המחבר מבקש להודות מתיו קלי, Tiziana Cogliati, ג&#39;סיקה Gumerson, Uwe Wolfrum, רבקה לברון, ויולה קרצ&#39;מר וזואי מאן להערכה ביקורתית שלהם של כתב היד. עבודה זו מומנה על ידי בפרס Sofja Kovalevskaya (קרן Humbodlt) ואת יוהנס-גוטנברג אוניברסיטת מיינץ, גרמניה.

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The author declares no competing financial interests.

בעת הכנת רקמת שבלול לניתוח, יש כמה נקודות מפתח כדי לזכור. ראשית, בדלי רקע גנטי יכולים לשנות את הפנוטיפ השבלול, מה שהופך אותו הצורך לנתח ולהשוות שולט להמלטה בלבד. שנית, הסרה מלאה של קרום tectorial נדרש לקבל את התמונות הטובות ביותר עם אימונוהיסטוכימיה והוא חיוני SEM. קרום tectorial...

cilia ראשי הם נספחי microtubule מבוסס ארוכים שמרחיבים מפני שטח של רוב בתאי יונקים. cilia הראשי מבולבלים לעתים קרובות עם cilia ניע, אשר יש תמיד מרובים לכל תא, ואשר מטרתו הוא להעביר נוזל על פני משטחים הממברנה. cilia היסודי, לעומת זאת, לאמץ תפקידים חושיים ובשל כך הן גם התייחסו cilia החושית כמו. לאחר שאבד עליהם כלח, אברון זה כבר &quot;מחדש&quot; לאחרונה כתוצאה ההתאגדות שלה עם שפע של מחלות גנטיות האדם 1. באופן אידיאלי מיקומו בתור אברון איתות, את cilium העיקרי הוכח לווסת מסלולים רבים איתות, שרבים מהם חשובים לא רק הומאוסטזיס רקמה ומחלות, אלא גם במהלך הפיתוח 2. אחד מסלולי איתות הראשונה הראו להיות הקשורות בתפקוד cilia היה מסלול איתות Wnt קאנונית, הידוע גם בשם הקוטביות בתא מישוריים (PCP) מסלול <sup<html> &gt; 3. מפל איתות זה בתחילה זיהה תסיסנית, הוא קריטי עבור עובר; בפרט עבור תהליכי התכנסות ארכה את הכיוון הנכון של תאי המטוס של אפיתלים 4. את האיתות הרציפה של גרעין המוגדר של חלבונים רגולטוריים מתרגם רמזים כיוונית אשר בסופו של הדבר להוביל rearrangements cytoskeletal ולגרום הקיטוב המתואם של תאי אפיתל במטוס 5. תהליך ההתכנסות והסיומת הוא דרוש לחלוטין את צינור השבלול להאריך ועבור דפוסים הסלולר נכונים 6. כמו זה מוסדר באמצעות שפעול מסלול PCP, אחד פנוטיפים הבולטים ביותר של מוטציות שבלול PCP הוא צינור שבלול יקוצר epithelia החושית מאורגנת 7. באופן דומה, מוטנטים עכבר, אשר חסר cilia, גם תערוכה כגון פנוטיפ התכנסות ורחב 8,9, למרות בדיוק איך זה מוסדר נותר הובהר. ve_content &quot;&gt; מכיוון תהליכי התכנסות והרחבה הם קריטיים עבור התולדה של צינור השבלול, דפוסים הסלולר של epithelia החושית בתוך צינור השבלול, השבלול המתפתח הוא איבר אידיאלי שבו לבחון איתות PCP במהלך ההתפתחות החולייתנים. הביטאון קורטי, השם שניתן האפיתל החושית המיוחדת המרפדת את צינור השבלול, מורכבת ותאי תומכים שאינם חושיים ותאי שיער mechanosensory אשר חייב להיות מכוונות באופן אחיד עבור השבלול לתפקד 10. תאי שיער mechanosensory נקראים כך בגלל חבילות stereociliary שמרחיבות מצלחת cuticular (למשטח פסגה) של כל תא שיער חושי 11. אלה משמשות מתמרים עיקריים של mechanosensation ולמרות המינוח שלהם כמו stereocilia, מורכבות למעשה של microvilli מבוסס נימה תקטין שונה. בתוך כל שיער בצורת שברון צרור, שלוש שורות של stereocilia מאורגנים רשות הורה מאוד ורגילttern באופן במקרה דמוי מדרג. Cilia microtubule מבוססי ריאל, כינה kinocilia, נדרשים לפיתוח והכיוון של חבילות stereociliary 12. עם כל תא שיער, kinocilium אחת מחוברת פיזית את צרור stereocilia, הממוקמת בסמוך מרכזי לשורה הגבוהה של stereocilia. הפונקציה מדויק של kinocilium אינה ברורה, אחת ההשערות היא כי kinocilium &#39;מושך&#39; את stereocilia לכושר כשהם מתבגרים מן microvilli 12. בבעלי החוליות, kinocilia בשבלול נוכח רק זמנים לסגת מתאי השיער בעכברים לפני תחילת שמיעת 11,13,14. אובדן מוחלט של השיער בתוצאות שבלול המתפתח צינוריות שבלול מקוצר קשות, יצרו אי ו MIS מוכווני חבילות stereociliary, וכן גופים הבסיס ממוקם mis 8,9. Cilium תפקודית אינה מורכבת רק של axoneme ריסי. חלבונים רבים הקשורים ciliaפונקציה להתרחש מתחמים מקומיים תחומיים משנה הקשורות cilia כמו גוף בסיסי, אזור מעבר, או ריסי axoneme 15. הגוף הבסיסי, נגזר אמא צנטריול של centrosome, היא גם המרכז מתארגן microtubule עבור microtubules הארכה הרחק cilium לתוך גוף תא יכול לווסת סחר תאי וכן סחר ריסים. אזור מעבר הריסים הוא אזור אחר שבו פונקצית ריסי מוסדר מבחינת ארגון יבוא ויצוא של תרכובות ריסי 16. מחקרים רבים זיהו קשר בין הריסים ו קאנונית Wnt (איתות PCP), אם כי המנגנון המדויק אינו ברור 17. יתירות של גני ריסים ו PCP ואת הרגישות של קוטביות בתא לאי תקינות הסלולר כללית, להקשות לקשר מוטציה ישירות גירעונות ספציפיים PCP. אחד פסקי לקריאה של איתות PCP הוא המיקום של הגוף הבסיסי ו primarcilium y, ולכן ההפרדה העיקרית מן הפגמים המשניים הם מאתגר. כמה מחקרים מוטנטים דג הזברה והעכבר הציעו שום קשר בין הריסים ו Wnt איתות 18-20. אי-התאמות בנתונים עשויות לשקף מינים, רקמות, או בדלים זמניים תלויים תרומות ריסים לקראת איתות Wnt. יתר על כן, היענות Wnt רגיל עלולים להיעזר במידת גופים הבזליים להישאר פונקציונליים. תובנה עמוקה יותר לתוך התפקיד של חלבונים ריסי במסלולי איתות הסלולר תופעות ביולוגיות אחרות חיונית להבנת ביולוגיה תאית והתפתחותית, וכן לפיתוח אסטרטגיות טיפול ממוקד.

<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.

שבלול Dissection והכן רקמות לאחר הסרת המוח, דיסקציה sagittal באמצע הקו לתפקיד ראש עכבר P0, המבוך הגרמי, במבט מאחור, ניתן דמיינו (איור 1A, חץ לבן) והוסר. איור 1B מציגה את המבוכים גרמ...

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

הערכת פנוטיפים מישורים-Cell-קוטביות ב Ciliopathy עכבר Mutant שבלול

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

10.3K Views.  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.

Video: 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.

תרבות של עוברי Explants שבלול העכבר וג&#39;ין העברה על ידי Electroporation

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

Developmental Biology

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.

בידוד LacZ-להביע תאים פנימית עכבר רקמות האוזן באמצעות זרימת 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 ...

Neuroscience

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

לטווח ארוך זמן פקיעת הדמיה של Explants שבלול העכבר

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

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.

ההר שלם Dissection וImmunofluorescence של מבוגרי עכבר השבלול

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.

הקפאה וצביעה חיסונית של רקמת האוזן הפנימית של עכבר: משלב עוברי לשלב בוגר

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.

מורפולוגית ופונקציונלית הערכה של הסרט סינפסות בתדר ספציפי אזורים של העכבר שבלול

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.

הכנה לפני שבלול בעכבר למבוגרים

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.

צמחי שבלול שלמים של ילודים כמודל במבחנה

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.

דיסקציה של בולה השמיעה של עכברים אחרי לידה: בידוד של עצמות באוזן התיכונה ניתוח היסטולוגית

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 ...

Biology

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>נתיחת השק האנדולימפטי מעכברים

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.

נתיחת השק האנדולימפטי מעכברים

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