저자는 통찰력 토론과 제안의 TrkA taulacZ 마우스 박사 아네트 마르쿠스 박사 루이스 레이 차트에 감사의 말씀을 전합니다. 이 작품은 버크 재단뿐만 아니라 화이트 홀 재단 연구비 2010-08-61, 생명 재단 (WFL-US-028 / 14) 날개에서 연구 지원 사업 시작 기금에 의해 지원되었다,에서 ZB1-1102-1을 부여 크리스토퍼 &amp; 데이나 리브 재단​​과 JZ에 국립 안과 연구소의 보조금 1R01EY022409과 3R01EY022409-01S1. KJO는 골드 스미스의 동료입니다.

참고 : 모든 절차는 실험 동물의 사용 및 관리에 대한 NIH 가이드 준수합니다. 동물 프로토콜은 웨일 코넬 의과 대학에서 IACUC 승인을 받았다. 1. 조직 준비 <ol><li> 자궁 경부 전위 15 시간 초과 - 임신 여성을 안락사. 개별적으로 차가운 인산염 완충 식염수 (PBS)로 채워진 6 잘 접시의 우물에서 시간 초과 - 임신 여성과 장소 배아에서 E18 배아 - 배아 E16를 해부하다. </li><li> 차가운 PBS에서 배아를 씻어. </li><li> 후속 유전자형을위한 사전 준비의 proteinase K 용액 배아과 장소 양막의 작은 부분을 제거한다. 양막이 끊어지면 대안, 배아의 꼬리 끝의 일부를 제거하고 K 테 용액에 넣 </li><li> 모든 곤충 핀 (측면 당 적어도 100)를 이용하여 배아 주위 피부에 구멍을 찔러. </li><li> (PFA)을 신선한 2 % 파라 포름 알데히드를 추가 천천히 PBS를 제거하고우물에 pH가 7.4. </li><li> 조심스럽게 4 ° C에서 2 시간 동안 흔들어. </li><li> PBS에 두 번 배아를 씻어 염색까지 4 ° C에서 PBS에 저장합니다. </li></ol> 2. 유전자형 <ol><li> 제조업체의 지침에 따라 단백질 분해 효소 K 혼합물에 양막 또는 배아 꼬리를 담근다. </li><li> 10 분 동안 56 ° C에서 품어. </li><li> 시중에서 판매하는 DNA 정제 키트를 사용하여 DNA를 추출합니다. </li><li> 유전자 특이 센스 및 안티센스 프라이머 (10 μM)의 각각 0.5 μL, 2 μL의 dNTP 믹스와 결합, 게놈 DNA 용액 정제 총 200 μL의 0.5 μl를 타고 (dATP 존재의 2.5 mM의,의 dCTP, dGTP의 및 dTTP) 0.2 μL의 Taq 중합 효소 (5 U / μL) 및 2 ㎕의 PCR 완충액 (10 배)은 20 ㎕의 총 부피로 H 2 O를 추가한다. 참고 : 프라이머 서열은 다음과 같습니다 (표 1) 의 TrkA : TrkA_F : GCG GGC GCC GCC GCG ATG, TrkA_R : GAA GCC GCC TGC GCG GCT CTG CCA GGG TG; PCR의 파편, 표준 길이 : 400 염기쌍 (BP). 백스 : In5R : GTT GAC CAG AGT GGC GTA GG, Ex5F : TGA TCA GAA CCA TCA TG, NeoR : CCG CTT CCA TTG CTC AGC GG; PCR 단편 길이 : 야생형, BP (304); Bax의 널 (null), 507 bp의. TauLacZ : lacZup : ACA ACG TCG TGA GTG GGA AAA, lacZdown : ATC AAC ATT AAA TGT GAG CGA G; PCR 단편 길이 : 360 bp의. </li><li> 의 TrkA에 대해 다음 프로그램으로 PCR을 실행 1 단계 : 1 분 95 ° C, 2 단계에서 30 초 72 ° C : 68 ° C, 4 단계에서 20 초 : 95 ° C, 3 단계에서 10 초. 40주기 4 - 반복 2 단계를 반복합니다. <ol><li> LacZ를하고 백스를 들어, 다음 프로그램으로 PCR을 실행 1 단계 : 1 분 95 ° C에서, 2 단계 : 10 초 95 ° C, 단계에서 3 : 1 분 54 ° C, 단계 4 : 72에서 1 분 35 사이클 - 4 ° C를 반복 2 단계를 반복합니다. </li></ol></li><li> DNA 래더의 차선을 20 분 동안 1X 트리스 - 아세테이트 - EDTA 완충액에서 100 V에서 2 % 아가 로스 겔에서 실행 PCR 샘플 길이 단편을 평가. </li><li> 야생형의 TrkA, T의 존재에 대해 분석 배아RKA - tauLacZ 및 Bax의 널 (null) 대립 유전자. 