이 프로토콜은 제인 다드, 토마스 Jessell와 앤드류 럼즈던의 연구소에서 개발되었다. 방법은 콜라겐의 척수의 문화 explants에 콘레드 바슬러, 앤 Calof, 토마스 에들런드, 필 해밀턴, Domna Karagogeos, 아리엘 루이즈 I Altaba 및 Toshiya 야마다 등 많은 사람들이 결정. Marysia Placzek과 마크 테시어 - 라빈은 축삭지도 분자를 식별하는 수단으로 체외의 explants에서에서 축삭 성장을 사용할 수있는 기술을 개척. 버틀러 실험실에서 작품은 NIH / NINDS에서 천과 R01 NS063999의 3 월부터 기금에 의해 지원됩니다.

1 부 : Transfected COS 세포 집계의 준비 공중 방울을 사용하여 <ol><li> 종자 COS - 7 (COS) 35mm 문화 요리 세포. 그들이 제조 업체의 프로토콜에 따라 Lipofectamine2000을 사용하여 세포에 플라스미드 표현의 80 % confluency, transfect 1μg에 도달했을 때. </li><li> 방울 매달려 준비하려면, transfection 미디어를 기음과 1ml 1X의 인산 버퍼 솔루션 (PBS)로 transfected COS 세포를 헹굴. 15 분 동안 효소가없는 세포 분리 매체의 0.5ml로 세포를 처리합니다. 반응을 중지 Opti - 멤 + 1X 페니실린 / 스트렙토 마이신 / 글루타민 (P / S / G) + 10% 태아 소 혈청 (FBS)의 솔루션의 1ml을 추가합니다. </li><li> 문화 요리의 표면에서 그들을 제거하고 15ml 원뿔 튜브로 전송하는 세포를 씹다. 펠렛 세포에 2000K에서 2 분 동안 회전. Opti - 멤 + 1X P / S / G + 10% FBS의 100μl에 뜨는 및 resuspend 펠렛을 제거합니다. </li><li> 접시의 바닥 위에 뚜껑 장소 반전, 35mm 문화 접시 뚜껑의 안쪽에 여러 20μl 방울을 스팟과 세포 집계까지 몇 시간 동안 37 ° C 배양기에서 두십시오. </li></ol> 2 부 : 도설 척수 Explants의 작성 <ol><li> 어머니의 자궁에서 E11 랫 배아를 해부하다 필요한까지 L15 매체 얼음 계속. </li><li> 날카롭게 텅스텐 바늘로 즉시 forelimb 버드 아래의 지역에서, 각 배아의 줄기에서 4-6 세그먼트 섹션을 제거합니다. 플라스틱 피펫을 사용하여, 4 잘 Nunc 요리를 잘 하나에서 조직 조각을 수집하고 얼음 계속. </li><li> 모든 배아 세그먼트가 해부를하고있을 때, 1ml L15의 솔루션 + Nunc 요리 잘 순식간에 1mg dispase에 조각을 전송할 수있는 플라스틱 피펫을 사용합니다. 오분 이상없이 오래 실온에서 알을 품다. 이상 dispase있는 조직 조각을 품어하지 마십시오. </li><li> 부화하는 동안, 페트리 접시와 혼합하는 소용돌이에 L15 약 10ml에 가열 inactivated 정상 염소 혈청 (HIGS)의 0.5ml를 추가합니다. 삼분의 일에 잘 Nunc 요리의 솔루션을 1ml를 전송하고 부화가 끝나면이 솔루션에 조직 조각을 전송할 수 있습니다. 얼음 계속. 참고 : 조직 조각이 시간 동안 얼음에 &quot;나머지&quot;를 허용하는 경우 이후의 절개가 쉬울 것입니다. </li></ol> 파트 3 : 마중물 콜라겐 <ol><li> 360μl 콜라겐을 40μl 10X 최소 필수 매체를 추가 튜브를 flicking 또는 간단히 vortexing하여 신속하게 철저하게 섞는다. picofuge에 빠른 속도로 스핀 다운. 이 솔루션은 노란색 것입니다. 최대한 얼음에 보관. </li><li> (아래 참고 참조) 약간 주황색 콜라겐 솔루션을 설정하는 충분한 0.8M 나트륨 중탄산염을 (NaHCO 3) 추가합니다. 다시 말하지만, flicking에 의해 신속하게 그리고 철저하게 혼합하고, picofuge에 잠시 스핀 다운. 솔루션은 혼합 후 분홍색 남아있을 경우, 당신은 너무 NaHCO 3 추가하고 콜라겐의 새로운 튜브로 다시 시작해야합니다. </li><li> 이 시점에서 콜라겐 솔루션은 준비하는 것입니다. 그것은 얼음에 액체 유지되지만, 실온에 데려왔을 때 약 5 분 (분홍색으로 변했) 내에 고체화됩니다. </li><li> 4 잘 Nunc 요리의 각 우물에 콜라겐의 스팟 20μl. 당신의 피펫의 팁을 사용하여 작은 &quot;패드&quot;를 형성하는 콜라겐을 확산. 상온에서 설정 콜라겐을 보자. </li></ol> 참고 : NaHCO 3의 정확한 금액은 콜라겐의 각 배치에 대해 titrated해야합니다. 낮은 시작 (11μl)와 솔루션은 오렌지의 약간 그늘 때까지 다음 0.5 - 1μl stepwise를 추가합니다. &quot;오른쪽&quot;금액은 보통 콜라겐 분홍색을 돌려 것이다 NaHCO 3의 작은 금액보다 1ul 적습니다. NaHCO 3의 금액은 선형 위 또는 아래로 확장되지 않습니다. 4 부 : 도설 척수의 Explants과 콜라겐 매트릭스에서 COS 세포 집계와 함께 그들의 위치의 해부 <ol><li> 갓 둘레에 텅스텐 바늘과 고급 포셉 한 쌍의 (# 5 # 55)를 사용하여 척수를 둘러싼 mesoderm를 제거합니다. </li><li> 텅스텐 바늘로 바닥 판에 평행하게 절단하는 것은 신중하게 척수의 배면의 다섯째를 제거합니다. </li><li> 모서리가로 깨끗하게 가능한 절단하고 있는지 만드는 등의 척추 explant를 이등분하다. 