우리는 초파리 균주를 제공하기 위해 비엔나 초파리 리소스 센터 및 블루밍턴 초파리 재고 센터 (NIH P40OD018537)을 인정합니다. 우리는 영상 전문가 지원을 위해 대한 현미경 이미징 센터에서 잭 프랜슨합니다. 이 연구는 독일의 정신 지체 네트워크에 의해, 두 DCN / Radboud 대학 의료 센터의 박사 장학금으로, VIDI 및 과학 연구에 대한 네덜란드기구 (NWO)에서 TOP 보조금 (917-96-346, 912-12-109)에 의해 지원되었다 교육 및 연구 (BMBF)의 독일 연방 정부의 NGFN + 프로그램 및 AS에 대한 유럽 연합의 FP7 대규모 통합 네트워크 Gencodys (건강-241995)에 의해 투자. 자금 제공자는 연구 설계, 자료 수집 및 분석, 게시하는 결정, 또는 원고의 준비를 전혀 역할을 없었다.

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저자는 공개 관심의 충돌이 없습니다.

&quot;초파리 NMJ Morphometrics&quot;와 &quot;초파리 NMJ 부통 Morphometrics은&quot;시냅스 형태의 평가에 관심이 연구자를위한 강력한 도구입니다. NMJ 매개 변수의 수동 평가는 힘든 것입니다; 이 매크로는 15 분에 숙련 된 연구원을 절약 할 것으로 추정된다 / NMJ 수동 영상 분할에 보냈다. 조건 또는 유전자형에 따라 평가 시냅스 1 ~ 2 수십, 이것은 신속하게 아주 작은 규모의 연구에서, 저장 시간의 상당한 양을 요약한다. 대형 화면을 수행 할 때, 수동 평가 및 정량화에 비해 높은 처리량 분석을 사용할 때의 이득은 엄청난 수있다. 증가 된 처리량에 더하여, 매크로 용이 객관적인 분석을 제공한다; 그들은 그렇지 않으면 눈을 멀게 실험뿐만 아니라 여러 연구자가 분석에 관여 할 때 발생하는 대인 관계의 차이를 필요로 개인 편견을 제외 할 수 있습니다. 마지막으로, 매크로는 민감하고 정확한을 제공극적인 NMJ 결함보다는 미묘한 원인이 지금까지 연구자의 눈에 의해 진가를 인정받지 못한 남아있는 시냅스 규제의 식별을 허용 NMJ 기능 alysis. 검증 절차 및 매크로에 사용되는 알고리즘에 대한 자세한 정보는 게시 Nijhof 등의 알에서 발견된다. (17). 매크로의 기능을 적절하게 근육이어서 4에 초파리 melanogaster의 NMJs의 형태 적 특징을 측정하기 위해 확인 된, 상기 매크로이 유기체의 다른 근육에 시냅스를 분석하기에 적합 있다고 입증되었다. 이 매크로는 다른 초파리 종과 더 곤충을 포함한 다른 종의 유사한 구조와 NMJ의 형태 학적 매개 변수를 측정 할 수있는 가능성이 높습니다. 진화에 매우 먼 심지어 NMJs는, 예를 들면, 마우스의 NMJs는 매우 유사한 구조 형태를 보여 (29). 매크로는 다른 종에서 NMJ 준비에 테스트하지 않은 있지만, 잠재적 인 사용자는 이러한 목적으로 매크로를 테스트하는 것이 좋습니다. 사용자가 / 정의 이미지에 가장 적합한 매크로 설정을 선택할 수있는 다양한 자동 임계 값 및 알고리즘을 탐구하는 것이 매우 중요합니다. 매크로 평가는 수동 평가 비교할 때 이러한 설정으로, 약 95 %의 정확도가 얻어진다. 매크로 설정을 조정하면 제대로 세그먼트는 이미지의 100 %가 매우 힘든 또는 불가능 절차가 될 수 있습니다. 그들의 수는 5 % 이하 경우에 따라서 적절하게 분할되지 이미지를 배제하는 것이 좋습니다. 