백스 - / -, 실험 질문에 따라 : - - /와의 TrkA taulacZ / taulacZ 백스 : 실험 배아의 유전형의 TrkA taulacZ / WT 있습니다. tauLacZ 기자가없는 배아 축삭 염색 부정적으로 사용되므로 염색 과정을 교정하는 부정적인 컨트롤을 제공 할 수 있습니다. </li></ol> 3. 배아 염색 <ol><li> 3.8 - 섹션 3.2에서 아래와 같이 일부 수정과의 lacZ 염색 상용 키트를 사용합니다. 여기에, 밀리 포아 키트를 사용합니다. </li><li> 실온에서 부드럽게 흔들어, 적어도 1 시간 동안 버퍼의 배아를 담근다. </li><li> 직접 실온에서 부드럽게 흔들어, 적어도 15 분 동안 버퍼 B의 배아를 체험. </li><li> 배아는 버퍼 A / B, 37 ° C에서 미리 따뜻한 조직 염기 용액에있는 동안. 배아 당 조직 염기 용액 5 mL를 사용합니다. </li><li> 배아 버퍼 A / B에있는 동안, 신선한 5-BR 준비omo- 100 % 디메틸 술폭 시드 (DMSO)에 40 ㎎ / ㎖의 농도로 -3- 인돌 α-D-갈 락토 (X-GAL) -4- 클로로. </li><li> 1 mg / ml의 농도로 예열 된 티슈베이스 용액에 X-gal을 희석시키고 유리 섬광 바이알로 X-GAL / 티슈베이스 혼합물을 5 mL를 추가한다. </li><li> X-GAL / 티슈베이스를 함유하는 혼합물을 유리 섬광 바이알로 배아를 전송합니다. 파라 필름으로 뚜껑을 밀봉 파란색 얼룩의 존재를 확인, 37 ℃에서 하룻밤 배양한다. </li><li> 신선한 4 % PFA, pH가 7.4 후위 배아. 조심스럽게 4 ° C에서 4 시간 동안 흔들. </li><li> 차가운 PBS에 두 번 배아를 씻어. </li></ol> 4. 조직 탈수 및 지우기 <ol><li> 유리 바이알에서 흔들면서 실온에서 30 분 동안 50 % 메탄올 / 50 % PBS 액에 넣어 배아. </li><li> 유리 바이알에서 흔들면서 실온에서 30 분 동안 75 % 메탄올 / 25 % PBS 탈수 계속. </li><li> 90 % 탈수 계속 메탄올 / 10 % P유리 바이알에서 흔들면서 실온에서 30 분 동안 BS. </li><li> 유리 바이알에서 흔들면서 실온에서 30 분 (2 배), 100 % 메탄올 탈수 계속. </li><li> 1 (V : V) 벤조일 알코올 벤조일 벤조 에이트의 혼합물 배아 탈수하는 동안, 1을 제조. 참고 : 벤조일 알코올과 벤조일 벤조 에이트는 독성이다. </li><li> 1 : 1 배아를 담가 (V : V) 100 % 메탄올의 혼합물 : 15 분 동안 100 % BABB. BABB 플라스틱을 용해하기 때문에 만 유리를 사용합니다. </li><li> 완전히 투명 조직 100 % BABB에서 배아를 담그고 삭제 배아에서 X-여자 염색을 시각화 할 수 있습니다. 참고 : 가능한 한 빨리 X-여자의 얼룩은 시간이 지남에 따라 퇴색하기 때문에 이미지. </li></ol> 5. 전체 마운트 이미지 <ol><li> 사진 사용하여 금속 집게에 대한 적절한 각도로, 100 % BABB에 몰입 배아를 안정화. 점등 미만 광용 광원의 조합을 사용 밝히는. </li><li> 해부 현미경으로 이미지를 수집디지털 카메라로 복. 0.5 mm 떨어져 Z 축을 따라 다섯 연속 초점면에 여러 시야 노출을 가져 가라. 노출 시간과 F-정거장 조명 및 카메라 사양에 따라 다를 수 있으며 각 설정에 최적화 될 필요가있다. </li><li> 오버레이 된 영상을 가공 및 포토 몽타주를 생성하는 기준 화상 처리 소프트웨어를 사용한다. </li></ol>

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The authors declare that there are no competing interests.

