoreceptive neuromasts, comprising hair cells, that compose the lateral line system. The hair cells then transmit fluid motion information from the sensor to the brain via the sensory afferent neurons. Concurrently, corollary discharge of the motor command is relayed to hair cells to prevent sensory overload. Accounting for the inhibitory effect of predictive motor signals during locomotion is, therefore, critical when evaluating the sensitivity of the lateral line system. We have developed an in vivo electrophysiological approach to simultaneously monitor posterior lateral line afferent neuron and ventral motor root activity in zebrafish larvae (4-7 days post fertilization) that can last for several hours. Extracellular recordings of afferent neurons are achieved using the loose patch clamp technique, which can detect activity from single or multiple neurons. Ventral root recordings are performed through the skin with glass electrodes to detect motor neuron activity. Our experimental protocol provides the potential to monitor endogenous or evoked changes in sensory input across motor behaviors in an intact, behaving vertebrate.

We describe a protocol to monitor changes in the afferent neuron activity during motor commands in a model vertebrate hair cell system.

Aktivität von posterioren Lateral Line Afferent Neuronen beim Schwimmen im Zebrafisch

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates ...

In Vivo Surface Electrocardiography for Adult Zebrafish

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of zebrafish not only as a research model for human cardiac electrophysiology and myopathies but also as a surrogate model in high throughput pharmaceutical screening for potential cardiotoxicities to humans, such as QT prolongation. As such, in vivo electrocardiography for adult zebrafish is an electrical phenotyping tool that is necessary, if not indispensable, for cross-sectional or longitudinal in vivo electrophysiological characterizations. However, too often, the lack of a reliable, practical, and cost-effective recording method remains a major challenge preventing this in vivo diagnostic tool from becoming more readily accessible. Here, we describe a practical, straightforward approach to in vivo electrocardiography for adult zebrafish using a low-maintenance, cost-effective, and comprehensive system that yields consistent, reliable recordings. We illustrate our protocol using healthy adult male zebrafish of 12-18 months of age. We also introduce a rapid real-time interpretation strategy for quality validation to ensure data accuracy and robustness early in the electrocardiogram recording process.

Here, we present a reliable, minimally invasive, and cost-effective method to record and interpret electrocardiograms in live anesthetized adult zebrafish.

In Vivo Surface Elektrokardiographie für erwachsene Zebrafische

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of ...

Biologie

Adult Zebrafish Electrophysiology: A Method to Measure the Extracellular Electrical Activity of Neurons In Vivo

Source: Johnston, L., et al. <a href="https://www.jove.com/v/51065/" target="_blank">Electrophysiological Recording in the Brain of Intact Adult Zebrafish</a>. &#160;J. Vis. Exp. (2013)
The protocol describes the procedure to perform electrophysiology in adult zebrafish. This method can be used for measuring neural activity in vivo.

Elektrophysiologie adulter Zebrafische: Eine Methode zur Messung der extrazellulären elektrischen Aktivität von Neuronen in vivo

Source: Johnston, L., et al. Electrophysiological Recording in the Brain of Intact Adult Zebrafish. &#160;J. Vis. Exp. (2013)
The protocol describes the ...

Zebrafisch

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