필요한 경우, 모서리를 다시 잘라. 약에 가져온 유리 모세관가 장착되어 입을 피펫을 사용하여 별도의 콜라겐 패드에 각 지느러미 척추 explant을 전송합니다. 지름 0.5mm. </li><li> 지느러미 척추 explant의 측면 가장자리로 거의 같은 넓이의 정사각형으로 transfected COS 세포의 집계를 잘라. </li><li> 입 피펫을 사용하여 지느러미 척추 explant과 콜라겐 패드에이 사각형 중 하나를 전송합니다. </li><li> 입 피펫으로 입 피펫을 사용하여 초과 액체를 대기음. 기음 이상하지 마십시오. </li><li> 패드에 준비하는 콜라겐의 4μl를 적용하고, 텅스텐 바늘을 사용하여직접 조직을 건드리지 않고 explants 통해 콜라겐 이동합니다. explants을 둘러싸고있는 콜라겐의 텅스텐 바늘을 이동하여, 동양 explants 있도록 COS 세포의 집계가 등의 척추 explant의 측면 가장자리에 인접한입니다. </li><li> 콜라겐 설정한 후 explants가 콜라겐에 완전히 쌌다되는 것을 보장하기 위해 콜라겐의 4μl의 응용 프로그램을 반복합니다. </li><li> 조직 문화 후드에서 30-40시간에 대한 Opti - 멤 + 1xP/S/G 문화 0.5ml를 추가합니다. </li><li> 얼음 접시를 유지, 1xPBS에 0.​​5ml 4 % paraformaldehyde로 45 분간 explants을 수정. </li><li> 1xPBS의 차단 솔루션 + 0.1 % 트리톤 - X100 1퍼센트의 HIGS (PBTN)의 0.5ml로 두 번 씻으십시오. 4 블록 ° C 시간 중 적어도 몇, 야간 부화가 immunohistochemistry에 의해 처리 후 바람직하고 있습니다. </li></ol> 제 5 부 : Immunohistochemistry에 의해 Commissural Axons의 시각화 <ol><li> PBTN에서 차단 후, 포셉 및 48 잘 접시에 장소 4 잘 접시의 explants을 잘라 버릴거야. </li><li> 각 잘하는 기본 항체의 200μl를 추가하고 4 박 떠나 ° C. commissural axons을 시각화하기 위해 마우스 방지 Tag1 항체의 1시 6분 솔루션을 사용합니다. transfection가 성공 여부를 결정하기 위해, 또한 신호 분자가 에피토프이 태그되었는지 중 신호 분자가 테스트하거나 관련 에피토프되는 대한 일차 항체를 포함합니다. </li><li> 4 PBTN 5 X 1시간 ° C. 각 샘플을 와시 </li><li> 각 잘하는 이차 항체의 200μl를 추가하고 4 박 떠나 ° C. 안티 Tag1 항체가 기본 항체로 사용했다면, FITC 또는 Cy3 중 하나에 결합 염소 항 마우스 IgM 항체 보조를 사용합니다. 가벼운 민감한 차 항체를 사용하면 FITC 또는 Cy3로 결합하는 경우,이 시점부터는 호일로 덮여 48 잘 요리를 보관. </li><li> 각 샘플 5 4에서 X 1시간 PBTN 각 ° C. 와시 </li><li> , 3 잘 우울증 슬라이드에 explant을 전송 Vectashield 매체와 coverslip에 마운트를 초과 액체를 제거합니다. 4 ° C 또는 -20 ° C. 중 하나에서 빛이 금고에 저장 </li></ol> 6 부 : Explants 대표 이미지 성공적인 실험에서, explant와 C​​OS의 집계가 서로 인접 유지하고 콜라겐이 세트로 떨어져 표류하지 않습니다. axons의 최소한의 전면에 자라난 것이있을 것이다; 중요한 axonal 전면에 자라난가 등의 척추 explant 너무 오래 교양있는 것을 나타냅니다. explant에서 성장하고 더 blebs있을 수 없다, 조직 문화 매체와 직접 접촉으로 온다면 blebbing이 발생합니다. axons을 방향으로 신호 분자의 기능은 공촛점 현미경을 사용하여 평가됩니다. 축삭의 reorientation의 범위는 COS 세포 집계 3 가장 가까운 axons의 reorientation의 평균 각도를 측정하여 계량하실 수 있습니다. 그림 1에 표시된, COS 세포는 표현 myc - 태그 BMP7 (파란색)가 지붕 판의 explant가 commissural axons를 (그림 들어갈 수있는 방식과 유사 BMP7의 원본 (그림 1B)에서 멀리 Tag1 + commissural axons (녹색)를 방향 1A). 제어 벡터를 표현 COS 세포는 commissural axons 3,4의 방향에 영향을 미치지 않습니다. 이러한 explants 또한 모터 뉴런과 지느러미 interneurons을 장식 전사 인자 Isl1 / 2 (빨간색)에 대한 항체로 분류되었습니다. 이 결과는 최초로 표시했다는 BMP 가족 mediates의 일원 지붕 판 chemorepellent 3 활동. <img src="/files/ftp_upload/1853/1853fig1tmb.jpg" border="0" /> 그림 1 : 제발 <a href="https://www.jove.com/files/ftp_upload/1853/1853fig1.jpg">여기를 클릭</a> 그림 1의 더 큰 버전을 볼 수 있습니다.

