네이 부여 EY014850 지원.

1. Sylgard 덮인 요리, 실험 챔버 및 면도날의 준비 <ol><li> Sylgard의 엘라스토머 코팅 35mm 팰컨 배양 접시가 하나의 photoreceptor 세포를 얻기 위해 외딴 망막의 적절한 자르고 필요합니다. 탄성체는 공급 업체의 지침과 소량이 계층과의 엉덩이를 커버하기 위해 각각의 그릇에 부어에 따라 준비가되어 있습니다. 요리는 코팅 후 커버 교체하고 그들을 저장합니다. 몇 일 시간에 탄성이 견고하고 요리를 준비하고 있습니다. </li><li> 그들이 이미징 실험 과정 동안 고정되도록 절연 photoreceptors은 실험 챔버의 바닥에 붙어해야합니다. 이것은 폴리 - L - 라이신 또는 폴리 - L - ornithine로 코팅하여 실의 바닥을 달성입니다. 챔버 당 하나의 하나의 0.01 % 용액 200 μL를 추가하고, 먼지에서 보호하기 위해 종이 타월로 챔버 스를 커버. 솔루션은 건조 후, 닫힌 상​​자에 증류수 및 저장과 실 씻으십시오. 이주 이내에 사용하십시오. </li><li> 실험의 끝에 회의소를 청소하려면, 석유 침지 렌즈와 세포 부스러기로부터 기름을 제거하기 위해 100 % 에탄올과 그들을 씻으십시오. 세포 파편을 제거하려면, 면화 - 스쳐 applicators을 사용하여 조심스럽게 챔버의 바닥을 씻어. 후, 증류수로 세척하고 다시 코팅하기 전에 챔버 스가 건조하게. </li><li> 금속 커터와 함께 작은 조각 (8 일 블레이드에서)에 양날 면도날 컷. </li></ol> 2. 솔루션의 준비 <ol><li> 솔루션의 구성은 종류에 따라 다릅니다. 양서류 들어, 링어가 (mmol / L)을 가지고 : 110 NaCl, 2.5 KCl, 1.6 MgCl 2, 1 CaCl 2, 5 HEPES, 산도 = 7.55 있습니다. 산도는 NaOH와 최종 값을 조정해야합니다. 포유류의 경우 링어가 (mmol / L)을 가지고 : 130 NaCl, 5 KCl, 0.5 MgCl 2, 2 CaCl 2, 25 hemisodium - HEPES, 산도 = 7.40 있습니다. 링어의 솔루션은 몇 달 동안 실온에서 잘 밀봉 보관하실 수 있습니다. </li><li> 박테리아 성장을 피하기 위해 -20 ° C에서 보관 주식 포도당 용액, 1 몰 / L,. </li><li> 실험 당일, 5 mmol / L.의 최종 농도 링어의 솔루션으로 포도당을 추가 하루의 끝에서, 링어의 그것이 박테리아를 성장 있기 때문에 포도당이 포함된 폐기하십시오. </li></ol> 3. 망막의 분리 <ol><li> 망막의 적절한 절단을 위해 그것은 동물이 희생되기 전에 최소한 2-3시간에 대한 어두운 적응하는 것이 중요합니다. 동물은 어두운 방에 적당한 통풍이 컨테이너에 어두운 적응해야합니다. </li><li> 절반 링어의 솔루션을 가진 두 개의 35mm 페트리 요리를 입력합니다. </li><li> 희미한 붉은 불빛 아래에있는 동물을 희생하고 눈을 제거합니다. 그 후, 모든 절차는 해부 현미경 또는 비디오 모니터와 카메라를 사용하여 적외선에 따라 수행됩니다. </li><li> 눈 바깥 표면으로부터 남은 조직을 제거합니다. 잘라 앞쪽에 부분을 제거한 후, 링어의 가득 배양 접시 중 하나에 세안 컵을 전송하기만하면됩니다. </li><li> 유리체를 제거하고 신중하게 떨어져 곤란이나 첨부 파일을 절단하여 세안 컵의 나머지 부분에서 망막을 분리. 부드럽게 망막을 채취해서 세안 컵에서 완전히 분리. </li><li> 플라스틱 전송 피펫 두 번째 페트리 접시에 망막을 전송합니다. 빛을 꽉 상자에서 망막을 포함하고있는 음식 보관하십시오. </li></ol> 4. 단일 photoreceptor 세포의 분리 <ol><li> 모든 절차는 적외선에 따라 수행됩니다. 망막의 작은 조각을 잘라 Sylgard 덮인 요리에 플라스틱 피펫으로 전송합니다. 망막의 조각을 포함하고있는 솔루션의 볼륨은 250 μL에 대해해야합니다. </li><li> 블레이드 홀더와 면도날의 작은 부분을 잡고 - 블레이드의 가장자리는 홀더에 약 45 °의 각도로해야합니다. Sylgard 계층에 대한 망막의 조각을 평평하게하고 Sylgard 계층에 붙어 유지하면서 날개가 잘게 망막의 조각을 잘라 사용합니다. </li><li> 실험 챔버에 세포를 포함하는 솔루션을 200 μL를 전송 - Sylgard 덮인 접시에 망막의 나머지 조각을 두십시오. 빛을 꽉 상자에서 분리된 세포와 챔버 보관하십시오. </li><li> 링어의로부터 2-3 ML를 추가 후, 해결하기 위해 세포를 10 분 기다립니다. 