배아의 TrkA taulacZ 마우스의 상술 한 X-GAL 염색 과정은 그대로 고정 배아에서 장거리 축삭 돌기 신속하고 상세한 시각화한다. 이 마우스가 축삭의 성장과 생존의 연결 모두에 기여할 수있는 메커니즘을 신호의 프로빙을 허용 백스 널 배경 때문에. 관심의 유전자 변형 또는 녹아웃 쥐와 교미하는 축삭 표현형의 종합적인 평가를 할 수 있으며 더 자세한 방식으로 축삭의 성장 신?...

정확한 신경 네트워크의 설립은 신경계의 기능에 필수적인 복잡한 발달 과정이다. 이 과정에서 장애는 인간의 신경 질환 1-3에 연루 된 신경 세포의 기능 장애로 이어집니다. 포유 동물에서 축삭 성장과 타겟 신경 분포의 기본 분자 메커니즘을 연구하기 위해, 우리는 두 가지 유전자 변형 마우스 라인의 조합을 사용하여 감각 뉴런의 TrkA는 발현의 축삭 궤적을 시각화하는 프로토콜을 개발했다. 의 TrkA는 신경 성장 인자 수용체 NGF 용이며 통각 감각 뉴런 (4)의 기능적인 마커이다. 의 TrkA 매우 초기 개발 과정에서 통각 신경 세포에서 발현 및 NGF에 의존하는 신경 세포의 생존, 축삭 성장, arborization 및 대상 신경 분포 5-9을 매개한다. 생쥐의 TrkA taulacZ에서는 야생형의 TrkA 유전자 taulacZ 식 C로 대체추정의 TrkA - 신경 세포의 축삭 형태 (11) 염색 β-여자 (X-GAL)에 의해 가시화 될 수 있도록 assette 10. 이형의 TrkA taulacZ / WT 라인을 사용하여, 우리는 규제 또는 생체 내에서 감각 구 심성 돌기의 발달을 방해 할 수있는 요소를 검사 할 수 있습니다. 더욱이 식의 TrkA 따라서 NGF / 시그널링의 TrkA 부재 메커니즘을 촉진 축삭 성장을 평가하기 위해 사용될 수 동형의 TrkA taulacZ / taulacZ 마우스에 존재하지 않는다. 통각 신경 NGF /의 TrkA뿐만 아니라 축삭 성장에 대한 시그널링에 의존하기 때문에, 또한 생존을 위해, 우리는, 그렇지 세포 죽음에서 그들을 구출 배아 DRG 신경 세포 사멸을 억제하는 것으로, 친 아폽토시스는 Bax 유전자가없는, 다른 마우스 선을 채용 시그널링의 TrkA의 부재하에 관찰 하였다. 백스 - / - (12)는 이와 같이 배경 molecu 허용특히 축삭의 성장 7-9,13-15에 영향을 미치는 신호 전달 경로의 LAR 해부. 의 TrkA에서 - / - : 백스 - / - 마우스는 DRG 뉴런은 생존하지만, 피부에 감각 구 심성 신경 분포가 완전히 (14, 15)를 폐지한다. 우리는 선택적으로 축삭 돌기의 발달에 각각의 기여를 결정하는 신호 전달 경로 활성화 할 수 있습니다. 이 방법의 유틸리티는 다른 유전 수정이의 TrkA taulacZ / taulacZ에 사육하는 경우가 축삭 성장 표현형의 변화에 대한 평가를 할 수 있다는 것입니다 백스 - / - 또는의 TrkA taulacZ / WT : 백스 - / - 배경.

