저자는 자금에 대한 RIKEN 주셔서 감사합니다. 우리는 또한 유전과 immunohistochemistry 프로토콜에 대한 토론 Cagri Yalgin, 캐롤라인 Delandre, 그리고 제이 패리쉬 감사드립니다.

시약의 1.Preparation <ol><li> 칼슘 + + 무료 HL3.1 식염수 28 준비합니다. <ol><li> MM 년 : 70 NaCl, 5 KCl, 20 MgCl 2, 10 NaHCO 3, 5 HEPES, 115 자당, 5 trehalose, 산도 7.2. 소독 필터와 4 저장 ° C. 참고 : 칼슘 + + 무료 솔루션 해부하는 동안 근육 수축을 방지합니다. </li></ol></li><li> 폴리 - L - 라이신 (PLL) coverslips하십시오. <ol><li> -20 ° C.에 4.2ml 물에 100mg PLL을 해산하고 Eppendorf 튜브 및 동결에 300μl aliquots을 </li><li> 코팅 coverslips 먼저 해동 나누어지는가 ddH 2 O에서 10ml로 불러 및 코닥 포토 플로의 20μl를 추가하기 전에, 마지막 PLL 농도는 0.7 MG / ML입니다. </li><li> PLL 솔루션에서 30 분에 대한 잠수함의 coverslips은 제거하고 건조 후 물, 건조와 함께 간략하게 린스. 두 번 반복합니다. 치료 coverslips 지난 약 달. </li></ol></li></ol> 2. 유전 십자가 <ol><li> 티O는 MARCM의 클론을 생성합니다. 여기 팬 - DA 드라이버 11,27을 사용하여 예제 교차은 다음과 같습니다 : FRT2A X hsFLP; Gal4 109 (2) 80, UAS - mCD8 : GFP, 욕조 - Gal80 FRT2A/SM5-TM6B </li><li> 클론을 Flp 아웃 생성합니다. 여기 클래스 IV - 특정 드라이버 8을 사용 예제 교차은 다음과 같습니다 : PPK - Gal4 X yw, hsFLP, UAS - FRT - CD2, Y + - 중지 - FRT - mCD8 : : GFP </li></ol> 3. 배아의 수집 <ol><li> 25 수집 병에 Drosophila의 십자가 유지 ° C와 효모 붙여넣기 30,31의 얇은 레이어와 사과 쥬스 한천 플레이트 29 확산에 배아를 수집합니다. </li></ol> 4. 배아의 열 충격 치료 <ol><li> 배아가 마련되었습니다되었을 사과 쥬스 한천 플레이트에 반대 빈 접시를 놓습니다. Parafilm (그림 2)와 두 접시 주위에 인감. </li><li> 열충격 동안 잠수함의 일전자 물에 목욕의 판 및 금속 무게를 사용하여 누르십시오. </li><li> MARCM의 배아에 대한 <ol><li> 2 시간에 대한 배아를 수집하고 둘러싸인 10cm 배양 접시에있는 부화가 추가로 2 시간 25 ° C에서 조직을 moistened. 참고 : 열 충격 프로토콜을 조정하는 것은 클론이 생성되는 빈도를 바꿀지도 모르겠어. </li><li> 38에 1 시간을위한 물 목욕의 단일 절연 뉴런, 잠수함 및 열 충격을 목표로 클론의 작은 숫자 ° C.에 대한 </li><li> 클론의 큰 번호를, 38에서 45mins에 대한 열 충격 ° C는 추가 30 분 후 다시 RT 30 분, 열 충격에 복구할 수 있습니다. </li></ol></li><li> 24 시간에 대한 배아를 Flp 아웃 모으고, 38에서 1 시간에 대한 열 충격 ° C. </li></ol> 5. 클론에 대한 심사 <ol><li> 열 충격 후, 물을 꽉 배열의 뚜껑을 제거하고 moistened 조직으로 둘러싸인 10cm의 배양 접시에있는 사과 주스 한천 플레이트를 배치합니다. 문화 돌아다니다까지 배아 25시 이후의 유충 ° CING 3 층 instar. 참고 : 문화 환경, 특히 영양이 다 돌기 아버의 형태 32,33을 변경 표시되었습니다. 성장하는 애벌레 항상 효모 붙여넣기에 대한 액세스 권한이 있는지 확인, 필요에 따라 붙여 효모를 모니터링하고 보충. </li><li> 프로토콜 이후에이 시점에서, 부드럽게 곤충 집게를 사용하여 애벌레를 조작. </li><li> 간단히 부드럽게 수돗물의 애벌레를 린스, 그리고 한천 플레이트에 놓습니다. 