이 단계에서 분리된 세포는 염색 로딩 프로토콜에 따라 특정 형광 염료 (예 : Fura - 2)로 로드할 수 있습니다. </li><li> 세포는 현재 실험에 대한 현미경 스테이지로 이동하실 수 있습니다. </li></ol> 5. 형광 이미징 <ol><li> epifluorescence 현미경의 무대에 챔버를 전송합니다. 조정 솔루션 재관류, 온도 프로브, 적외선 조명 등 </li><li> 커튼을 닫고 실험을 시작합니다. 현미경 케이지 내부의 적외선 켜고, 실험 챔버의 하단에 초점을, 그리고 세포를 찾기 위해 무대를 이동합니다. </li></ol> 6. 대표 결과S : 그림. 1 도롱뇽 (Ambystoma tigrinum) 망막에서이 프로토콜을 취득한 건강한 고립로드 앤드 콘 photoreceptors의 형태를 보여줍니다. 샐러맨더 세포 때문에 그들의 큰 크기와 망막의 분리 후 몇 시간 동안 생존 능력의 단일 세포 형광 이미징 연구에 대한 광범위하게 사용되었습니다. 또한, 샐러맨더 망막에서 하나는 정기적으로 막대와 원뿔 photoreceptors 모두를 얻을 수 있습니다. 세포의 건강에 중요한 기준이 손상 타원체 (그림 1), mitochondria가 집중하고있는 세포의 일부의 존재입니다. 세포 DAPI 광학에 따라 볼 때, mitochondria의 농도는 NADH의 존재로 인해 강한 형광 신호 (그림 2) 제공합니다. 손상 타원체의 부족 실험. 그림 일반적으로 부적당 손상된 세포의 기호입니다. 3 부어 세포 기관과 응축 핵으로 손상된 샐러맨더로드 photoreceptor를 보여줍니다. 이러한 세포는 DAPI 광학에 따라 훨씬 낮은 형광 디스플레이지만, 타원체 영역 (산화 플라빈의 세포핵과 flavoproteins에서 발생)에 강력한 유행 신호를 보여 FITC 광학 아래의 조회. 격리된 photoreceptors의 건강을위한 또 다른 기준은 빛의 자극에 의해 즉시 자신의 외부 세그먼트에있는 모든 트랜스 레티놀을 (비타민 A) 생성하는 능력이 있습니다. 비타민 A의 생성은 그대로 대사 기계에 의존 NADPH의 상당한 양의 필요합니다. 그림 4와 5는 비타민 그대로 개구리와 마우스 막대의 photoreceptors 각각의 바깥쪽 세그먼트의 형성을 보여줍니다. <img src="/files/ftp_upload/2789/2789fig1.jpg" alt="그림 1"/> 그림 1. 건강한 하나의 막대와 원뿔 photoreceptors. 세포는 호랑이 샐러맨더 망막으로부터 격리되었다. Phototransduction은 외부 세그먼트에서 일어나는와 타원체는 밀도가 mitochondria들이 즐비합니다. 로즈는 희미한 빛 비전, 밝은 빛이 비전에 대한 콘 책임이 있습니다. <img src="/files/ftp_upload/2789/2789fig2.jpg" alt="그림 2"/> 그림 2. 샐러맨더로드 및 원뿔형 생활의 형광. 이들은 각각 ellipsoids에 강한 NADH의 형광없이 중요한 비타민들은 바깥 세그먼트에서 형광을 보여주는, 어두운 조정 세포 수 있습니다. 형광 이미지의 촬영은 어두운 - 적응 기간 후 가시 광선에 처음으로 노출을 나타냅니다. <img src="/files/ftp_upload/2789/2789fig3.jpg" alt="그림 3"/> 그림 3. 손상된 샐러맨더로드 photoreceptor. 부어 세포 본체와 응축 핵이 손상을 나타내는 있습니다. 세포는 산화와 최소한의 NADH 신호 (DAPI 광학 장치)가있다지만, 훨씬 강력한 유행 신호 (FITC 광학)입니다. <img src="/files/ftp_upload/2789/2789fig4.jpg" alt="그림 4"/> 그림 4. NADH와 레티놀과 함께 개구리 막대. 이것은 타원체 지역에서 강력한 NADH의 형광을 보여주는 건강한 개구리 막대의 photoreceptor입니다. 빛의 노출 전에 바깥 부문 최소한의 형광이 있습니다. 빛의 노출에 따라, 때문에 비타민 A의 형성에 외부 세그먼트 형광의 상당한 증가가 <img src="/files/ftp_upload/2789/2789fig5.jpg" alt="그림 5"/> 그림 5. 레티놀과 마우스 막대. 이것은 마우스 막대의 photoreceptors의 타원체 영역 강한 형광 신호를 표시하지 비타민 A의 형성에 의한 빛의 노출 후 상당한 외부 세그먼트 형광을 보여주는 건강한 마우스 막대의 photoreceptor입니다.