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whole-mount imaging of mouse embryo sensory axon projections

배아 전장 신경 돌기의 시각화는 신경 네트워크가 개발 포유류 방법을 이해하는 것이 필수적이다. 여기에서 우리는 현장에서 여러 유전자 ​​조작 마우스 라인을 사용하여 자신의 표현형 특성을 평가하기 후근 신경절 (DRG) 축삭 돌기의 부분 집합에 레이블을하는 방법을 설명합니다. 양성 뉴런의 TrkA 통증 신호의 송신에 전용 침해 수용체 뉴런이다. 우리는 그대로 마우스 배아의 모든의 TrkA 양성 주변 축삭의 궤도에 라벨의 TrkA taulacZ 마우스 라인을 사용한다. 우리는 더 독립적으로 신경 세포의 생존에 유전자 조작의 가능한 효과의 성장과 관련된 질문을 평가하기 위해, 기본적으로 신경 세포의 사멸을 폐지 백스 널 배경 위에의 TrkA taulacZ 라인을 번식. 이어서, 관심의 유전자 변형 마우스의 TrkA taul와 교배ACZ / Bax의 널 라인은 본 발명에 개시된 기술을 사용하여 연구를위한 준비가되었다. 이 프레젠테이션은 전체 마운트 준비에 전체 길이 축삭 궤적의 시각화 수 있도록 해부, 조직 준비, 염색 및 청소시 상세한 마우스 사육 계획에 대한 자세한 내용은 유전자형이 포함되어 있습니다.

의 TrkA WT / taulacZ의 유전자형 : 백스 - / -과의 TrkA taulacZ / taulacZ : 백스 - / - 배아가 명확하게 표준 PCR 유전형에 의해 결정될 수있다 (그림 1). X-여자 염색 디스플레이는 통상적으로 염색 배아 (그림 2, 3A)에 피하 주변 축삭 아버을 자세히 설명하고, 조직 제거한 후 배아에 걸쳐 (그림 3B, 4). - / - 라인 ?...

Watch this Scientific Journal Video about Whole-mount Imaging of Mouse Embryo Sensory Axon Projections at JoVE.com

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

마우스 배아 감각 축삭 돌기 전체 마운트 이미지

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

whole-mount-imaging-of-mouse-embryo-sensory-axon-projections

Research

JoVE Journal

Neuroscience

10.0K 조회수.  Weill Medical College of Cornell University. We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

비디오: Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

여러 마우스 Neuroanatomical 자기 공명 영상

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

연구

신경과학

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

망막 안료 상피의 전체 마운트를위한 마우스 아이의 해부

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

전체 마운트 근육 분석에 의한 재현성 마우스 좌골 신경 크러시와 중생의 후속 평가

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

혈관을 조사하기 위해 신생아 마우스 망막의 전체 마운트 면역 형광 염색<em&gt; 생체 내</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

에있는 후각 수용체 뉴런의 전체 마운트 Immunolabeling<em&gt; 초파리</em&gt; 안테나

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel cell clusters), sebaceous glands, nerves and nerve endings, and capillaries. The spatial arrangement of these structures is tightly controlled on a microscopic scale - as seen, for example, in the orderly arrangement of cell types within a single hair follicle - and on a macroscopic scale - as seen by the nearly identical orientations of thousands of hair follicles within a local region of skin. Visualizing these structures without physically sectioning the skin is possible because of the 2-dimensional geometry of this organ. In this protocol, we show that mouse skin can be dissected, fixed, permeabilized, stained, and clarified as an intact two dimensional object, a flat mount. The protocol allows for easy visualization of skin structures in their entirety through the full thickness of large areas of skin by optical sectioning and reconstruction. Images of these structures can also be integrated with information about position and orientation relative to the body axes.

Mammalian skin contains a diverse array of structures - such as hair follicles and nerve endings - that exhibit distinctive patterns of spatial organization. Analyzing skin as a flat mount takes advantage of the 2-dimensional geometry of this tissue to produce full-thickness high-resolution images of skin structures.

마우스의 피부와 모낭 패터닝 및 감각 축삭 형태학의 분석에의 응용의 평면 마운트 이미징

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

이미징 CA<sup&gt; 2 +</sup마우스 망막의 광 수용체 콘 축삭 터미널에서&gt; 역학

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

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 ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

광학 일관성 단층 촬영: 마우스 망막 신경 절 세포에서 Vivo에서 화상 진 찰

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

후각 감각 축색 돌연변이 분자 식별 코드의 시각화를위한 뒷 전구의 4 배 면역 면역 측정법

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...