참고 :이 단계는 배경 애벌레 표면에 음식이나 먼지로부터 자동 형광을 줄여줍니다. </li><li> 강력한 형광 해부 현미경으로 애벌레를 검사합니다. 본문 벽에 GFP - 긍정적인 뉴런 / 세포와 애벌레를 식별합니다. </li></ol> dendrites 6.에서는 생체내 이미징 <ol><li> 80 % 글리세롤의 작은 방울로 우울증 슬라이드 유리에 유충을 넣습니다. 유충를 무력화하기 위해 슬라이드에 coverslip 놓으십시오. 공기 유충과 coversli 사이에 남아 없다는 것을 확인P. </li><li> 부드럽게 관심의 신경 세포의 시각화 수 있도록 유충을 롤 coverslip 밀어. 이미지 mCD8 : 공촛점 현미경을 통해 GFP - 라벨이 돌기 아버. </li></ol> 7. 애벌레 해부 시작하기 전에 참고 : DA 신경 세포의 dendrites는 절개의 개시 이후 급격히 저하. 좋은 dendrite의 형태를 보장하기 위해 5 분 미만으로 각각의 유충을 해부하다. <ol><li> 다음과 같은 수정 이전 34 설명된대로 Sylgard 접시에 3 층 instar의 애벌레를 헤매고 해부하다 : <ol><li> DA 신경 세포 축삭 테르의 이미징 들어, CNS는 그대로이고 segmental 신경이 깨진되지 않도록. 애벌레를 연 후, 먼저 조심스럽게 몸 벽에 tracheal 연결을 절단하고 부드럽게 전체 창자를 제거합니다. </li><li> 혼자 이미징 dendrites 경우, 아첨이 장착 있도록 CNS를 제거합니다. </li></ol></li></ol> 8. Fixat애벌레 필레의 이온과 차단 <ol><li> 여전히 Sylgard 판에 고정된 유충으로 부드럽게 회전 흔드는에서 RT에서 20mins 위해 PBS에 4퍼센트 PFA에서 문제를 해결. </li><li> 고정 후 애벌레 조직의 성분 (지방 몸, 기관 등)를 제거합니다. </li><li> Sylgard 접시와 뿌리에 3 배 상업 중심지 (0.1 % 트리톤 X - 100 PBS)를 PBST로 씻으십시오. 축삭의 테르를 검사하면, Sylgard 판에 필렛을 유지. (얼룩의 dendrites 경우이 단계 34에서 0.5 ML 관에 애벌레 필레을 전송 가능합니다) </li><li> 뿌리에 PBST에 5퍼센트 정상 당나귀 혈청 (NDS)에 RT에서 20mins에 대한 애벌레를 차단합니다. </li></ol> 9. 애벌레 필레의 염색법 <ol><li> 차 항체에 차단 솔루션과 부화를 둘러싸인 작은 파티 간다 구 컨테이너에 ° C가 조직을 moistened 4 박 (NDS / PBST 5 %)를 제거합니다. </li><li> 4 ° C에서 튜브를 제거하고 RT에서 추가 시간을 품다. </li><li> PBST에서 배의 상업 중심지 씻으십시오. 보조 antib 추가ody은 (5 %에서 NDS / PBST) 및 커버 형광단 사진 - 표백 34 방지할 수 있습니다. </li><li> 두 시간 동안 RT에서 부화하거나, 또는 야간 4 ° C RT에서 한 시간 따라갔다. </li><li> PBST에서 애벌레 배의 상업 중심지를 씻어 및 장착을 참조하십시오. </li></ol> 10. dendrite 아버의 시험 애벌레 필레의 마운트 <ol><li> 해부 가위 또는 머리 (입 후크 포함), 34 (spiracles 포함) 뒷부분을 차단하기 위해 메스를 사용하여 가능한 한 평면 각 애벌레 필릿를 탑재합니다. </li><li> 슬라이드 아래 표피 사이드에있는 애벌레 필릿를 놓고, 80 % 글리세롤에 마운트하고, &#39;빠른&#39;마운트 34에 대한 매니큐어로 coverslip의 측면을 봉쇄. </li><li> 영구 장착하고 명확한 이미지 <ol><li> PLL의 coverslip에 PBS의 드롭에 해부 애벌레 등심 근육 쪽을 아래로 놓고 빨리 coverslip을 준수합니다. </li><li> howeve, 장착 후 가능한 한 많은 액체로 제거R은 완전히 건조하는 것을 허용하지 않습니다. </li><li> 35 % 에탄올, 50 % 70 %, 95 %, 각 10 분 (2 백퍼센트 EtOH 솔루션 자주 변경해야 결국 2X 100 % 에탄올 다음 : 에탄올 시리즈를 통해 coverslip을 (첨부 애벌레 등심)를 타고 ) 결국, 크실렌의 2 배 상업 중심지를 씻는다. </li><li> 깨끗한 슬라이드 DPX mountant (Distyrene의 가소제의 크실렌)의 방울을 넣고 조심스럽게 위에 등심 쪽 다운 coverslip을 믿는다. 