Die Autoren danken Dr. Candace Hsieh für Anregungen und Unterstützung danken, mit bildgebenden Verfahren und Dr. Doris Wu für Kompetenz auf chick Innenohr Anatomie während der frühen Embryogenese. Diese Arbeit wurde von der NSF IOS-0642346, NIH T32-DC010775, NIH T32-GM008620, NIH R01-DC010796 und DOE GAANN P200A120165 unterstützt.

Ein. Bereiten Sie die folgenden Dissection Tools und Reagenzien <ul><li> Künstlicher zerebrospinaler Flüssigkeit (ACSF; 130 mM NaCl, 3 mM KCl, 1,2 mM KH 2 PO 4, 20 mM NaHCO 3, 3 mM HEPES, 10 mM Glucose, 2 mM CaCl 2, 1,3 mM MgSO 4) kontinuierlich mit 95% O infundiert 2/5% CO 2 bei Raumtemperatur. Infusionslösung, zu 2/3 eine 500 ml Nalgene Weithals-Glas mit einem Loch im Deckel gebohrt füllen. Tank wird über einen Schlauch zu einem Glasfuß Gasspüler, der die aCSF dringt durch das Loch im Glas Deckel befestigt werden. Passen Druck, um einen konstanten Strom von Blasen, die etwa 1/3 der Flüssigkeit zu erreichen, sind aber nicht stark genug, um zu bewirken spritzt über die Felge zu erhalten. Submerge 5 ml Glasampullen (bis 6) in das Glas zu Nalgene einzelnen Proben voneinander und aus dem Gasstrom Turbulenzen zu trennen. ACSF sollte mindestens 20 Minuten vor jedem Gewebe Inkubationszeit Sauerstoff versorgt werden. </li><li> Kleine Silikon-Dissektion Teller (35 mm x 10mm Petri beschichtet mit Sylgard Elastomere Kit) mit zwei Dissektion Stifte in kleine sechseckige Polystyrol gelegt (47 mm unten) Wiegeschiffchen. </li><li> Zwei feinen Spitze-Zange, eine gekrümmte Spitze-Zange, einer 50 ml-Becher, und einen Behälter für das Tissue Abfall. </li><li> Gezogen Glasmikropipette (1,2 OD / 0,9 ID) zerbrochen zu etwa 20-50 um die Öffnung an der Spitze und mit Rhodamin Dextran Amin (RDA, MW = 3.000, Invitrogen) in einer 6,25% igen Lösung mit 0,4% Triton X-100 in PBS . Bringen Sie durch feine Schlauch an picospritzer und Mikromanipulator stabilisieren. </li></ul> 2. Micro-Dissektion Basilar Papilla am E8 4,5 Reveal <ol><li> Mit gebogenen Pinzette, schneiden E8 Embryo direkt unter dem Hirnstamm, indem Kopf direkt auf silikonbeschichteten Dissektion Gericht in Wiegeschiffchen. </li><li> Mit Dissektion Stifte, positionieren Sie den Kopf mit einem ventral so das Ohr auf der Seite von Interesse ist leicht sichtbar, geneigt der Kopf leicht nach dorsal und zeigt 10-30 Grad weg von center (Abbildung 2A). Unmittelbar sichern den Kopf mit Stiften. Wenn bilateralen Ablaufverfolgung gewünscht wird, in der Mitte der Kopf bei 0 Grad, oder eine vollständige ventral, so dass beide Seiten zugegriffen werden kann. Ähnliches gilt bei einer linksseitigen Injektion, sichern den Kopf bei -10 bis -30 Grad von der Mitte, so dass die linke Seite zugänglich ist. </li><li> Mit dem 50-ml-Becher, übertragen 30 ml des oxygenierten aCSF in die Schüssel, voll Eintauchen des Gewebes. </li><li> Fassen und schälen sich den Unterkiefer mit feiner Spitze Pinzette, dann entfernen Sie das untere Augenlid auf der Seite von Interesse. </li><li> Entfernen Sie den darüberliegenden Gefäßgewebe, einschließlich aller übrigen Gaumen und Kehle. Diese weichen Geweben sollte abzuschälen enthüllt die glatte Oberfläche zu entwickeln Chondrocranium mit den unverwechselbaren Vertebralarterien an seiner Oberfläche. Zu diesem Zeitpunkt sollte die charakteristischen weißen otoconial Masse sichtbar sein. </li><li> Vorsichtig zu sezieren die Haut um den äußeren Gehörgang, Knorpel des Kiefergelenks und des Mittelohrs, während die inner Ohr intakt. Verwenden Sie die Pinzette vorsichtig weggeschnitten diese knorpeligen