이미지의 품질이 낮 으면 분명히, 매크로는 만족스럽지 못한 영상 분할의 높은 비율을 생성합니다. 낮은 품질의 이미지는 유사하게 수동 평가에 영향을 미칠 것이다, 따라서 매크로의 성능에 링크 할 수 없습니다. 그들은 이미지 설계된대로 그럼에도 불구하고 매크로 오히려 견고높은 함량 현미경 (17) (샘플의 다수의 이미지를 허용하는 자동 형광 현미경) s의 생성. 중요한 점은 사용자가 시각적으로 매크로에 의해 생성 된 모든 결과의 이미지를 검사한다는 것이다. 이것은 감지하고 만족스럽지 분할로 사진을 제외 할 수 있습니다. 이 프로토콜 (6)에서, 사용자는 &quot;분석&quot;서브 매크로 실행시 정확한 이미지 분할의 설정을 조정하는 방법에 대해 안내된다. 빠르게 매크로의 요구 사항 및 방법 매크로 설정 &quot;매크로 설정을 Examples_adjusting&quot;라는 폴더를 조정하는 방법에 익숙해 매크로 저장소 https://figshare.com/s/ec634918c027f62f7f2a에 포함되어 있습니다. 예제 (고 함량 / 촛점 / 형광 현미경) 다른 현미경 플랫폼에서 얻어진 화상과 다른 immunostainings 열세 하위 각각이 제공된다. PDF 파일 권리 &quot;예 가이드는&quot;동일한에 포함되어 있습니다각 예제에 필요한 설정이 예상 결과와 결과 이미지를 제공하는 텍스트 문서와 함께 제공되는 폴더. 매크로는 티파니 파일을 분리로 저장된 이미지를 처리하도록 설계되었습니다, 그럼에도 불구하고 일부 사용자는 다른 형식으로 자신의 이미지를 저장 한 수 있습니다. 상세한 지침 문서 &quot;예 안내서&quot;어떻게 매크로로 이미지를 가져 - 다음 웹 사이트 https://figshare.com/s/ec634918c027f62f7f2a (21)는 세 개의 예제 파일 (3 예 1) &quot;초파리 NMJ&quot;라는 이름의 폴더가 티파니 분리 된 파일로 저장되지 않은 경우도 같은 폴더에서 찾을 수 있습니다. NMJ 지역, NMJ 주변, 튼스의 수, NMJ의 부통 지역, NMJ 길이, NMJ 긴 지점의 길이, 이슬라 번호 : 함께 &quot;초파리 NMJ Morphometrics&quot;와 &quot;초파리 NMJ 부통 Morphometrics&quot;매크로는 10 개 개의 다른 NMJ 기능을 정량화NDS, 지점의 수, 분기점의 수 및 활성 영역의 수. 이것은 단지 하나 또는 몇 시냅스 기능 (30), (31)을 평가할 수 있습니다 지금까지 사용 가능한 도구를 통해 큰 장점을 제공한다. Multiparametric 정량 분석은 시냅스 생물학의 여러 측면까지를 제어하는 새로운 규제를 식별하기 위해 새로운 발견, 예를 들어,에 대한 큰 잠재력을 맺는다. 또한 동일 또는 중복 NMJ 기능을 coregulate 따라서 일반적인 분자 경로에서 작동 할 가능성이 유전자를 결정하는 데 필요한 해상도를 제공합니다. 마지막으로, 유전자 시냅스 파라미터는 조정 된 형태 학적 상관 관계를 확인 교란 조건 (17)으로 서로 상호 얼마나 다른지 조사 할 수있는 가능성을 연다. 함께 찍은,이 프로토콜은 &quot;초파리 NMJ Morphometrics&quot;두 매크로를 사용하는 방법을 보여줍니다 및고 처리량 방법으로 열 개 형태 NMJ 기능 대물 민감한 정량화를 수행 &quot;초파리 NMJ Bouton은 Morphometrics&quot;.