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N., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interphotoreceptor+Retinoid-Binding+Protein+Is+the+Physiologically+Relevant+Carrier+That+Removes+Retinol+from+Rod+Photoreceptor+Outer+Segments.">Interphotoreceptor Retinoid-Binding Protein Is the Physiologically Relevant Carrier That Removes Retinol from Rod Photoreceptor Outer Segments.</a> Biochemistry. 46, 8669-8679 (2007).</li><li>Kolesnikov, A. V., Ala-Laurila, P., Shukolyukov, S. A., Crouch, R. K., Wiggert, B., Estevez, M. E., Govardovskii, V. I., Cornwall, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+Cycle+and+Its+Metabolic+Support+in+Gecko+Photoreceptors.">Visual Cycle and Its Metabolic Support in Gecko Photoreceptors.</a> Vision Res. 47, 363-374 (2007).</li><li>Chen, C., Blakeley, L. R., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+All-Trans+Retinol+after+Visual+Pigment+Bleaching+in+Mouse+Photoreceptors.">Formation of All-Trans Retinol after Visual Pigment Bleaching in Mouse Photoreceptors.</a> Invest Ophthalmol Vis Sci. 50, 3589-3595 (2009).</li></ol>

관심 없음 충돌 선언하지 않습니다.

건강 분리된 세포가 얻은 정보가 아닌 경우, 문제는 망막의 분리 또는 건강 또는 그 돼지와도 자리잡고 있습니다. 일반적으로, 눈 및 유리체의 전면을 제거 후, ​​망막이 쉽게 안료 상피를 리프트. 그렇지 않으면, 세안 컵의 주변부터 해제로 껍질보십시오. 아직 분리하기 어려운 경우에는 가능성이 가능성이 동물은 시간의 적절한 기간 동안 어두운 적응되지 않았음을, 또는 빨간 불빛이 너무 밝?...

<table border="1"><tbody><tr><th> 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 </th></tr><tr><td> 어두운 방 (100-150피트 2) </td><td></td><td></td><td></td></tr><tr><td> 레드 불 19 </td><td></td><td></td><td> 온라인 상점 </td></tr><tr><td> 적외선 광원 및 적외선 이미지 뷰어 </td><td> FJW 광학 시스템, 주식 회사 </td><td></td><td></td></tr><tr><td> 현미경 19 해부 </td><td></td><td></td><td> 적외선 시청자와 함께 복 </td></tr><tr><td> Epifluorescence 현미경은 빛을 꽉 케이지 19 동봉된 </td><td></td><td></td><td></td></tr><tr><td> 해부 도구 (가위, 집게, 블레이드 홀더) </td><td> Roboz 또는 파인 과학 도구 </td><td></td><td></td></tr><tr><td> Sylgard의 