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이 분석을 수행하는 성공을 결정하는 중요한 요인이 처음이고, 조직이 너무 연장 기간 동안 dispase로 치료해서는 안됩니다, 이러한 치료는 조직이 매우 끈적되고 및 생존 감소가 발생하게됩니다. 둘째, 콜라겐은 완벽하게 준비하는과 최대한 얼음에 보관해야합니다. 이 설정 시작하면 그것은 처리하기 위해 점점 더 어려워 될 것입니다, 핑크 차례 즉. 세는 텅스텐 바늘은 항상 매우 날카로운 보관해...

assaying the ability of diffusible signaling molecules to reorient embryonic spinal commissural axons

척추 척수 1 도설 commissural axons은 축삭지도 신호를 식별하는 귀중한 모델 시스템을왔다. 여기, 우리는 시험 관내 분석, commissural axons의 방향 2 외부와 내장 신호의 효과를 연구하기 위해 광범위하게 사용되고 있습니다 &quot;reorientation 분석&quot;에 대해 설명합니다. 에 chemorepellent BMP를 포함하여,이 분석은 제인 다드, 토마스 Jessell와 앤드류 럼즈던의 실험실에서 수많은 사람들에 의해 개발되었습니다 (자세한 내용은 응답을 참조)이 분석의 버전은 주요 축삭지도 분자의 reorientation 활동을 증명하는 데 사용되었다 지붕 판 3,4 및 Netrin1 5 소닉 헤지 호그 (쉬) 척수의 바닥 판에 6 chemoattractive 활동. 척수의 지느러미 3 분의 2로부터 2-3 세그먼트로 구성된 Explants은 배아 일 (E) 11 쥐에서 해부하고 입체 콜라겐 젤 7 양식입니다. E11 등의 척추 explants은 당단백질, Tag1 8 그들 axonal 표현식에서 확인할 수 있습니다 새로 태어난 commissural 뉴런을 포함합니다. 문화 30-40시간 과정 동안 commissural 축삭의 궤도는 생체내에서 볼 비슷한 시간 코스 이들 지느러미 explants에 recapitulated 있습니다. 이 axonal 탄도는 테스트 조직이나 지느러미 explant의 측면 가장자리 중 하나를 접촉 후보 신호 분자를 표현 COS 셀 집합 중 하나를 배치하여 도전을 수 있습니다. 추가 조직의 주변에 연장 Commissural axons은 내생 지붕 판과 이소성 조직 측면에서 모두 신호의 영향 아래에서 성장합니다. commissural axons 이러한 상황에서 reoriented 아르 정도는 계량하실 수 있습니다. 이 분석을 사용하여 그것은 모두 commissural 궤도 9 직접이 신호의 필요성뿐만 아니라 commissural axons에게 3,4를 방향에 특정 신호의 자족을 확인 할 수 있습니다.