이러한 지적 장애, 자폐증 스펙트럼 장애와 같은인지 장애, 정신 분열증은 종종 비정상적인 시냅스 기능 1, 2, 3을 특징으로한다. 시냅스의 형태와 기능은 밀접하게 얽혀있다; 형태 적 결함이 시냅스 오작동을 일으킬 수 있고, 역으로, 비정상적인 시냅스 전달은 시냅스 성숙 및 형태 4, 5, 6에 영향을 미칠 것이다. 모델 생물의 수는 더 나은 시냅스 생물학을 이해하고 시냅스 변화가 건강과 질병 7, 8, 9의 뇌 기능에 미치는 영향을 밝혀하기 위해 사용되어왔다. 초파리 NMJ은 광범위하게 연구하고 글루타메이트 SY에 대한 생체 내 모델에서 잘 확립napse 생물학 10, 11. 지난 수십 년 동안,이 모델은 NMJs 사이의 형태 학적 차이를 감지 할 목적으로,뿐만 아니라 대규모 유전 화면에 대한 생리 학적 유전자에 초점을 맞춘 연구에 사용되어왔다. 특히, 순방향 유전자 스크린 시냅스 개발 기초 많은 중요한 레귤레이터 및 메커니즘을 식별하여 12, 13, 14, 15, 16를 작동 하였다. 그러나, 이러한 화면의 대부분 NMJ 단말기 형태의 시각적 평가 및 정성 시냅스 이상 검출 또는 몇몇 형태 적 특징의 반 정량적 점수에 의존. 결과적으로, 인간의 눈에 비 명백하다 오히려 미묘한 시냅스 형태 학적 이상이 간과하기 쉬운. 위해서하는 것은 포괄적으로 양적 차이는 감지 할 수 있어야합니다NMJ 정확하게 관심의 형태 학적 매개 변수의 체계적인 정량화에 의해 평가되어야한다. 수동으로 NMJ 기능을 측정하는 것은 여러 NMJ 관심의 기능 및 / 또는 대규모 유전 검사를 수행 할 때이있다, 특히 힘든입니다. multiparametric 높은 처리량 형태소 해석을 지원하기 위해 객관적 정량을 달성하기 위해, 두 매크로 &quot;초파리 NMJ Morphometrics&quot;및 &quot;초파리 NMJ Bouton은 Morphometrics는&quot;17 개발되었다. 두 매크로는 오픈 소스 이미지 분석 소프트웨어 피지 (18)에서 실행, 모두 공 초점 및 nonconfocal 이미지를 정량화 할 수 있습니다. &quot;초파리 NMJ Morphometrics&quot;방안은 큰 1 시냅스 마커 디스크 (굴림-1) 또는 연접 고추 냉이 퍼 옥시 다제 (HRP)로 면역 염색 NMJ 단자는 활성 영역 마커 bruchpilot (BRP)와 CO는 표지. 그것은 구 형태 parame을 정량화TERS (이하에서 설명) NMJ 영역 NMJ 경계, 튼스 수, NMJ 길이 NMJ 긴 분기 길이, 섬의 수, 지점의 수, 분기점 및 시냅스 말단 활성 영역 번호의 수 (도 1) . 튼스의 수를 결정하는 알고리즘이 매크로에 존재하지만, 정확성 (17)에 대한 기준을 충족하지 않았다. 적절 튼스의 수를 평가하기 위해, 특별히 반대 Synaptotagmin (SYT를) 또는 항 시스테인 문자열 단백질 (CSP)으로 면역 염색 NMJ 제제를 사용 튼스 정량화하도록 설계된 &quot;초파리 NMJ Bouton은 Morphometrics&quot;매크로를 사용할 필요가있다, 및 BRP와 공동 immunolabeled. 은 &quot;초파리 NMJ Bouton은 Morphometrics&quot;매크로는 다음과 같은 매개 변수를 정량화 : 활성 ZO의 튼스의 수, NMJ의 부통 지역, NMJ 길이, NMJ 긴 지점의 길이, 섬의 수, 지점 수, 분기 지점의 수와 수를NES (도 2). 매크로 3 서브 매크로 구성 : (I)는 &quot;스택 변환&quot;모든 가능한 이미지 파일을 식별하고, Z-hyperstacks 두 채널의 최대 강도 예측을 생성한다. 출력으로,이 매크로는 &quot;stack_image_name&quot;와 &quot;flatstack_image_name&quot;라고 시냅스 당 두 개의 새로운 파일을 생성합니다. II) &quot;ROI 정의&quot;를 연속하여 모두 최대 투영 화상 &quot;flatstack_image_name을&quot;수동으로 열어 관심의 특정 시냅스 단말이 존재하는 관심 (ROI)의 영역을 정의하는 요청들을 제공한다. 이것은 이미지 (11)에 존재할 수있는 인접한 근육 및 / 또는 (예를 들면 1 초 등) 시냅스 말단 다른 유형의 시냅스 연결의 배제를 허용하도록 구현 하였다. (III) &quot;분석&quot;완전히 ROI의 경계 내에서 이미지의 모든 지역에 분석을 자동으로 적용합니다. 같이모든 측정 수치가 주석 될 &quot;RESULTS.TXT&quot;및 매크로에 의해 생성 된 원본 영상 분할이 설명 될 것이다 &quot;res_image_name.tif&quot;이 단계의 결과, 사용자는 두 개의 새로운 파일을 구하는 것이다. NMJ 윤곽은 NMJ 골격과 BRP-양극 활성 영역의 수 : 화상 분석 중에 세 구조는 각각의 시냅스 말단으로부터 유도된다. NMJ 개요는 NMJ 영역과 경계를 결정하기 위해 사용되며, 후속 유역 분리 튼스의 수를 제공한다. 골격에서 다섯 개 NMJ 기능이 도출된다 : 총 NMJ 길이, 두 끝점을 연결하는 가장 긴 연속 경로의 길이의 합 (긴 가지 길이), NMJ 당 연결되지 않은 구획의 수를 ( &quot;섬&quot;이라 ), 브랜치 수 및 분기점 (하나 개의 분기점이 세 개 이상의 분기 접속)의 수. 활성 영역의 수를 카운트하여 BRP 채널의 판단BRP 양성 명소. (주석 NMJ 윤곽 (황색 선)은 NMJ 골격 (청색 라인) 및 (백색 병소로 표시) BRP-양극 활성 영역의 개수는 결과 영상에 표시되는 상기 파라미터의 측정은로 처리된다. TXT) 출력 파일 (도 3). 초파리 NMJ Morphometrics &quot;및&quot;초파리 NMJ Bouton은 Morphometrics 초파리 NMJ Morphometrics &quot;및&quot;초파리 NMJ Bouton은 Morphometrics &quot;&quot;제 설명 광범위 Nijhof 외. (17)에 의해 확인되었다.이 논문은 매크로를 사용 NMJ 형태를 분석하는 방법에 초점을 맞추고있다. &quot; 그럼에도 불구하고, 매크로를 이용한 분석에 앞서, NMJ 해부 및 immunostainings은 이러한 중요한 단계이다. 수행해야하고, 면역 조직 화학에 사용되는 마커의 조합은 매크로 분석에 적합 할 필요가있다. 이러한 단계를 간략하게 언급되어있는 SE이 프로토콜 ction 1, 이러한 절차를 실행하도록 상세히 설명 프로토콜을 참조로 사용자를 안내.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Immunostainings</td>