엘라스토머 </td><td> 에식스 (샬럿, NC) </td><td> Sylgard 184 엘라스토머 키트 </td><td></td></tr><tr><td> 폴리 - L - ornithine (0.01 %) </td><td> 시그마 - 알드리치 </td><td> P4957 </td><td></td></tr><tr><td> 폴리 - L - 라이신 (0.1 %) </td><td> 시그마 - 알드리치 </td><td> P8920 </td><td> 0.01 %로 희석 </td></tr><tr><td> 실험 챔버 </td><td> 워너 인 스트 루먼트 (햄든, CT) </td><td> D3512P </td><td></td></tr><tr><td> 배양 접시, 플라스틱 pipettes </td><td> 피셔 사이 언티픽 </td><td></td><td></td></tr></tbody></table>

preparation of living isolated vertebrate photoreceptor cells for fluorescence imaging

척추 망막에서 phototransduction, 전기 신호에 빛의 변환,로드 앤드 원추 photoreceptor 세포 1-4에 의해 수행됩니다. 로드의 photoreceptors는 희미한 빛, 밝은 빛에서 원뿔의 비전에 대한 책임이 있습니다. Phototransduction는 photoreceptor 세포, 영상 안료, 기본 조명 감지기의 높은 농도를 포함하는 전문 구역의 바깥 부분에서 이루어진다. 망막 CIS, 단백질, 옵신에 첨부된 - 시각 색소는 발색단, 11로 구성되어 있습니다. 시각 색소에 흡수 광자는 11 발색단을 isomerizes - CIS를 모든 트랜스 있습니다. 이 photoisomerization는 막 잠재력에 변화 culminating, 그리고 전기 신호에 빛의 자극의 전달에 대한 데리고 반응의 폭포를 시작 시각 색소의 conformational 변화를 제공합니다. 빛의 자극에서 세포의 복구 빛으로 활성 중간체의 비활성화 및 멤브레인 잠재력의 회복을 포함한다. CA 2 +는 phototransduction에 관련된 효소의 몇 가지의 활동을 modulates, 그 농도가 빛의 자극에 따라 줄어 듭니다. 이러한 방법으로, CA 2 + 배경 빛으로 자극하고 적응에서 세포의 회복에 중요한 역할을합니다. 모든 트랜스 5-7로 CIS 발색단 - 복구 프로세스의 또 다른 필수적인 부분은 11 photoisomerization로 빛을 감지하는 동안 파괴되고있는 시각 색소의 재생이다. 이 중생은 APO - 단백질 옵신 뒤에두고, photoactivated 안료의 모든 트랜스 망막의 릴리스와 함께 시작됩니다. 출시된 모든 트랜스 망막이 빠르게 모든 트랜스 레티놀과 옵신에 NADPH를 이용한 반응 감소 것은 신선한 11 결합 - CIS 망막은 시각 색소를 개혁하기 위해 외부 세그먼트에 왔어요. 모든 트랜스 레티놀은 다음 전문 캐리어 Interphotoreceptor의 Retinoid 바인딩 단백질 (IRBP)에 의해 외부 세그먼트와 인접 세포 밖으로 전송됩니다. 단일 photoreceptor 세포의 형광 이미지들은 생리 및 세포 생물학을 연구하는 데 사용할 수 있습니다. CA 2 +에 민감한 형광 염료 8-12뿐만 아니라 내부 세그먼트 칼슘 2의 역할 칼슘 2 + 점포 조명에 자세히 외부 세그먼트 칼슘 2 + 변화와 응답 사이의 상호 작용을 검사하는 데 사용할 수있는 + 항상성 13,14 . 형광 염료는 MG 2 측정에 사용할 수 있습니다 + 농도 15, 산도, 그리고 수성 및 멤브레인 구획 16 추적기로. 마지막으로, 모든 트랜스 레티놀 (비타민 A)의 본질적인 형광은 단일 photoreceptor 세포 17-19 년에 형성 및 제거의 속도론을 모니터링하는 데 사용할 수 있습니다.