Strukturen. </li><li> Entfernen Vertebralarterien und Bündelung Blut mit einem sanften Kehrbewegung mit den feinen Pinzette Tipps. Blut kann darüber hinwegtäuschen, Ansicht und Koagulation kann die Injektionspipette Öffnung stecken. </li><li> Das Cochlea Kanal mit benachbarten Sinnesepithel (basilaris Papilla) und akustische Ganglion liegt direkt unterhalb der Chondrocranium 6 und kann als länglicher fingerartigen Vorsprung aus dem Innenohr ventral, mit der am weitesten distalen Spitze (Apex) in der Nähe des vorderen visualisiert Mittellinie 5. Es kann eine kleine Ansammlung von Blut (rote) werden aus der Dissektion und / oder otolithic Verkalkungen (weiß) im Scheitelpunkt des Cochlea Kanal (2B), die in Visualisierung unterstützen kann. An der Spitze ist die lagenar Makula, dachte eine sensorische Patch der vestibulären Funktion 7,8 sein. </li></ol> 3. Selektive Markierung von CN VIII Auditory Fibers <ol><li&gt; Legen Sie die Injektionspipette in der Nähe des Embryos Fläche an der Ductus cochlearis und basiläre Papille Region. </li><li> Langsam senken die Mikropipette zum ersten Punktion der Schädel. Diese Oberkopf ist dünner als die Umgebung und erfordert weniger Kraftaufwand zu durchdringen. Die Mikropipette sollte nun im Inneren des Ductus cochlearis sein. Optional: eine kleine Injektion von Tracing Farbstoff kann hier gemacht werden, um in die Visualisierung der basilaris Papilla unterstützen. </li><li> Ohne Neupositionierung der Mikropipette weiter senken, bis es nur Verstöße gegen die tief Rand des Ductus cochlearis (Abbildung 1). Durchführen einer Injektion und wiederholen hier in mehreren Bereichen entlang der Länge des basilaren Papille (2C, 2D) mit Ausnahme des am meisten proximalen Spitze, wo der vestibulären Lagena befindet. Mit dem picospritzer zwischen 10-30 psi eingestellt ist, werden mehrere Injektionen gemacht. Das Volumen des injizierten Farbstoff Fluids variiert und hängt von dem Ausmaß der gewünschten Etikettierung. Jede Injektion sollte in einem Fluoreszenzfarbstoff-la führenBeled Ort von etwa 200 bis 400 Mikrometer Durchmesser (2D). </li><li> Sobald die Injektion abgeschlossen ist, verwenden Pinzette, um die Probe oberhalb des Hirnstamms durchschneiden und verwenden Sie eine Pipette, um den kaudalen Teil in einen kleinen Topf legen im Behälter aCSF kontinuierlich mit 95% O 2/5% CO 2 durchströmt. </li><li> Für jedes Embryo erlauben 20-30 min zur Farbstoffübertragung, abhängig vom Abstand der zentralen Tracing gewünscht. </li><li> Entfernen Gewebe und die Vorbereitungen für histologische Schnitte. In dem hier gezeigten Beispiel wird Gewebe in 4% Paraformaldehyd (in 1X PBS) über Nacht, 30% Saccharose (in 1X PBS) über Nacht eingetaucht. </li></ol> 4. Gegenfärbung und Analyse <ol><li> Gegenfärbung ist nützlich, um zu helfen orient der Beobachter an anatomische Lage sowie zu identifizieren bestimmte Regionen von Interesse. Eine wirksame Gegenfärbung in dieser Zubereitung ist Neurofilament Immunmarkierung (Figuren 1, 3B, 3E), die stark l MitAbels Axonen und ermöglicht Abgrenzung der axonalen Bahnen im ZNS. </li><li> Analyse sollte während der markierten Region durchgeführt werden, wie die Vorsprünge können mehrere hundert Mikrometer oder mehr entlang der Achse rostrokaudalen erstrecken. Einige Beispiele für grundlegende Untersuchungen sind: Targeting Fehlerraten, Timing und / oder Veränderungen der Divergenz der Projektionsmuster. RDA-markierte Axone visualisiert im peripheren und zentralen Nervensystems mit hoher Auflösung (3A, 3D). </li></ol>