어둠 속에서 계속 및 이미징하기 전에 설정하는 DPX 한 하루를 기다립니다. </li></ol></li></ol> 11. 축삭 테르의 시험 애벌레 필레의 마운트 <ol><li> segmental 신경 해부, 고정 기간 동안 그대로 PNS와 CNS 간의 실행 그리고 주변 감각 신경 세포 본체에서 VNC로 axons의 추적 수 있도록 염색법 유지. </li><li> 우울증 슬라이드에 80 % 글리세롤의 각 애벌레 등심 표피 쪽을 탑재합니다. DA 세포 본체에서에 axons를 추적공촛점 현미경으로 VNC. 참고 : VNC의 기원 세그먼트에 각 바디 벽 세그먼트 프로젝트에 PNS의 신경. </li></ol> 12. 대표 결과 : 대표 결과는 수치 3-5에 표시됩니다. 그림. 3 공촛점 현미경 생체내에서 캡처한 클래스 IV DA 신경 세포의 전체 아버를 보여줍니다. 그림. 4 제대로 형태를 보존하기 위해 수정되었습니다 immunohistochemically 분류 클래스 III 신경 세포의 dendrite 아버의 일부까지 가까이를 보여줍니다. 관련 삽입은 (안티 GFP 항체와 함께 모두 스테인드) 실패 절개 및 고정 후 발생할 수있는 저하를 보여줍니다. 그림. 5 CNS의 단일 클래스 IV 축삭 말단 (안티 - GFP, 녹색)을 보여줍니다, 모든 클래스 IV의 테르는 반 CD2 (마젠타)와 공동으로 얼룩진 있습니다. <img alt="그림 1" src="/files/ftp_upload/3111/3111fig1.jpg" /> 한 프로토콜 개요를 그림. A) 수집 후 열 충격 배아. B)<html> 라이브 유충에 다음 GFP 양성 DA 신경 세포 복제와 유충 및 이미지 GFP - 라벨 dendrite 아버을 선택합니다. C) 유충을 해부하다, 그리고 immunohistochemically 등심을 얼룩. D) 스테인드 필릿를 탑재하고 이미지를 돌기 아버과 DA 신경 세포 클론의 axonal 예상합니다. <img alt="그림 2" src="/files/ftp_upload/3111/3111fig2.jpg" /> 열 충격에 대한 사과 쥬스 한천 플레이트 2 준비 그림. 접시 (흰색 화살표, AB)를 타고, (빨간색 화살표 B) 위에 두 번째 페트리 접시를 추가하고 Parafilm (파란색 화살표, B)와 주변 인감. <img alt="그림 3" src="/files/ftp_upload/3111/3111fig3.jpg" /> mCD8의 dendrite 아버의 생체내 이미지 그림 3 : 제 3 instar의 유충의 클래스 IV (v&#39;ada) 뉴런을 나타내는 GFP - 표시 MARCM 클론 (2.1에서와 같이 유전자형). 스케일 바 50μm이다. d/3111/3111fig4.jpg &quot;/&gt; 그림 4 mCD8 : 성공 절개 및 고정 (녹색) 이후 좋은 형태를 보여주는 안티 - GFP 항체 물들일 클래스 III 신경 세포 (ddaA)의 GFP - 표시 MARCM 클론. 실패 해부와 고정 (마젠타) 이후 발생 삽입 보여줍니다 dendrite 저하 (흰색 화살표). 스케일 바 25μm이다. <img alt="그림 5" src="/files/ftp_upload/3111/3111fig5.jpg" /> 그림 5 제 3 회 instar 애벌레 VNC. 하나의 클래스 VI (vdaB) 축삭 말단 (2.2에서와 같이 유전자형)의 Flp 아웃 클론은 안티 - GFP 항체 (녹색)를 사용하여 감지됩니다. 안티 - CD2는 모든 클래스 IV 테르 (마젠타)를 표시합니다. 스케일 바 25μm이다.