Watch this Scientific Journal Video about Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging at JoVE.com

Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

방법은 형광 이미징에 대해 서로 다른 척추 동물에서 하나의 생활 photoreceptor 세포의 준비를 위해 설명되어 있습니다. 방법은 이미지와 같은 NADH이나 비타민 A와 같은 내생 fluorophores의 형광을 위해 사용하거나, CA에 민감한 exogenously 추가 형광 염료의 수<sup&gt; 2 +</sup&gt; 또는 기타 요인.

형광 이미징을위한 절연 척추 Photoreceptor 세포를 생활 준비

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells1-4.  ...

preparation-living-isolated-vertebrate-photoreceptor-cells-for

Research

JoVE Journal

Neuroscience

12.2K 조회수.  Medical University of South Carolina. 방법은 형광 이미징에 대해 서로 다른 척추 동물에서 하나의 생활 photoreceptor 세포의 준비를 위해 설명되어 있습니다. 방법은 이미지와 같은 NADH이나 비타민 A와 같은 내생 fluorophores의 형광을 위해 사용하거나, CA에 민감한 exogenously 추가 형광 염료의 수 2 + 또는 기타 요인.

비디오: Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

유전적으로 망막 신경절 라벨 셀에서 라이트 응답을 기록하기 위해 절연 망막 준비

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

연구

신경과학

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

손상의 감각 오르간 전구체 세포 라이브 셀 이미징 Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

내생 NADH 형광의 2 광자 이미징에 의한 마우스 대뇌 피질의 Microregional Hypoxia의 탐지

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

칼슘 이미징에 대한 급성 Subventricular 지대 슬라이스의 작성

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

네 차원 형광 이미징과 신경 말단 생활에 엔도 좀 역학의 시각화

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 &#181;m thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 &#181;m is discarded before 200 &#181;m of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 &#176;C. Pieces of outer layer are incubated in 500 &#181;l of Ringer&#39;s solution with 2 units of activated papain for 20 min at 37 &#176;C. The reaction is stopped by adding 500 &#181;l 10% fetal calf serum (FCS) in Dulbecco&#39;s Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 &#181;l of DMEM and seeded at 1 x 105 cells/cm2. The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.

Neural retina of a mouse aged 8 days is on top of a 4% gelatin block. After isolation of the photoreceptor layer (200 &#181;m) by vibratome, the photoreceptors are seeded after mechanical and enzymatic dissociation for culture. The photoreceptor layer can be used for molecular, biochemical analyses or transplantation.

