우리는 기꺼이 스크립트에 박사 존 헤이즈 감사, Drs. 에릭 브레 디하고 공 촛점 현미경과 지원 크리스토퍼 델 흑인, 드류 휴즈, 프로젝트를 개발하고 예비 데이터를 제공에서 그의 작품을위한 로라 Odorizzi, 알렉스 Garafalo, 레베카 Lowden, 그리고 리즈 MacMurray, 통계 분석에 대한 유용한 제안 박사 그렉 스미스. 이 작품은 MSS에 대한 NIH 보조금 (NINDS IR15N5067566-01)와 윌리엄과 메리의 대학에 하워드 휴즈 의학 연구소 과학 교육 교부금에 의해 지원되었다.

관심 없음 충돌이 선언 없습니다.

모든 vertebrates에서 보존 아르는 잘 특징 세포 유형으로, 망막은 셀 타입 사양과 차별화에 관한 분자 - 세포 프로세스를 공부에 유용한 모델을 제공합니다. 기본 세포 배양은 유전자 발현, 단백질 역학, 그리고 칼슘 및 단일 셀 해상도 수준의 전기 활동을 포함한 과정의 광범위한 조사를위한 강력한 방법을 갖게. 여기에 우리가 추정 망막 개발하고 기본 세포 배양의 매우 초기 단계에서 쉽게 액세스 ...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td></td></tr><tr><td></td><td></td><td></td><td> Dissections 및 배양을위한 </td></tr><tr><td> BD 팔콘 쉬운 그립 조직 문화 요리, 35mm </td><td> 어부 </td><td> 08-772A </td><td></td></tr><tr><td> 일회용 폴리스티렌 페트리 요리, 60 X 15mm </td><td> 어부 </td><td> 0875713A </td><td></td></tr><tr><td> 35mm Nunclon 표면 배양 접시 (Airvent 포함) </td><td> 어부 </td><td> 12-565-91 </td><td></td></tr><tr><td> 뒤몽 좋은 집게 (Dumostar # 55) </td><td> 어부 </td><td> NC9341917 </td><td></td></tr><tr><td> Cellattice 마이크로 지배 플라스틱 coverslip, 25mm </td><td> 어부 </td><td> 50-313-17 </td><td></td></tr><tr><td> 에틸 m-Aminobenzoate Methanesulfonate 소금 (MS-222) </td><td> MP Biomedicals, LLC </td><td> 103106 </td><td></td></tr><tr><td> Gentamicin 황산 </td><td> 엔조 생명 과학 </td><td> 380-003 - G025 </td><td></td></tr><tr><td> Gibco 트립신 (1:250) 파우더 </td><td> 생명 기술 </td><td> 27250-018 </td><td></td></tr><tr><td> 클로스 트리 디움 histolyticum에서 Collagenase B </td><td> 로슈 </td><td> 11088831001 </td><td></td></tr><tr><td> 페니실린 - 스트렙토 마이신 </td><td> Gibco </td><td> 15140-122 </td><td></td></tr><tr><td> 나일 블루 황산 (선택 사항) </td><td> Pfaltz &amp; 바우어 </td><td> N05550 </td><td></td></tr><tr><td> 500 ML 진공 필터 / 저장 병 시스템, 0.22 μm 기공 33.2 cm 두 CN 막 </td><td> 코닝 </td><td> 430758 </td><td></td></tr><tr><td></td><td> &lt;/ TD&gt; <td></td><td> 칼슘 이미징을위한 </td></tr><tr><td> Fluo-4는 DMSO의 1 ㎜ 솔루션, 영구 세포 AM </td><td> 생명 기술 </td><td> F-14217 </td><td></td></tr><tr><td> 물에 Pluronic F-127 10 % 솔루션 </td><td> 생명 기술 </td><td> P-6866 </td><td></td></tr><tr><td> LSM 510 공 촛점 현미경 시스템 </td><td> Zeiss </td><td> 모델 중단 </td><td></td></tr><tr><td> 시약 차단 </td><td> 로슈 </td><td> 11096176001 </td><td></td></tr><tr><td></td><td></td><td></td><td> 