Watch this Scientific Journal Video about Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons at JoVE.com

Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

이 분석은 commissural axons를 방향에 대한 신호 분자의 능력, 여기에 본 Morphogenetic의 단백질 7 (BMP7)을 평가합니다. 배아 등의 척수의 explant는 COS 세포의 secreting 후보 성장 요인의 집계에 인접한 양식입니다. explant 내에서 성장 Reoriented commissural axons은 immunohistochemistry에 의해 시각입니다.

배아 척수 Commissural Axons을 방향을 Diffusible 신호 분자의 능력을 시금

Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals.  Here, we ...

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Biology

8.8K 조회수.  University of Southern California. 이 분석은 commissural axons를 방향에 대한 신호 분자의 능력, 여기에 본 Morphogenetic의 단백질 7 (BMP7)을 평가합니다. 배아 등의 척수의 explant는 COS 세포의 secreting 후보 성장 요인의 집계에 인접한 양식입니다. explant 내에서 성장 Reoriented commissural axons은 immunohistochemistry에 의해 시각입니다.

비디오: Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

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

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues1 and survival factors2 located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons3. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons4. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro5,6.
Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. 
SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol7) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival8.

We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads.

Neurite 가지의 Assays에 대한 해부와 칙 Statoacoustic 신경절과 콜라겐 젤에서 척수의 Explants의 문화

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

마우스 배아 척수의 전체 마운트 준비 분기 Axonal 공부 DRG 뉴런의 DiI - 라벨링

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

해부 및 제브라 피쉬 배아의 측면 장착 : 척수 개발의 분석

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

세포 배양과 병아리 배아에서 축슨 유도 수용체의 역학을 평가하기 위해 pHluorin의 사용

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

Culture of Isolated Floor Plate Tissue and Production of Conditioned Medium to Assess Functional Properties of Floor Plate-released Signals

During development, progenitors and post-mitotic neurons receive signals from adjacent territories that regulate their fate. The floor-plate is a group of glial cells lining the ependymal canal at ventral position. The floor-plate expresses key morphogens contributing to the patterning of cell lineages in the spinal cord. At later developmental stages, the floor-plate regulates the navigation of axons in the spinal cord, acting as a barrier to prevent the crossing of ipsilateral axons and controlling midline crossing by commissural axons1. These functions are achieved through the secretion of various guidance cues. Some of these cues act as attractants and repellents for the growing axons while others regulate guidance receptors and downstream signaling to modulate the sensitivity of the axons to the local guidance cues2,3. Here we describe a method that allows investigating the properties of floor-plate derived signals in a variety of developmental contexts, based on the production of Floor-Plate conditioned medium (FPcm)4-6. We then exemplify the use of this FPcm in the context of axon guidance. First, the spinal cord is isolated from mouse embryo at E12.5 and the floor-plate is dissected out and cultivated in a plasma-thrombin matrix (Figure 1). Second two days later, commissural tissue are dissected out from E12.5 embryos, triturated and exposed to the FPcm. Third, the tissue are processed for Western blot analysis of commissural markers.

The floor plate is a crucial structure of the developing spinal cord, providing diffusible signals to progenitors and differentiating neurons. We describe a method to produce floor-plate conditioned medium, to apply it to fresh spinal cord tissue, and to assess the consequences on proteins of interest by biochemistry.

바닥 플레이트 출시 신호의 기능적 특성을 평가하기 위해 에어컨 중간의 절연 층 플레이트 조직과 생산의 문화

During development, progenitors and post-mitotic neurons receive signals from adjacent territories that regulate their fate. The floor-plate is a group of ...

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

척수 전기 생리학

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

생물학

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

활용 칙 신경 튜브에서 뉴런의 유전 정의된 그룹 Axonal 경로를 벗기기 비켜에서 Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

접근 방법은 등쪽 뿌리 신경절 뉴런의 정렬 및 수초를 강화하는

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

배아 운동 뉴런의 Axonal 투영 패턴의 시각화<em&gt; Drosophila</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...