에 vibratome 단면 마우스 망막 광 수용체의 문화를 준비하는 방법

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in ...

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 ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

살아있는 세포에 Sod1 돌연변이 단백질 집계 형성의 높은 콘텐츠 정량화에 대 한 분석 결과 개발

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Reliable Isolation of Central Nervous System Microvessels Across Five Vertebrate Groups

Isolation of microvessels from the central nervous system (CNS) is commonly performed by combining cortical tissue from multiple animals, most often rodents. This approach limits the interrogation of blood-brain barrier (BBB) properties to the cortex and does not allow for individual comparison. This project focuses on the development of an isolation method that allows for the comparison of the neurovascular unit (NVU) from multiple CNS regions: cortex, cerebellum, optic lobe, hypothalamus, pituitary, brainstem, and spinal cord. Moreover, this protocol, originally developed for murine samples, was successfully adapted for use on CNS tissues from small and large vertebrate species from which we are also able to isolate microvessels from brain hemisphere white matter. This method, when paired with immunolabeling, allows for quantitation of protein expression and statistical comparison between individuals, tissue type, or treatment. We proved this applicability by evaluating changes in protein expression during experimental autoimmune encephalomyelitis (EAE), a murine model of a neuroinflammatory disease, multiple sclerosis. Additionally, microvessels isolated by this method could be used for downstream applications like qPCR, RNA-seq, and Western blot, among others. Even though this is not the first attempt to isolate CNS microvessels without the use of ultracentrifugation or enzymatic dissociation, it is unique in its adeptness for the comparison of single individuals and multiple CNS regions. Therefore, it allows for investigation of a range of differences that may otherwise remain obscure: CNS portions (cortex, cerebellum, optic lobe, brainstem, hypothalamus, pituitary, and spinal cord), CNS tissue type (gray or white matter), individuals, experimental treatment groups, and species.

The goal of this protocol is to isolate microvessels from multiple regions of the central nervous system of lissencephalic and gyrencephalic vertebrates.

5개의 척추동물 단에 걸쳐 중추 신경계 마이크로혈관의 믿을 수 있는 격리

Isolation of microvessels from the central nervous system (CNS) is commonly performed by combining cortical tissue from multiple animals, most often ...

Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics Under Physiological Conditions

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks of Parkinson's disease. Yet, much of its biological activity, as well as its aggregation, centrally involves the soluble monomer form of the protein. Elucidation of the molecular mechanisms of aSyn biology and pathophysiology requires structurally highly resolved methods and is sensitive to biological conditions. Its natively unfolded, meta-stable structures make monomeric aSyn intractable to many structural biology techniques. Here, the application of one such approach is described: hydrogen/deuterium-exchange mass spectrometry (HDX-MS) on the millisecond timescale for the study of proteins with low thermodynamic stability and weak protection factors, such as aSyn. At the millisecond timescale, HDX-MS data contain information on the solvent accessibility and hydrogen-bonded structure of aSyn, which are lost at longer labeling times, ultimately yielding structural resolution up to the amino acid level. Therefore, HDX-MS can provide information at high structural and temporal resolutions on conformational dynamics and thermodynamics, intra- and inter-molecular interactions, and the structural impact of mutations or alterations to environmental conditions. While broadly applicable, it is demonstrated how to acquire, analyze, and interpret millisecond HDX-MS measurements in monomeric aSyn.

The structural ensemble of monomeric alpha-synuclein affects its physiological function and physicochemical properties. The present protocol describes how to perform millisecond hydrogen/deuterium-exchange mass spectrometry and subsequent data analyses to determine conformational information on the monomer of this intrinsically disordered protein under physiological conditions.

생리학적 조건하에서의 알파-시뉴클레인 구조역학 연구를 위한 밀리초 수소/중수소-교환 질량 분광법

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks ...