Name: evaluation of planar-cell-polarity phenotypes in ciliopathy mouse mutant cochlea
Uploaded: 2016-02-21T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Evaluation of Planar-Cell-Polarity Phenotypes in Ciliopathy Mouse Mutant Cochlea at JoVE.com

Der Autor möchte Matthew Kelley, Tiziana Cogliati, Jessica Gumerson, Uwe Wolfrum, Rivka Levron, Viola Kretschmer und Zoe Mann und für die kritische Bewertung des Manuskriptes. Diese Arbeit wurde durch den Sofja Kovalevskaja-Preis (Humbodlt Foundation) und der Johannes-Gutenberg-Universität, Mainz, Deutschland finanziert.

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The author declares no competing financial interests.

Bei Cochlea-Gewebe für die Analyse vorzubereiten, gibt es ein paar wichtige Punkte zu beachten. Zum einen Hintergrund in der genetischen Unterschiede können die Cochlea-Phänotyp zu ändern, ist es notwendig zu analysieren und zu vergleichen, nur littermate Kontrollen machen. Zweitens vollständige Entfernung des Deckmembran ist erforderlich, um die besten Bilder mit Immunhistochemie zu erhalten, und ist wesentlich für SEM. Die Deckmembran ist eine undurchsichtige Struktur und kann die Zellen der Sinnesepithel direkt...