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Studien der frühen Entwicklung des VIII. Nerven wurden zum Teil wegen der Schwierigkeiten bei der Ermittlung embryonalen Axone aus mehreren verschiedenen Ganglien begrenzt. Mehrere Studien haben die molekularen Signale Führung auditorischen und vestibulären sensorischen Zellen und Ganglienzellen Schicksale während der frühen Entwicklung, 5,11,12 erforscht, aber die Prozesse, die die zentrale Innervation müssen noch bestimmt werden. Berichte von akustischen Ganglienzellen Projektionen beschreiben typisch...

<table border="1"><tbody><tr><td> Name des Reagenz / Material </td><td> Firma </td><td> Catalog Number </td><td> Kommentare </td></tr><tr><td> Polystyrol Wiegen Dish </td><td> Fisher Scientific </td><td> 02-202-101 </td><td></td></tr><tr><td> Petrischale, 35 X 10 mm </td><td> Fisher Scientific </td><td> 50820644 </td><td> Verwenden, um Silikon-Dissektion Gericht zu machen </td></tr><tr><td> Sylgard Silikon-Elastomer Kit </td><td> World Precision Instruments </td><td> SYLG184 </td><td> Coat Petri zu machen Dissektion Gericht </td></tr><tr><td> Dissection Pins </td><td> Verschiedene </td><td></td><td> Hält Embryo im Ort während der Präparation </td></tr><tr><td> NaCL </td><td> Verschiedene </td><td></td><td> Teil aCSF Rezept </td></tr><tr><td> KCl </td><td> Verschiedene </td><td></td><td> Teil aCSF Rezept </td&gt; </tr><tr><td> KH 2 PO 4 </td><td> Verschiedene </td><td></td><td> Teil aCSF Rezept </td></tr><tr><td> NaHCO 3 </td><td> Verschiedene </td><td></td><td> Teil aCSF Rezept </td></tr><tr><td> Glucose </td><td> Verschiedene </td><td></td><td> Teil aCSF Rezept </td></tr><tr><td> CaCl 2 </td><td> Verschiedene </td><td></td><td> Teil aCSF Rezept </td></tr><tr><td> MgSO 4 </td><td> Verschiedene </td><td></td><td> Teil aCSF Rezept </td></tr><tr><td> Behälter für aCSF. Vorschlagen transluzenten Weithals-Nalgene Flasche, 500 ml (16 oz) mit Deckel. </td><td> CPLabSafety </td><td> QP-PLC-03717 </td><td> Bohrloch Öffnung an der Oberseite des Deckels für Glas bubling Stammzellen zu durchdringen Flüssigkeit </td></tr><tr><td> Leere 5 ml Glasfläschchen oder vergleichbaren transparenten Flasche </td><td> American Pharmaceutical Partners, Inc </td><td> 6332300105 </td><td&gt; während aCSF Inkubation verwenden, um Proben getrennt von einander und von der sprudelnden Strom </td></tr><tr><td> Tank von Carbogen (95% O 2/5% CO 2) durch einen Schlauch zur Waschflasche verbunden </td><td> Verschiedene </td><td></td><td> Bringen Sie durch einen Schlauch zu Glasfuß Bubbler zur Infusion in aCSF </td></tr><tr><td> Glasfuß Bubbler </td><td> Verschiedene </td><td></td><td> Um Carbogen in aCSF einzuflößen </td></tr><tr><td> Curved-Spitze Pinzette </td><td> World Precision Instruments </td><td> 501008 </td><td> Um Embryo Kopf vom Ei entfernen </td></tr><tr><td> Zwei feine Spitze Pinzette </td><td> World Precision Instruments </td><td> 501985 </td><td> Für Mikro-Dissektion </td></tr><tr><td> 50 ml Becherglas </td><td> verschiedene </td><td></td><td></td></tr><tr><td> Rhodamin Dextran Amine (RDA) </td><td> Invitrogen </td><td> verschiedene </td><td> Fluorescent Axon Tracer </td></tr><tr><td> Triton X-100 </td><td> ICN Biomedicals </td><td></td><td></td></tr><tr><td> Phosphate Buffered Saline, (1X PBS) </td><td> Verschiedene </td><td></td><td> Standard-Lab-Reagenz </td></tr><tr><td> Thin Wall Glaskapillaren 1,2 OD, 0,9 ID 4 &quot;(100 mm) Länge </td><td> World Precision Instruments </td><td> TW120F-4 </td><td> Legen Sie mit RDA. Jede Kapillare macht zwei Glasmikropipetten </td></tr><tr><td> Needle / Pipette puller </td><td> David Kopf Instruments </td><td> Modell 720 </td><td> Einstellungen verwendet: Heat 16,4, Solenoid 2,2 </td></tr><tr><td> Picospritzer </td><td> Parker Instrumentation </td><td> verschiedene </td><td> Bringen Sie durch feine Schlauch an Glasmikropipette </td></tr><tr><td> Micromanipulator </td><td> Narishige </td><td> verschiedene </td><td></td></tr><tr><td> Dissection Mikroskop mit Fluoreszenz </td><td> Verschiedene </td><td></td><td></td&gt; </tr><tr><td> 4% Paraformaldehyd </td><td> Verschiedene </td><td></td><td> Standard-Lab-Reagenz </td></tr><tr><td> anti-Neurofilament-Antikörper, optional </td><td> Millipore </td><td> AB1991 </td><td> Folgen histologischen Protokoll vom Hersteller empfohlen </td></tr><tr><td> Kryostat und zugehörige Materialien für das Schneiden </td><td> Leica </td><td> verschiedene </td><td></td></tr><tr><td> Epifluoreszenz-Mikroskop zum Abbilden </td><td> Zeiss, verschiedene </td><td></td><td></td></tr></tbody></table>