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관심 없음 충돌 선언하지 않습니다.

Drosophila 애벌레 DA 신경 세포 모델은 메커니즘을 조사 한 우수한 유전자 시스템을 제공하는 제어 신경 세포의 형태 및 회로 형성. MARCM은 일반적으로 라벨 및 돌연변이 DA 신경 세포의 클론을 생성하는 데 사용됩니다. MARCM 위해 우리는 두 팬 - 신경 (예 : Gal4 c155) 또는 DA 뉴런 특정 드라이버를 사용합니다. 팬 - 신경 드라이버를 사용하면 직접 공개 주식 센터에서 널리 사용...

<table border="1"><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> SZX16 형광 해부 현미경 (GFPHQ 필터 포함) </td><td> 올림포스 산 </td><td> SZX16 </td><td></td></tr><tr><td> 곤충 집게 라이브 </td><td> FST </td><td> 26030-10 </td><td></td></tr><tr><td> 26mm X 76mm 우울증 슬라이드 유리 </td><td> Toshinriko (주) </td><td> T8 - R004 </td><td></td></tr><tr><td> Sylgard 184 (또는 Silpot 184) </td><td> 다우 코닝 </td><td> 3097358-1004 </td><td></td></tr><tr><td> 폴리 - L - 라이신 </td><td> 시그마 </td><td> P - 1524 </td><td> 이 제품은 가장 효과적인 입증되었습니다 </td></tr><tr><td> DPX 장착 매체 </td><td> 시그마 </td><td> 44,581 </td><td></td></tr><tr><td> 토끼 방지 GFP </td><td> Invitrogen </td><td> A - 11122 </td><td> 희석 1:500 </td></tr><tr><td> 쥐 방지 CD8 </td><td> Caltag </td><td> 5H10 </td><td> 희석 1:200 </td></tr><tr><td> 마우스 안티 - CD2 </td><td> ABD serotec </td><td> MCA443R </td><td> 희석 1:700 </td></tr><tr><td> 마우스 방지 Fasciclin2 </td><td> DSHB </td><td> 1D4 </td><td> 희석 1시 10분 </td></tr></table>

morphological analysis of drosophila larval peripheral sensory neuron dendrites and axons using genetic mosaics

신경 시스템 개발은 정확한 신경 클래스 특정 돌기 개발 및 axonal 배선 다음 신경 세포의 위치와 정체성의 정확한 사양을 필요로합니다. 최근 Drosophila 애벌레 말초 신경계 (PNS)의 돌기 arborization (DA) 감각 뉴런은 신경 세포의 분화 모두 일반 클래스 구체적인 메커니즘을 명료하게하다하는 강력한 유전자 모델이되고 있습니다. 네 가지 주요 DA 신경 세포 클래스 (I - IV) 1이 있습니다. 그들은 증가 dendrite 아버 복잡한 순서에 따라 이름이, 그들의 분화 20-10의 유전자 컨트롤에 클래스 구체적인 차이점을 가지고 있습니다. 1) 그것이 검찰 신경 dendrite 아버가 확산 과일 파리에서 사용할 수있는 강력한 유전자 도구, 2)를 활용할 수 있기 때문에 검찰 감각 시스템은 돌기 형태 11-13의 통제 뒤에 분자 메커니즘을 조사하기 위해 실용적인 모델입니다 광학 CLE 아래에 단지 2 차원에AR 애벌레의 표피)는 쉽게 생체내, 3 고해상도로 시각화하고 있습니다 돌기 형태의 클래스 특정 다양성)은 단순한 대 고도로 분지 돌기 나무의 형성을 제어하는 핵심 요소를 찾을 수 비교 분석을 용이하게, 4 틀에 박힌 돌기 아버 다른 검찰 뉴런의 모양은 morphometric 통계 분석을 용이하게합니다. DA 신경 세포 활동 애벌레 운동 중앙 패턴 발생기 14-16의 출력을 수정합니다. 다른 신경 세포 DA 수업은 독특한 감각 modalities를 가지고 있고, 자신의 정품 인증 14,16-20 다른 행동 반응을 elicits. 또한 다른 클래스는 복부 신경 코드에 Drosophila 애벌레 중추 신경계 (VNC) 21에 stereotypically axonal 전망을 보낼 수 있습니다. 이러한 예측은 돌기 분야 7,22의 본문 벽에 DA 뉴런 감각 양상과 위치를 모두 지형 표현과 종료, 23. DA axonal 추정치 그래서 시험은 지형 매핑 7,22,23뿐만 아니라, 간단한 회로 modulating 애벌레의 운동 14-17의 배선을 기본 메커니즘을 명료하게하다하는 데 사용할 수 있습니다. 우리는 여기서 MARCM (Repressible 세포 마커와 함께 모자이크 분석) 1,10,25과 Flp 아웃 22,26,27 기술 (그림 1.에 요약)를 통해 24 마킹 검찰의 뉴런을 생성하고 유전자 모자이크를 분석하는 실용적인 가이드를 제시한다.

Watch this Scientific Journal Video about Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics at JoVE.com

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

의 돌기 arborization 감각 뉴런<em&gt; Drosophila</em&gt; 애벌레 주변 신경 시스템은 일반 신경 세포의 분화의 신경 세포 수준의 특정 메커니즘 모두를 명료하게하다 유용 모델입니다. 우리는 돌기 arborization의 신경 세포 유전자의 모자이크를 생성하고 분석하는 실용적인 가이드를 제시한다.

의 형태학의 분석 Drosophila</em유전 모자이크 사용하기> 애벌레 주변 감각 신경 세포의 Dendrites 및 Axons

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

morphological-analysis-drosophila-larval-peripheral-sensory-neuron

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Neuroscience

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

15.2K 조회수.  RIKEN Brain Science Institute. 의 돌기 arborization 감각 뉴런 Drosophila 애벌레 주변 신경 시스템은 일반 신경 세포의 분화의 신경 세포 수준의 특정 메커니즘 모두를 명료하게하다 유용 모델입니다. 우리는 돌기 arborization의 신경 세포 유전자의 모자이크를 생성하고 분석하는 실용적인 가이드를 제시한다.

비디오: Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

Drosophila 애벌레 Segmental Axons 이내 시냅틱 Vesicles의 생체내 시각화에

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

연구

신경과학

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval body wall contains several cell types that are models to study cell and tissue morphogenesis. For example tracheae are used to examine tube morphogenesis1, and the dendritic arborization (DA) sensory neurons of the Drosophila larva have become a primary system for the elucidation of general and neuron-class-specific mechanisms of dendritic differentiation2-5 and degeneration6.
The shape of dendrite branches can vary significantly between neuron classes, and even among different branches of a single neuron7,8. Genetic studies in DA neurons suggest that differential cytoskeletal organization can underlie morphological differences in dendritic branch shape4,9-11. We provide a robust immunological labeling method to assay in vivo microtubule organization in DA sensory neuron dendrite arbor (Figures 1, 2, Movie 1). This protocol illustrates the dissection and immunostaining of first instar larva, a stage when active sensory neuron dendrite outgrowth and branching organization is occurring 12,13.
In addition to staining sensory neurons, this method achieves robust labeling of microtubule organization in muscles (Movies 2, 3), trachea (Figure 3, Movie 3), and other body wall tissues. It is valuable for investigators wishing to analyze microtubule organization in situ in the body wall when investigating mechanisms that control tissue and cell shape.

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

To understand how complex cell shapes, such as neuronal dendrites, are achieved during development, it is important to be able to accurately assay microtubule organization. Here we describe a robust immunohistological labeling method to examine microtubule organization of dendritic arborization neuron sensory dendrites, trachea, muscle, and other Drosophila larva body wall tissues.

Immunohistological 감각 신경 세포의 Dendrites에 Microtubules의 라벨링, Tracheae, 그리고 근육에 Drosophila 유충 바디 벽

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

사용하는 마우스 대뇌 피질과 해마의 시각화 및 Dendrites와 쪽이의 유전자 조작<em&gt; utero에서</em&gt; Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

Axons, 수석 및 뇌 슬라이스의 개별 뉴런의 수지상 쪽의 전압에 민감한 염료 기록

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

의 애벌레 먹이 회로의 기능적 분석 초파리</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

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 ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

생화학 적 분석에 대한 감각 뉴런에서 생산 및 축삭의 분리는 다공성 필터를 사용하여

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in communication with the epidermal cells they innervate and thus their analysis benefits from in situ visualization of both neuronally and epidermally expressed proteins by immunofluorescence. Traditional methods of preparing larval fillets for immunofluorescence experiments leave intact the muscle tissue that covers most of the body wall, presenting several challenges to imaging neuronal and epidermal proteins. Here we describe a method for removing muscle tissue from Drosophila larval fillets. This protocol enables imaging of proteins that are otherwise obscured by muscle tissue, improves signal to noise ratio, and facilitates the use of super-resolution microscopy to study da neuron development.

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall.

제거<em&gt; 초파리</em감각 뉴런과 표피 세포의 면역 형광 분석을위한 애벌레 필렛에서&gt; 근육 조직

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

선형 아가로 오스 채널을 사용하여 공부하기<em&gt; 초파리</em&gt; 애벌레 크롤링 동작

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

초파리 melanogaster Larval 및 성인 두뇌의 변화 분석

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

의 형태학의 분석 Drosophila 애벌레 주변 감각 신경 세포의 Dendrites 및 Axons