Primäre Zilien sind lang Mikrotubuli-basierte Fortsätze, die von der Oberfläche der meisten Säugerzellen erstrecken. Primäre Zilien sind oft mit motile Zilien, von denen verwechselt gibt es immer mehrere pro Zelle, und deren Zweck es ist Fluids über Membranoberflächen zu bewegen. Primäre Zilien, dagegen annehmen sensorischen Rollen und sind somit auch als Sinneshärchen bezeichnet. Nachdem lange vergessen hat diese Organellen vor kurzem als Ergebnis seiner Verbindung mit einer Vielzahl von menschlichen genetischen Erkrankungen ein &quot;wiederentdeckt&quot; worden. Idealerweise als Signalorganelle angeordnet ist, hat der primäre cilium wurden zahlreiche Signalwege gezeigt zu regeln, von denen viele nicht nur wichtig in Gewebshomöostase und Krankheit, sondern auch während der Entwicklung 2. Einer der ersten Signalwege gezeigt mit Zilien Dysfunktion assoziiert zu sein war der nicht-kanonischen Wnt-Signalwegs, auch bekannt als die planare Zellpolarität (PCP) Weg <sup<html> &gt; 3. Diese Signalkaskade zunächst in Drosophila identifiziert, ist entscheidend für die Embryogenese; insbesondere zur Konvergenz und Ausdehnung Prozesse und für die korrekte Ausrichtung der Zellen in der Ebene der Epithelien 4. Die sequentielle Signalisierung einer Kernsatz von regulatorischen Proteinen übersetzt Richtungshinweise, die letztlich zu Zytoskelett-Umlagerungen führen und 5 in die koordinierte Polarisation von Epithelzellen in einer Ebene zur Folge haben. Der Prozess der Annäherung und Erweiterung ist absolut für den Schneckengang erforderlich zu verlängern und für die korrekte zelluläre Muster 6. Da dies über die Aktivierung des PCP Wegs reguliert wird, eines der markantesten Phänotypen der Mutanten Cochlea PCP ist eine verkürzte Schneckengang mit desorganisiert Sinnesepithelien 7. Ebenso Mausmutanten, die Wimpern fehlen, zeigen auch eine solche Konvergenz und Erweiterung Phänotyp 8,9, obwohl genau, wie dies geregelt ist noch nicht geklärt werden. ve_content &quot;&gt; Da Prozesse Konvergenz und Erweiterung entscheidend sind für das Auswachsen der Schneckengang und Mobil Strukturierung der Sinnesepithelien im Schneckengang, die Entwicklung von Cochlea ist eine ideale Organ, in dem PCP-Signalisierung während der Wirbeltierentwicklung zu untersuchen. Die Orgel von Corti, der Begriff der Fach Sinnesepithel gegeben, die Linien der Schneckengang, der nicht-Sinnesstützzellen und mechanosensorischen Haarzellen besteht, die einheitlich für die Schnecke ausgerichtet sein muss 10 zu funktionieren. die mechanosensorischen Haarzellen sind so, weil die angerufene stereociliary Bündel, die von der Kutikula Platte (Spitzenfläche) jedes Haarsinneszelle 11. Diese dienen als Primärwandler Mechanosensation und trotz ihrer Nomenklatur als Stereozilien verlängern, sind tatsächlich von modifizierten Aktin-Filament-basierte Mikrovilli umfasste. Innerhalb jeder winkelförmige Haar Bündel, drei Reihen von Stereozilien sind in einem hoch geordneten und regelmäßigen pa organisiertttern in einem Treppenhaus artig. Echt Mikrotubuli-basierte Zilien, bezeichnet Kinozilien, werden für die Entwicklung und die Ausrichtung der stereociliary Bündel 12 erforderlich. Bei jeder Haarzelle wird eine Einzel Kinozilium physisch an den Stereozilien Bündel befestigt, befindet sich auf dem höchsten Reihe von Stereozilien zentral nebeneinander. Die genaue Funktion des Kinozilium ist unklar, und eine Hypothese ist, dass die Kinozilium die Stereozilien in Form &quot;zieht&quot;, wie sie von Mikrovilli 12 reifen. Bei Wirbeltieren sind Kinozilien in der Cochlea vorhanden nur vorübergehend und von den Haarzellen in Mäusen zurückzuziehen vor dem Einsetzen 11,13,14 hören. Vollständiger Verlust von Zilien in den Entwicklungs Cochlea führt zu stark verkürzt Cochlea Kanäle, mis geformt und mis orientierten stereociliary Bündel sowie Fehl positioniert Basalkörpern 8,9. Eine funktionelle Cilium ist nicht nur der Ciliar- Axonem umfasste. Viele Proteine ​​mit Zilien verbundenFunktion kommen in Komplexen lokalisiert Zilien bezogene Sub-Domains wie beispielsweise die Basaltemperatur, Übergangszone oder Ciliar- Axonem 15. Der Grundkörper, von der Mutter centriole der Zentrosom abgeleitet, ist auch ein Mikrotubuli-Organisationszentrum für Mikrotubuli aus dem Flimmer in dem Zellkörper weg erstreckt und intrazellulären Transport sowie Ciliar- Handel regulieren. Die Ciliar- Übergangszone ist ein weiterer Bereich, in dem Ziliarfunktion in Bezug auf 16 der Organisation von Import und Export von Ciliar- Verbindungen reguliert wird. Mehrere Studien haben einen Zusammenhang zwischen Wimpern und nicht-kanonische Wnt (PCP-Signalisierung) identifiziert, obwohl der genaue Mechanismus 17 ist unklar. Redundanz von Ciliar- und PCP-Gene und die Empfindlichkeit der Zellpolarität zu verallgemeinern Zellanomalien, machen es schwierig, direkt eine Mutation an PCP spezifische Defizite zu verknüpfen. Einer der Lese outs von PCP-Signalisierung ist die Positionierung der Basaltemperatur und primary Flimmer, damit die primären von den sekundären Defekte Trennung ist eine Herausforderung. Einige Studien im Zebrafisch und Maus-Mutanten haben keinen Zusammenhang zwischen Wimpern und Wnt-Signal 18-20 vorgeschlagen. Abweichungen in den Daten zurückgelegt werden Spezies, Gewebe oder zeitliche abhängige Unterschiede in Ciliar- Beiträge an Wnt-Signal. Darüber hinaus könnten normale Wnt Reaktionsfähigkeit beibehalten werden, wenn Basalkörpern funktionsfähig bleiben. Ein tiefer Einblick in die Rolle von Ciliar- Proteinen in zellulären Signalwegen und andere biologische Phänomene ist von entscheidender Bedeutung für das Verständnis der zellulären und Entwicklungsbiologie, sowie für die Entwicklung von zielgerichteten Behandlungsstrategien.

<table><tbody><tr><td>Tools/Equipment</td><td></td><td></td><td></td></tr><tr><td>Silicone elastomere - Sylgard 184</td><td>Sigma-Aldrich</td><td>761028-5EA</td><td>See Note 2</td></tr><tr><td>Micro dissecting scissors-straight blade</td><td>Various</td><td></td><td></td></tr><tr><td>Fine forceps (no. 5 and 55) and blunt forceps</td><td>Various</td><td></td><td></td></tr><tr><td>Dissecting microscope.</td><td>Various</td><td></td><td></td></tr><tr><td>Uncoated glass microscope slides</td><td>Various</td><td></td><td></td></tr><tr><td>Microscope cover slips (22 mm &amp;times; 40 mm &amp;times; 0.15 mm)</td><td>Various</td><td></td><td></td></tr><tr><td>Transfer pipettes</td><td>Various</td><td></td><td></td></tr><tr><td>Minutien pins</td><td>&amp;nbsp;Fine Science Tools</td><td>26002-10</td><td></td></tr><tr><td>SEM sample holder</td><td>tousimis</td><td>8762</td><td></td></tr><tr><td>Scanning electron microscopy studs</td><td>TED PELLA</td><td>16111</td><td></td></tr><tr><td>PELCO Tabs: Carbon adhesive</td><td>TED PELLA</td><td>16084-3</td><td></td></tr><tr><td>Fluorescent Microscope</td><td>Various</td><td></td><td></td></tr><tr><td>Critical Point Dryer</td><td>Various</td><td></td><td></td></tr><tr><td>Scanning Electron Microscope</td><td>Various</td><td></td><td></td></tr><tr><td>Glass microscope slides</td><td>Various</td><td></td><td></td></tr><tr><td>Glass coverslips</td><td>Various</td><td></td><td></td></tr><tr><td>Kimwipe Tissue&amp;nbsp;</td><td>Various</td><td></td><td></td></tr><tr><td>Fine Paint Brush</td><td></td><td></td><td></td></tr><tr><td>Reagents</td><td></td><td></td><td></td></tr><tr><td>1&amp;times; Phosphate buffered saline (PBS)</td><td>Gibco/Life Technologies</td><td>10010023</td><td></td></tr><tr><td>Paraformaldehyde&amp;nbsp; (PFA) (EM Grade Required for EM)</td><td>Various</td><td></td><td>Prepare a 4% solution in 1&amp;times; PBS made fresh each time. EM Grade Required for EM.</td></tr><tr><td>2.5% Glutaraldehyde Grade1</td><td>Sigma-Aldrich</td><td>G5882</td><td></td></tr><tr><td>Tris-HCl (pH 7.5)</td><td>Various</td><td></td><td></td></tr><tr><td>NaCl</td><td>Various</td><td></td><td></td></tr><tr><td>CaCl 2</td><td>Various</td><td></td><td></td></tr><tr><td>Triton X-100</td><td>Various</td><td></td><td></td></tr><tr><td>Normal Goat Serum</td><td>Various</td><td></td><td></td></tr><tr><td>AffiniPure Fab Fragment Donkey Anti-Mouse IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>715-007-003</td><td></td></tr><tr><td>Fluoromount-G Mounting media</td><td>SouthernBiotech</td><td>0100-01</td><td></td></tr><tr><td>10&amp;times; Hanks&amp;rsquo; Balanced Salt Solution (HBSS)</td><td>Gibco/Life Technologies</td><td>14065</td><td></td></tr><tr><td>Hepes</td><td>Gibco/Life Technologies</td><td>15630-080</td><td></td></tr><tr><td>Osmium tetroxide (OsO4 )</td><td>Sigma-Aldrich/Fluka Analytical</td><td>75632</td><td></td></tr><tr><td>Tannic acid</td><td>Sigma-Aldrich</td><td>403040</td><td></td></tr><tr><td>Ethanol 200 proof</td><td>Various</td><td></td><td></td></tr><tr><td>Antibodies</td><td></td><td></td><td></td></tr><tr><td>anti Arl13b</td><td>Protein Tech</td><td>17711-1-AP</td><td>Suggested concentration 1:1,000</td></tr><tr><td>anti acetylated tubulin (611-B1)</td><td>Sigma-Aldrich</td><td>T6793</td><td>Suggested concentration 1:800</td></tr><tr><td>anti gamma tubulin (GTU-88)</td><td>Sigma-Aldrich</td><td>T6557</td><td>Suggested concentration 1:200</td></tr><tr><td>anti Zo_1&amp;nbsp;</td><td>Invitrogen</td><td>40-2300</td><td>Suggested concentration 1:500</td></tr><tr><td>Myosin VI</td><td>Proteus Biosciences</td><td>25-6791</td><td>Suggested concentration 1:1000</td></tr><tr><td>Myosin VIIa</td><td>Proteus Biosciences</td><td>25-6790</td><td>Suggested concentration 1:1,000</td></tr><tr><td>anti Vangl2</td><td>Merk Millipore</td><td>ABN373</td><td>Suggested concentration 1:250</td></tr><tr><td>anti G&amp;alpha;i3</td><td>Sigma-Aldrich</td><td>G4040</td><td>Suggested concentration 1:250</td></tr><tr><td>Alexa Fluor&amp;reg; 488 Phalloidin</td><td>Invitrogen/Life Technologies</td><td>A12379</td><td>Suggested concentration 1:300 - 1,000</td></tr><tr><td>Alexa Fluor&amp;reg; 568 Phalloidin</td><td>Invitrogen/Life Technologies</td><td>A12380</td><td>Suggested concentration 1:300 - 1,000</td></tr></tbody></table>

evaluation of planar-cell-polarity phenotypes in ciliopathy mouse mutant cochlea

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest signaling pathways shown to be associated with ciliary function was the non-canonical Wnt signaling pathway, also referred to as planar cell polarity (PCP) signaling. One of the best places in which to study the effects of planar cell polarity (PCP) signaling during vertebrate development is the mammalian cochlea. PCP signaling disruption in the mouse cochlea disrupts cochlear outgrowth, cellular patterning and hair cell orientation, all of which are affected by cilia dysfunction. The goal of this protocol is to describe the analysis of PCP signaling in the developing mammalian cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy. Defects in convergence and extension are manifested as a shortening of the cochlear duct and/or changes in cellular patterning, which can be quantified following dissection from developing mouse mutants. Changes in stereociliary bundle orientation and kinocilia length or positioning can be observed and quantitated using either immunofluorescence or scanning electron microscopy (SEM). A deeper insight into the role of ciliary proteins in cellular signaling pathways and other biological phenomena is crucial for our understanding of cellular and developmental biology, as well as for the development of targeted treatment strategies.

Cochlea Dissection und Gewebepräparation Nach der Entfernung des Gehirns, eine Anzeige Mittellinie sagittale Sektion eines P0 Mauskopf, der knöchernen Labyrinth, von hinten gesehen, kann sichtbar gemacht werden (Abbildung 1A, weißer Pfeil) und entfernt. 1B zeigt die isolierten knöchernen Labyrinthe mit dem Cochlea-Haar nach oben, Bauch, (links) und von hinten, Rücken (rec...

Watch this Scientific Journal Video about Evaluation of Planar-Cell-Polarity Phenotypes in Ciliopathy Mouse Mutant Cochlea at JoVE.com

Evaluation of Planar-Cell-Polarity Phenotypes in Ciliopathy Mouse Mutant Cochlea

Bewertung der Planar-Cell-Polarity Phänotypen in Ziliopathie Maus-Mutanten-Cochlea

Primary cilia influence various signaling pathways. The mammalian cochlea is ideal for examining planar cell polarity (PCP) signaling. Cilia dysfunction affects cochlear outgrowth, cellular patterning and hair cell orientation, readouts of PCP. Our goal is to analyze PCP signaling in mouse cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy.

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest ...

evaluation-planar-cell-polarity-phenotypes-ciliopathy-mouse-mutant

Research

JoVE Journal

Medicine

10.4K Aufrufe.  Johannes Gutenberg-Universität Mainz. Primary cilia influence various signaling pathways. The mammalian cochlea is ideal for examining planar cell polarity (PCP) signaling. Cilia dysfunction affects cochlear outgrowth, cellular patterning and hair cell orientation, readouts of PCP. Our goal is to analyze PCP signaling in mouse cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy.

Serie: Evaluation of Planar-Cell-Polarity Phenotypes in Ciliopathy Mouse Mutant Cochlea

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

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

Isolating LacZ-exprimierenden Zellen aus Maus Innenohrgeweben mittels Durchflusszytometrie

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

Neurowissenschaften

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

Dreidimensionale organotypischen Kulturen der vestibulären und akustische Sinnesorgane

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

Ganze Berge Dissection und Immunfluoreszenz der erwachsenen Maus Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Cryosectioning and Immunostaining Mouse Inner Ear Tissue: From Embryonic to Adult Stages

This protocol details general histology methods for preparing inner ear samples from embryonic, neonatal, and adult mice. The purpose of this protocol is to provide a straightforward and standardized method for inner ear tissue processing for researchers, possibly new to the field, interested in working with the mouse cochlea. Included here are protocols for dissection, fixation, embedding, cryosectioning, and immunostaining. Section immunostaining is one of the best methods for examining individual cells within the context of the entire cochlea.
Key steps include dissecting the inner ear away from the skull, fixing the tissue using 4% paraformaldehyde, embedding with Optimal Cutting Temperature compound, cryosectioning, and immunostaining with antibodies targeting specific proteins expressed by the cochlea.
Included in our methods are special considerations made for the cochlea given its 
shape and structure.&#160;Reasonably good focus and dissection skills are needed, as tissue damage can affect the quality of immunostaining and consistency among samples. However, with the procedures we outline, most users can be trained in a few weeks to make beautiful preparations. Overall, this protocol offers a valuable way to promote research to understand auditory development, function, and disease in mouse models.

This protocol, intended for novice users of mouse inner ear tissue, comprehensively details steps for processing mouse inner ears at different developmental stages for section immunostaining.

Kryosektion und Immunfärbung von Innenohrgewebe von Mäusen: vom Embryonalstadium bis zum Erwachsenenstadium

This protocol details general histology methods for preparing inner ear samples from embryonic, neonatal, and adult mice. The purpose of this protocol is ...

Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have indicated that hair cell synapses may be the initial targets in sensorineural hearing loss (SNHL). Such studies have proposed the concept of cochlear &#34;synaptopathy&#34;, which refers to alterations in ribbon synapse number, structure, or function that result in abnormal synaptic transmission between IHCs and SGNs. While cochlear synaptopathy is irreversible, it does not affect the hearing threshold. In noise-induced experimental models, restricted damage to IHC synapses in select frequency regions is employed to identify the environmental factors that specifically cause synaptopathy, as well as the physiological consequences of disturbing this inner ear circuit. Here, we present a protocol for analyzing cochlear synaptic morphology and function at a specific frequency region in adult mice. In this protocol, cochlear localization of specific frequency regions is performed using place-frequency maps in conjunction with cochleogram data, following which the morphological characteristics of ribbon synapses are evaluated via synaptic immunostaining. The functional status of ribbon synapses is then determined based on the amplitudes of auditory brainstem response (ABR) wave I. The present report demonstrates that this approach can be used to deepen our understanding of the pathogenesis and mechanisms of synaptic dysfunction in the cochlea, which may aid in the development of novel therapeutic interventions.

This manuscript describes an experimental protocol for evaluating the morphological characteristics and functional status of ribbon synapses in normal mice. The present model is also suitable for noise-induced and age-related cochlear synaptopathy-restricted models. The correlative results of previous mouse studies are also discussed.

Morphologische und funktionelle Bewertung von Bandsynapsen an bestimmten Frequenzregionen der Maus cochlea

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have ...

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

Cochlea-Oberflächenvorbereitung in der Erwachsenenmaus

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...