selective tracing of auditory fibers in the avian embryonic vestibulocochlear nerve

Die embryonale Küken ist ein weit verbreitetes Modell für die Untersuchung der peripheren und zentralen Ganglienzellen Projektionen. Im auditorischen System würde selektive Markierung von auditiven Axone innerhalb des VIII. Hirnnerven verbessern das Studium der zentralen auditorischen Schaltungsentwicklung. Dieser Ansatz ist schwierig, weil mehrere Sinnesorgane des Innenohrs der VIII. Nerven 1 beitragen. Darüber hinaus haben Markern, die zuverlässig unterscheiden auditive gegenüber vestibulären Gruppen von Axonen innerhalb der Vogelgrippe VIII. Nerven noch nicht identifiziert werden. Auditive und vestibulären Wege nicht funktional in frühen Embryonen unterschieden werden, als sensorische hervorgerufenen Reaktionen nicht vorhanden sind, bevor die Schaltungen ausgebildet sind. Zentral vorstehenden VIII. Nervenaxone haben in einigen Studien verfolgt worden, aber auditiven Axon Markierung wurde durch Markierung von anderen VIII. Nerven Bauteile 2,3 begleitet. Hier beschreiben wir eine Methode zur anterograde Tracing von der akustischen ganglion selektiv label auditiven Axone innerhalb des sich entwickelnden VIII. Nerven. Erste, nach teilweiser Durchtrennung des vorderen Schädelbereich des 8-tägiger Hühnerembryos in sauerstoffhaltigen künstliche Zerebrospinalflüssigkeit eingetaucht wird der Kanal durch Cochlea anatomischen Landmarken identifiziert. Als Nächstes wird ein feiner gezogen Glasmikropipette positioniert, um eine kleine Menge an Rhodamin Dextran Amin in den Kanal und benachbart tiefen Bereich, wo die akustischen Ganglienzellen befinden injizieren. Innerhalb 30 Minuten nach der Injektion werden auditive Axone zentral in den Hinterhirn zurückverfolgt und können später visualisiert werden nach histologischen Vorbereitung. Diese Methode liefert ein nützliches Werkzeug für Entwicklungsstudien der peripheren zum zentralen auditorischen Schaltung Bildung.

Die Komponenten der VIII. Nerv und der Anatomie des Nerven selbst sind komplex und gewundenen (Figuren 1, 3). Durch selektives Tracing Fasern aus akustischen Ganglienzellen können Segmente des VIII. Nerven sowie primären auditorischen Afferenzen im Hirnstamm sauber zurückverfolgt werden und sich von ihren vestibulären Gegenstücke (Abbildungen 2, 3). Ebenso könnte diese Technik verwendet, um periphere Vorsprünge der akustischen Ganglienzellen (3G) studieren oder m...

Watch this Scientific Journal Video about Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve at JoVE.com

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

Hier beschreiben wir eine Technik, die von Mikrodissektion Fluoreszenzfarbstoff Injektion in den akustischen Ganglion frühen Hühnerembryonen zur selektiven Rückverfolgung von auditorischen Axon Fasern im Nerv und Rautenhirn gefolgt.

Selektive Tracing von Auditory Fibers im Avian Embryonale vestibulocochlearis

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

selective-tracing-auditory-fibers-avian-embryonic-vestibulocochlear

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

9.1K Aufrufe.  University of California, Irvine. Hier beschreiben wir eine Technik, die von Mikrodissektion Fluoreszenzfarbstoff Injektion in den akustischen Ganglion frühen Hühnerembryonen zur selektiven Rückverfolgung von auditorischen Axon Fasern im Nerv und Rautenhirn gefolgt.

Serie: Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Kultur von embryonalen Maus Cochlear Explantate und Gentransfer durch Elektroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Forschung

Entwicklungsbiologie

Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the postsynaptic activity of a single ribbon synapse 1-4. Ribbon synapses of sensory cells release neurotransmitter continuously, the rate of which is modulated in response to graded changes in IHC membrane potential 5. Ribbon synapses have been shown to operate by multivesicular release, where multiple vesicles can be released simultaneously to evoke excitatory postsynaptic currents (EPSCs) of varying amplitudes 1, 4, 6-11. Neither the role of the presynaptic ribbon, nor the mechanism underlying multivesicular release is currently well understood.
The IHC is innervated by 10-20 auditory nerve fibers, and every fiber contacts the IHC with a unmyelinated single ending to form a single ribbon synapse. The small size of the afferent boutons contacting IHCs (approximately 1 &mu;m in diameter) enables recordings with exceptional temporal resolution to be made. Furthermore, the technique can be adapted to record from both pre- and postsynaptic cells simultaneously, allowing the transfer function at the synapse to be studied directly 2. This method therefore provides a means by which fundamental aspects of neurotransmission can be studied, from multivesicular release to the elusive function of the ribbon in sensory cells.

Whole-cell patch-clamp recordings from auditory nerve fiber dendrites at the inner hair cell ribbon synapse in the mammalian cochlea.

Postsynaptische Recordings am afferenten Dendriten Kontaktaufnahme mit Cochlear inneren Haarzellen: Monitoring multivesikulären Veröffentlichung in einem Ribbon Synapse

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the ...

Neurowissenschaften

Preparation and Culture of Chicken Auditory Brainstem Slices

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at discreet periods of development 1-4 as well as mechanisms for temporal coding in the central nervous system 5-7.
Here we present a method to prepare chicken auditory brainstem slices that can be used for acute experimental procedures or to culture organotypic slices for long-term experimental manipulations.
The chicken auditory brainstem is composed of nucleus angularis, magnocellularis, laminaris and superior olive. These nuclei are responsible for binaural sound processing and single coronal slice preparations preserve the entire circuitry. Ultimately, organotypic slice cultures can provide the opportunity to manipulate several developmental parameters such as synaptic activity, expression of pre and postsynaptic components, expression of aspects controlling excitability and differential gene expression
This approach can be used to broaden general knowledge about neural circuit development, refinement and maturation.

The chicken auditory brainstem is comprised of nuclei responsible for binaural sound processing. A single coronal slice preparation maintains the entire circuitry while the cultured approach provides a unique preparation to study the development of neuronal structure and auditory function at the molecular, cellular and network levels.

Vorbereitung und Kultur von Chicken auditorischen Hirnstamm Slices

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

Gentransfer in das Huhn Hörorgan von<em&gt; In Ovo</em&gt; Micro-Elektroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

In Ovo Electroporation in the Chicken Auditory Brainstem

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that the chicken embryo is an effective research model for studying basic biological functions of auditory system development. More recently, the chicken embryo has become particularly valuable in studying gene expression, regulation and function associated with hearing. In ovo electroporation can be used to target auditory brainstem regions responsible for highly specialized auditory functions. These regions include the chicken nucleus magnocellularis (NM) and nucleus laminaris (NL). NM and NL neurons arise from distinct precursors of rhombomeres 5 and 6 (R5/R6). Here, we present in ovo electroporation of plasmid-encoded genes to study gene-related properties in these regions. We show a method for spatial and temporal control of gene expression that promote either gain or loss of functional phenotypes. By targeting auditory neural progenitor regions associated with R5/R6, we show plasmid transfection in NM and NL. Temporal regulation of gene expression can be achieved by adopting a tet-on vector system. This is a drug inducible procedure that expresses the genes of interest in the presence of doxycycline (Dox). The in ovo electroporation technique &#8211; together with either biochemical, pharmacological, and or in vivo functional assays &#8211; provides an innovative approach to study auditory neuron development and associated pathophysiological phenomena.

In Ovo Electroporation in the Chicken Auditory Brainstem

Auditory brainstem neurons of avians and mammals are specialized for fast neural encoding, a fundamental process for normal hearing functions. These neurons arise from distinct precursors of embryonic hindbrain. We present techniques utilizing electroporation to express genes in the hindbrain of chicken embryos to study gene function during auditory development.

In Ovo Electroporation im Hühnchen auditorischen Hirnstamm

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that ...

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

In Ovo und Ex Ovo Methoden zur Untersuchung der Entwicklung des Vogelinnenohrs

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as ...

Slicing the Embryonic Chicken Auditory Brainstem to Evaluate Tonotopic Gradients and Microcircuits

The chicken embryo is a widely accepted animal model to study the auditory brainstem, composed of highly specialized microcircuitry and neuronal topology differentially oriented along a tonotopic (i.e., frequency) axis. The tonotopic axis permits the segregated encoding of high-frequency sounds in the rostral-medial plane and low-frequency encoding in caudo-lateral regions. Traditionally, coronal brainstem slices of embryonic tissue permit the study of relative individual iso-frequency lamina. Although sufficient to investigate anatomical and physiological questions pertaining to individual iso-frequency regions, the study of tonotopic variation and its development across larger auditory brainstem areas is somewhat limited. This protocol reports brainstem slicing techniques from chicken embryos that encompass larger gradients of frequency regions in the lower auditory brainstem. The utilization of different slicing methods for chicken auditory brainstem tissue permits electrophysiological and anatomical experiments within one brainstem slice, where larger gradients of tonotopic properties and developmental trajectories are better preserved than coronal sections. Multiple slicing techniques allow for improved investigation of the diverse anatomical, biophysical, and tonotopic properties of auditory brainstem microcircuits.

Here we present a protocol for obtaining non-coronal auditory brainstem slices of the chicken embryo for the investigation of tonotopic properties and developmental trajectories within one brainstem slice. These slices include sagittal, horizontal, and horizontal/transverse sections encompassing larger tonotopic regions within an individual slice plane that the traditional coronal sections.

Schneiden des embryonalen Hühnerhirnstamms zur Bewertung tonotopischer Gradienten und Mikroschaltkreise

The chicken embryo is a widely accepted animal model to study the auditory brainstem, composed of highly specialized microcircuitry and neuronal topology ...

Evaluation of Auditory Brainstem Response in Chicken Hatchlings

The auditory brainstem response (ABR) is an invaluable assay in clinical audiology, non-human animals, and human research. Despite the widespread use of ABRs in measuring auditory neural synchrony and estimating hearing sensitivity in other vertebrate model systems, methods for recording ABRs in the chicken have not been reported in nearly four decades. Chickens provide a robust animal research model because their auditory system is near functional maturation during late embryonic and early hatchling stages. We have demonstrated methods used to elicit one or two-channel ABR recordings using subdermal needle electrode arrays in chicken hatchlings. Regardless of electrode recording configuration (i.e., montage), ABR recordings included 3-4 positive-going peak waveforms within the first 6 ms of a suprathreshold click stimulus. Peak-to-trough waveform amplitudes ranged from 2-11 &#181;V at high-intensity levels, with positive peaks exhibiting expected latency-intensity functions (i.e., increase in latency as a function of decreased intensity). Standardized earphone position was critical for optimal recordings as loose skin can occlude the ear canal, and animal movement can dislodge the stimulus transducer. Peak amplitudes were smaller, and latencies were longer as animal body temperature lowered, supporting the need for maintaining physiological body temperature. For young hatchlings (&#60;3 h post-hatch day 1), thresholds were elevated by ~5 dB, peak latencies increased ~1-2 ms, and peak to trough amplitudes were decreased ~1 &#181;V compared to older hatchlings. This suggests a potential conductive-related issue (i.e., fluid in the middle ear cavity) and should be considered for young hatchlings. Overall, the ABR methods outlined here permit accurate and reproducible recording of in-vivo auditory function in chicken hatchlings that could be applied to different stages of development. Such findings are easily compared to human and mammalian models of hearing loss, aging, or other auditory-related manipulations.