현장 하이브리드 화에 형광등 (물고기)의 경우 </td></tr><tr><td> 안티 Digoxigenin-POD, 팹 조각 </td><td> 로슈 </td><td> 11207733910 </td><td></td></tr><tr><td> 안티 - DNP-HRP 항체 </td><td> Perkin-엘머 </td><td> NEL747A </td><td></td></tr><tr><td> Cy3NHS 에스테르 </td><td> GE 헬스 케어 </td><td> PA13101 </td><td></td></tr><tr><td> NHS-Fluorescein </td><td> 열 과학 </td><td> 46,409 </td><td></td></tr><tr><td> 포름 아미드, Deionized </td><td> Amresco </td><td> 0606-950ML </td><td></td></tr><tr><td> Torula RNA 타입 IX </td><td> 시그마 - 알드리치 </td><td> R3629 </td><td></td></tr><tr><td> 돼지 창자 점막에서 헤파린 나트륨 소금, </td><td> 시그마 - 알드리치 </td><td> H3393-250KU </td><td></td></tr><tr><td> 고맙네 </td><td> 시그마 - 알드리치 </td><td> C3023 </td><td></td></tr><tr><td></td><td></td><td></td><td> 표 2. 특정 시약 및 장비. </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> 용액 </td><td> 참고 </td><td> 내용 </td></tr><tr><td> 세포 배양 매체 </td><td> 장과 스피처 2009 년 54 </td><td> 116 MM NaCl 0.67 MM KCl 이 MM CaCl 2 1.31 MM MgSO 4 4.6 MM 트리스 1퍼센트 (V : V) 페니실린 / 스트렙토 마이신 HCL와 7.8 산도를 조정합니다. 0.22 μm의 CN 필터를 통과하여 소독을 필터링 할 수 있습니다. 4 ° C.에 저장 </td></tr><tr><td> 칼슘 마그네슘 무료 매체 (CMF) </td><td> 번지 외 1994 년 42,. 번지와 스피처, 1995 55 </td><td> 116 MM NaCl 0.67 MM KCl 4.6 MM 트리스 0.4 MM EDTA (에틸렌 다이아 민 테트라 초산) 1퍼센트 (V : V) 페니실린 / 스트렙토 마이신 HCL와 7.8 산도를 조정합니다. 0.22 μm의 CN 필터를 통과하여 소독을 필터링 할 수 있습니다. 4 ° C.에 저장 </td></tr><tr><td> Maleic 산 버퍼 (적인 mAb) </td><td> Sive 외., 2000 53 </td><td> 100 MM maleic 산성 150 MM NaCl (산도 7.5). </td></tr><tr><td> 10X 마크의 수정일 Ringers (MMR), </td><td> Sive 외., 2000 53 </td><td> 100 MM NaCl MM KCl 1 ㎜ MgSO 4 이 MM CaCl 2 5 MM HEPES pH는 NaOH와 7.4로 조정 0.1X MMR은 50 MG / ML gentamicin 황산과 산도가 NaOH와 7.4로 조정이 포함되어 있습니다. </td></tr><tr><td> MEMFA 솔루션, 10X </td><td> Sive 외., 2000 53 </td><td> 0.1 M를 처리 (PH 7.4) 이 MM EGTA 1 ㎜ MgSO 4 3.7 %의 포름 알데히드 10X 솔루션은, 포름 알데히드없이, 4 ° C.에 저장할 수 있습니다 포름 알데히드는 (표준 37% 주식의 10분의 1 볼륨) 신선한 추가됩니다. </td></tr><tr><td> 현장 하이브리드 화 버퍼에서 </td><td> Siv전자 외., 2000 53 </td><td> 50 % 포름 아미드 5 배 SSC 1 MG / ML Torula RNA 100 MG / ML 헤파린 1X Denhart의 솔루션 0.1 % 십대 초반 20 0.1 % 챕스 10 MM EDTA (에틸렌 다이아 민 테트라 초산). </td></tr></tbody></table> 페니실린과 스트렙토 마이신은 우리의 문화 미디어에 사용되는 동안 솔루션. * Gentamicin, 항생제는 우리 MMR 솔루션에서 사용됩니다. </td></tr></tbody></table>