Whole Neonatal Cochlear Explants as an In vitro Model

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is characterized by the cumulative and irreversible loss of sensory hair cells and auditory nerves in the cochlea. Entire and vital cochlear explants are one of the fundamental tools in hearing research to detect hair cell loss and to characterize the molecular mechanisms of the inner ear cells. Many years ago, a protocol for neonatal cochlear isolation was developed, and although it has been modified over time, it still holds potential for improvement. 
This paper presents an optimized protocol for isolating and culturing whole neonatal cochlear explants in multi-well culture chambers that enables the study of hair cells and spiral ganglion neuron cells along the entire length of the cochlea. The protocol was tested using cochlear explants from mice and rats. Healthy cochlear explants were obtained to study the interaction between hair cells, spiral ganglion neuron cells, and the surrounding supporting cells. 
One of the main advantages of this method is that it simplifies the organ culture steps without compromising the quality of the explants. All three turns of the organ of Corti are attached to the bottom of the chamber, which facilitates in vitro experiments and the comprehensive analysis of the explants. We provide some examples of cochlear images from different experiments with live and fixed explants, demonstrating that the explants retain their structure despite exposure to ototoxic drugs. This optimized protocol can be widely used for the integrative analysis of the mammalian cochlea.

Whole Neonatal Cochlear Explants as an In vitro Model

The current protocol updates previous protocols and incorporates relatively straightforward approaches for culturing high-quality cochlear explants. This provides reliable data acquisition and high-resolution imaging in live and fixed cells. This protocol supports the ongoing trend of studying inner ear cells.

Ganze neonatale Cochlea-Explantate als In-vitro-Modell

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called the auditory bulla houses the ossicles, whereas the auditory capsule encloses the inner ear, namely the cochlea and semicircular canals. Murine ossicles are essential for hearing and thus of great interest to researchers in the field of otolaryngology, but their metabolism, development, and evolution are highly relevant to other fields. Altered bone metabolism can affect hearing function in adult mice, and various gene-deficient mice show changes in morphogenesis of auditory ossicles in utero. Although murine auditory ossicles are tiny, their manipulation is feasible if one understands their anatomical orientation and 3D structure. Here, we describe how to dissect the auditory bulla and capsule of postnatal mice and then isolate individual ossicles by removing part of the bulla. We also discuss how to embed the bulla and capsule in different orientations to generate paraffin or frozen sections suitable for preparation of longitudinal, horizontal, or frontal sections of the malleus. Finally, we enumerate anatomical differences between mouse and human auditory ossicles. These methods would be useful in analyzing pathological, developmental and evolutionary aspects of auditory ossicles and the middle ear in mice.

We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis.

Dissektion der Auditory Bulla in postnatale Mäuse: Die Isolierung der Gehörknöchelchen und histologische Analyse

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...

Biologie

Dissection of the Endolymphatic Sac from Mice

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the human phenotypes. Although important progress has been done in the understanding of the development and function of the neurosensory epithelia of the cochlea and vestibula, limited knowledge is available regarding the development, cellular composition, molecular pathways and functional characteristics of the endolymphatic sac. This is, in large part, due to the difficulty of visualizing and microdissecting this tissue, which is an epithelium comprised of only one cell layer. The study presented here describes an approach to access and microdissect the endolymphatic sac from the wild-type mouse inner ear at different ages. The result of a similar dissection is shown in a pendrin-deficient mouse model of enlargement of the vestibular aqueduct. A transgenic mouse with a fluorescent endolymphatic sac is presented. This reporter mouse can be used to readily visualize the endolymphatic sac with limited dissection and determine its size. It can also be used as an educational tool to teach how to dissect the endolymphatic sac. These dissection procedures should facilitate further characterization of this understudied part of the inner ear.

<meta charset="utf8"></head><body>Dissektion des endolymphatischen Sacks von Mäusen

This study describes how to dissect the endolymphatic sac from the inner ear of the mouse at different ages. The result of a similar dissection is shown in an Slc26a4-null mouse model of enlargement of the endolymphatic sac. A transgenic mouse with a fluorescent reporter expressed in the endolymphatic sac is presented as a model to readily visualize the endolymphatic sac, and educational tool.

Dissektion des endolymphatischen Sacks von Mäusen

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the ...