We have used standard auditory brainstem response (ABR) techniques and applied them to hatchling chickens, a precocious avian model for auditory function. The protocol outlines animal preparation and ABR acquisition techniques in detail, with steps that could translate to other avian or rodent models.

Bewertung der auditiven Hirnstammreaktion bei Hühnerschlüpflingen

The auditory brainstem response (ABR) is an invaluable assay in clinical audiology, non-human animals, and human research. Despite the widespread use of ...

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

Fragile X mental retardation protein (FMRP) is an mRNA-binding protein that regulates local protein translation. FMRP loss or dysfunction leads to aberrant neuronal and synaptic activities in fragile X syndrome (FXS), which is characterized by intellectual disability, sensory abnormalities, and social communication problems. Studies of FMRP function and FXS pathogenesis have primarily been conducted with Fmr1 (the gene encoding FMRP) knockout in transgenic animals. Here we report an in vivo method for determining the cell-autonomous function of FMRP during the period of circuit assembly and synaptic formation using chicken embryos. This method employs stage-, site-, and direction-specific electroporation of a drug-inducible vector system containing Fmr1 small hairpin RNA (shRNA) and an EGFP reporter. With this method, we achieved selective FMRP knockdown in the auditory ganglion (AG) and in one of its brainstem targets, the nucleus magnocellularis (NM), thus providing a component-specific manipulation within the AG-NM circuit. Additionally, the mosaic pattern of the transfection allows within-animal controls and neighboring neuron/fiber comparisons for enhanced reliability and sensitivity in data analyzing. The inducible vector system provides temporal control of gene editing onset to minimize accumulating developmental effects. The combination of these strategies provides an innovative tool to dissect the cell-autonomous function of FMRP in synaptic and circuit development.

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

Using in ovo electroporation, we devised a method to selectively transfect the auditory inner ear and cochlear nucleus in chicken embryos to achieve a cell-group-specific knockdown of fragile X mental retardation protein during discrete periods of circuit assembly.

Sezieren der zellautonomen Funktion des fragilen X-mentalen Retardierungsproteins in einem auditorischen Schaltkreis durch In-Ovo-Elektroporation

Fragile X mental retardation protein (FMRP) is an mRNA-binding protein that regulates local protein translation. FMRP loss or dysfunction leads to ...

Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter receptors that contribute to cell diversity across this population. This protocol outlines the steps necessary for successful dissecting, dissociating, and short-term culturing of the somata of inner ear bipolar neurons for the purpose of patch-clamp recordings. Detailed instructions for preparing vestibular ganglion neurons are provided with the necessary modifications needed for plating spiral ganglion neurons. The protocol includes instructions for performing whole-cell patch-clamp recordings in the perforated-patch configuration. Example results characterizing the voltage-clamp recordings of hyperpolarization-activated cyclic nucleotide-gated (HCN)-mediated currents highlight the stability of perforated-patch recording configuration in comparison to the more standard ruptured-patch configuration. The combination of these methods, isolated somata plus perforated-patch-clamp recordings, can be used to study cellular processes that require long, stable recordings and the preservation of intracellular milieu, such as signaling through G-protein coupled receptors.

Presented here are methods providing detailed instructions for dissecting, dissociating, culturing, and patch-clamp recording from vestibular ganglion and spiral ganglion neurons of the inner ear.

Isolierung und Kultivierung von vestibulären und spiralförmigen Ganglien-Somata von neonatalen Nagetieren für Patch-Clamp-Aufnahmen

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter ...