저자는 원고들의 중요한 평가 매튜 켈리, 티지 아나 Cogliati, 제시카 Gumerson, 우베 Wolfrum, Rivka Levron, 비올라 KRETSCHMER와 조이 맨을 감사하고 싶습니다. 이 작품은 Sofja Kovalevskaya에 상 (Humbodlt 재단)와 요하네스 구텐베르크 대학, 마인츠, 독일에 의해 투자되었다.

<ol>
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The author declares no competing financial interests.

분석을 위해 인공 조직을 준비 할 때, 명심 할 몇 가지 주요 사항이 있습니다. 우선, 유전 적 배경 차이는 필요한 분석에만 한배 새끼 컨트롤과 비교하게 인공 표현형을 수정할 수있다. 둘째, tectorial 막의 완전한 제거는 면역 조직 화학 최상의 이미지를 얻기 위해 필요한 및 SEM을 위해 필수적이다. tectorial 막은 불투명 한 구조 및 이미지가 더 도전하고, 그 아래에 직접 감각 상피의 세포를 모호하게 ...

차 섬모는 대부분의 포유 동물 세포의 표면에서 연장 긴 미세 소관 기반의 부속 기관입니다. 차 섬모는 종종있는 항상 셀 당 여러가, 운동성 섬모와 혼동하고, 그 목적은 막 표면에서 유체를 이동하는 것입니다 수 있습니다. 차 섬모 대조적으로, 감각 역할을 채택하고 그 결과 또한 감각 섬모라고합니다. 일단 오래 잊고,이 세포 기관은 최근 인간의 유전 질환 1의 다수와의 연결의 결과로 &#39;재발견&#39;되고있다. 이상적 신호 소기관으로 위치 된 일차 섬모 수많은 시그널링 조직 항상성 및 질병뿐만 아니라 중요 이는 많은 경로,뿐만 아니라 개발이 과정을 조절하는 것으로 나타났다. 섬모 기능 장애와 연관되는 것으로 도시 제 1 시그널링 경로 중 하나가 평면 셀 극성 (PCP) 경로라고도 비정규 Wnt 신호 전달 경로이었다 <sup<html> &gt; 3. 처음 초파리에서 확인이 신호 캐스케이드, 배아 중요하다; 통합 및 확장 프로세스 특히와 상피 (4)의 평면에서 세포의 올바른 방향에 대해. 조절 단백질의 집합 코어의 연속 신호는 궁극적으로 세포 골격 재 배열로 이어질 및 평면 5 상피 세포의 조정 편광 될 방향 신호를 변환합니다. 융합과 확장의 과정은 절대적으로 연장하는 인공 와우 덕트과 올바른 휴대 패턴 (6)이 필요합니다. 이것은 PCP 경로의 활성화를 통해 조절된다으로 달팽이관 PCP 돌연변이의 가장 눈에 띄는 표현형 하나 무질서 감각 상피 7 단축 인공 덕트이다. 규제이 밝혀지지 남아 정확히 어떻게하지만 마찬가지로, 또한, 속눈썹이 부족 마우스 돌연변이는 이러한 통합 및 확장 표현형 8,9을 나타낸다. 통합 및 확장 프로세스가 인공 와우 덕트의 부산물 및 인공 와우 덕트 내의 감각 상피의 세포 패터닝 중요하기 때문에 ve_content는 &quot;&gt;, 개발 달팽이관은 척추 개발하는 동안 PCP 신호를 검사 할 수있는 이상적인 기관이다.의 기관 코르티, 인공 와우 덕트, 균일 (10)를 작동하는 달팽이관을 위해 지향해야 비 감각지지 세포와 mechanosensory 유모 세포로 구성되어 줄 전문 감각 상피에 주어진 용어입니다. mechanosensory 유모 세포 그렇게 때문에의 호출 cuticular 판에서 연장 stereociliary 번들 각 감각 헤어 셀 (11)의 (선 단면). mechanosensation 및 stereocilia 그들의 명칭에도 불구하고 차 트랜스 듀서로서 이러한 행위는 실제로 수정 액틴 필라멘트 기반의 미세 융모로 구성된다. 각 갈매기 모양의 머리 내 번들은, stereocilia의 세 행이 높은 주문 및 일반 PA로 구성됩니다계단 경우와 같은 방식으로 ttern. 리얼 미세 소관 기반 섬모가 되나 kinocilia는 stereociliary 번들 (12)의 발전 방향에 대해 요구된다. 각 머리 셀에, 하나의 kinocilium 물리적 stereocilia의 가장 높은 행 중앙에 인접하여 위치, stereocilia 번들에 부착된다. kinocilium의 정확한 기능은 명확하지 않다, 하나의 가설은 미세 융모 (12)로부터 성숙 kinocilium 모양으로 stereocilia를 &#39;당겨&#39;이다. 척추 동물에서, 달팽이관에 kinocilia은 일시적으로 만 존재하고 11,13,14 청각의 발병하기 전에 쥐의 유모 세포에서 후퇴. 심각하게 단축 인공 와우 덕트의 개발 달팽이관 결과에 섬모의 전체 손실, 잘못을 형성하고 stereociliary 번들뿐만 아니라 잘못된 위치에 기초 몸 8,9 잘못이 지향. 기능 섬모는 단지 섬모 axoneme 구성되어 있지 않습니다. 섬모와 관련된 많은 단백질기능은 기초 신체, 전이 영역, 또는 섬모 axoneme 15 섬모 관련 하위 도메인에 지역화 단지에서 발생합니다. 중심체의 어머니 중심 소체에서 파생 된 기저 몸은 또한 세포체로 떨어져 섬모에서 연장 미세 소관에 대한 미세 소관 조직화 센터 및 세포 내 인신 매매뿐만 아니라 섬모 인신 매매를 규제 할 수 있습니다. 섬모 전이 영역은 섬모 기능이 섬모 화합물 (16)의 가져 오기 및 내보내기를 조직의 관점에서 규제되고 또 다른 영역이다. 정확한 메커니즘은 명확하지 않지만 17 여러 연구는 섬모 및 비표준의 Wnt (PCP 신호) 사이의 연결을 확인 하였다. 섬모와 PCP 유전자의 중복과 일반 세포의 이상으로 세포 극성의 감도, 어려운 직접 PCP 별 적자에 돌연변이를 링크 할 수 있습니다. PCP 시그널링 리드 아웃 중 하나가 기부 본체와 PRIMAR의 포지셔닝y를 섬모 따라서 보조 결함 차를 분리하는 도전이다. 제브라 피쉬와 마우스 돌연변이의 일부 연구는 18 ~ 20 신호 섬모와의 Wnt 사이에 연결을 제안 없었다. 데이터의 불일치 종, 조직, 또는 Wnt 신호으로 섬모 기여금 시간에 의존하는 차이를 반영 할 수있다. 기초 몸은 기능을 유지하는 경우 또한, 정상의 Wnt 응답 성이 유지 될 수 있습니다. 세포 신호 전달 경로와 다른 생물학적 현상의 섬모 단백질의 역할에 대한 깊은 통찰력 우리 세포 및 발생 생물학 이해뿐만 아니라 표적 치료 전략의 개발이 매우 중요하다.

<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.

와우 해부 및 조직 준비 뇌의 제거 후, (그림 1A, 흰색 화살표)를 시각화 및 제거 할 수 있습니다 뒤에서 본 P0 마우스 머리, 뼈 미로, 사후 중간 라인 시상 해부.도 1b는 인공 머리를 가진 격리 된 뼈 미로를 보여줍니다 위쪽, 복부 (왼쪽)와 뒤에서 마주 지느러미 (오른쪽). 타원형 창에 흰색 ?...

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

Ciliopathy 마우스 돌연변이 달팽이관의 평면 셀 극성 표현형의 평가

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

10.3K 조회수.  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.

비디오: 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.

Electroporation에 의한 배아 마우스 달팽이의 Explants의 문화 유전자 전송

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

연구

발생학

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.

분리 LacZ를 표현 유동세포계측법를 사용하여 마우스 내이 (内耳) 조직의 세포를

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 ...

신경과학

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.

Vestibular 및 청각 감각 기관의 3 차원 Organotypic 문화

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.

전체 마운트 해부 및 성인 마우스 달팽이관의 면역 형광

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.

냉동 절제 및 면역 염색 마우스 내이 조직: 배아에서 성체 단계까지

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.

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.

성인 마우스의 달팽이관 표면 준비

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.

체외 모델로서의 전체 신생아 인공와우 이식

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.

출생 후의 쥐의 청각 수포의 해부 : 중이의 뼈와 조직 학적 분석의 분리

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 ...

생물학

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>생쥐의 내림프낭 해부

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.

생쥐의 내림프낭 해부

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