dissection, culture, and analysis of xenopus laevis embryonic retinal tissue

신경 판의 앞쪽 영역이 척추 망막에 상승을 제공하는 과정은 임상과 기초 모두 연구의 주요 초점이되고 있습니다. 망막 질환을 이해하고 치료에 대한 명백한 의료 관련뿐만 아니라, 척추 망막의 개발을 이해 neuronal 세포 유형 결정과 차별화 1-16에 대한 중요하고 우아한 모델 시스템으로 제공하고 있습니다. 신경 망막은 틀에 박힌 층에 배치 여섯 개별 세포 유형 (신경절, 수평, amacrine photoreceptors, 양극성 세포와 뮐러 glial 세포), 대체로 모든 vertebrates 12,14-18 사이에 보존되어있는 패턴으로 구성됩니다. 그대로 개발 배아에서 망막을 공부하는 것은 분명이 복잡한 기관은 계층 구조로 forebrain의 돌출부에서 어떻게 전개되는지 이해가 필요합니다 동안 이리저리 혜택을 많은 질문이 있습니다m 추정 망막 세포의 주요 세포 배양 7,19-23을 사용하여 방식을 채용. 예를 들어, 서로 다른 단계에서 제거 dissociated 조직의 세포를 분석하는 것은 하나는 다른 발달 단계에서 개별 셀의 사양의 상태를 분별 할 수있다 즉, 주변 조직 8,19-22와의 상호 작용의 부재에있는 세포의 운명 ,24-33. 기본 세포 배양은 또한 탐정 특정 시약과 문화를 처리하고 단일 세포 수준 5,8,21,24,27-30,33-39에 결과를 분석 할 수 있습니다. Xenopus laevis의 연구 고전 모델 시스템은 초기 신경 발달 19,27,29,31-32,40-42은 망막 기본 세포 배양 10,38,43-45에 특히 적합한 시스템으로 제공하고 있습니다. 추정 망막 조직은 즉시 신경 유도 25,38,43 따라 개발 초기 단계에서 액세스 할 수 있습니다. 또한, 일을 부여태아의 각 셀 노른자의 공급을 포함에서 망막 세포가 버퍼 소금 솔루션으로 구성된 매우 간단한 정의 미디어에서 배양 할 수 있습니다, 따라서 부화 또는 기타 커 기반 제품 10,24,44-45의 혼란 효과를 제거 . 그러나, 주변 조직 및 후속 처리에서 망막 조직의 격리 도전하고 있습니다. 여기, 우리는 결과적으로, 단일 세포의 해상도 칼슘 활동 및 유전자 표현 분석됩니다 기본 셀 문화를 준비하는 데 사용됩니다 Xenopus laevis의 망막 세포의 분해 및 분리하기위한 방법을 제시한다. 본 논문에서 제시 한 주제 자발적인 칼슘 과도의 분석이지만, 기술은 연구 질문 및 접근 방법 (그림 1)의 다양한 배열 광범위하게 적용됩니다.

성공적으로 해부하는 광 소포 (단계 25)와 retinae (무대 35)의 예는 2E와 2J 그림에 표시됩니다. 이 프로토콜이 개발의 여러 단계에서 사용할 수 있지만, 더 실험에 대한 정확성을 보장하기 위해 만 망막 조직을 얻는 것이 중요합니다. 조심스럽게 모든 단계에서 표피를 제거하고 포셉의 망막 조직을 주사하지 않도록. 단계 35 세 이상에서, 렌즈는 망막 위에 맑은 층으로 간주 할 수 있으며, ?...

Watch this Scientific Journal Video about Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue at JoVE.com

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis는 세포의 운명 사양 및 기본 세포 배양의 개별 망막 세포의 생리 기능을 연구하기위한 이상적인 모델 시스템을 제공합니다. 여기 망막 조직을 해부하고 칼슘 활동 이미징 및 현장 하이브리드 화에 의해 분석 기본 셀 문화를 생성하는 기법을 제시한다.

의 해부, 문화, 및 분석<em&gt; Xenopus laevis</em&gt; 배아 망막 조직

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

dissection-culture-analysis-xenopus-laevis-embryonic-retinal

Research

JoVE Journal

Neuroscience

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

13.8K 조회수.  College of William and Mary. Xenopus laevis는 세포의 운명 사양 및 기본 세포 배양의 개별 망막 세포의 생리 기능을 연구하기위한 이상적인 모델 시스템을 제공합니다. 여기 망막 조직을 해부하고 칼슘 활동 이미징 및 현장 하이브리드 화에 의해 분석 기본 셀 문화를 생성하는 기법을 제시한다.

비디오: Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

배아 척수에서 뉴런의 Commissural 해부와 문화

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

연구

신경과학

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection 및 배아 마우스 Hippocampal과 두피 뉴런의 주요 문화

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

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 ...

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

마우스 배아의 Motoneurons의 렉틴 기반 격리와 문화

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

쥐 배아 신경 세포의 분리 및 문화 : 빠른 프로토콜

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

에서 신경 Explant의 문화<em&gt; Xenopus laevis</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

배아 전뇌에서 내피 세포의 분리 및 문화

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

테스트 시각 기능에 대한 간단한 행동 분석<em&gt;는 Xenopus laevis의</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

의 후각 망울에서 칼슘의 변경 사항을 모니터링을 통해 온도에 의한 신경 세포의 활동을 기록<em&gt; Xenopus의의 laevis의</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

준비 및 Xenopus laevis Tectal 신경의 전체 셀 패치 클램프 기록에 대 한 프로토콜

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...