			<td></td>
			<td></td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>Mouse anti-discs large 1</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>AFFN-DLG1-4D6</td>
			<td>1/25 (conjungated using the Zenon Alexa Fluor 528 Labeling Kit)</td>
		</tr>
		<tr>
			<td>Rabbit anti-horseradish peroxidase</td>
			<td>Jackson IR</td>
			<td>323-005-021</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Rabbit anti-Synaptotagmin</td>
			<td>Gift from Hugo Bellen</td>
			<td></td>
			<td>Jan-00</td>
		</tr>
		<tr>
			<td>Mouse anti-Cysteine string protein</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>DCSP-1(ab49)</td>
			<td>1/10 (conjungated using the Zenon Alexa Fluor 528 Labeling Kit)&#160;</td>
		</tr>
		<tr>
			<td>Mouse anti-Bruchpilot</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>nc82</td>
			<td>Jan-50</td>
		</tr>
		<tr>
			<td>Goat anti-mouse Alexa Fluor 488</td>
			<td>Life technologies</td>
			<td>A11029</td>
			<td>1/200</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit Alexa Fluor 568</td>
			<td>Life technologies</td>
			<td>A11011</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Zenon Alexa Fluor 568 Mouse IgG1 Labeling Kit</td>
			<td>ThermoFisher</td>
			<td>Z25006</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>ThermoFisher</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Material</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">Confocal microscope or fluorescence microscope</td>
			<td>Leica SP5</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Axio imager</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>Mac or Pc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Material</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

two algorithms for high-throughput and multi-parametric quantification of drosophila neuromuscular junction morphology

시냅틱 형태는 시냅스 효능에 밀접하게 관련되어, 많은 경우에 형태 학적 시냅스 결함은 궁극적으로 시냅스 오작동을 초래할. 초파리 애벌레 신경 근육 접합부 (NMJ), 글루타메이트 시냅스에 대한 잘 확립 된 모델은 광범위하게 수십 년 동안 연구되어왔다. NMJ 형태 학적 결함을 일으키는 돌연변이의 식별 시냅스 개발 및 기능을 조절하는 유전자의 레퍼토리를 한 것으로 밝혀졌습니다. 이들의 대부분은 초파리 NMJ의 형태 학적 이상을 감지하는 질적 접근 방식에 초점을 맞춘 대규모 연구에서 확인되었다. 질적 분석의 단점은 NMJ 형태에 기여 미묘한 플레이어 가능성이 주목 남아 있다는 것입니다. 정량 분석은 형태 학적 미묘한 차이를 검출하는 데 필요한 반면, 수고가 있기 때문에, 이러한 분석은 일반적으로 아직 수행되지 않는다. 이 프로토콜은 두 상세 이미지 분석 알고리즘의 설명 &quot;초파리 </em초파리 NMJ의 정량적 정확하고 객관적인 형태 학적 분석을 위해 피지 대응 매크로로서 사용할&gt; NMJ Morphometrics &quot;및&quot;초파리 NMJ Bouton은 Morphometrics &quot;이.이 방법은 일반적으로 사용되는 마커 immunolabeled NMJ 단자를 분석하기 위해 개발 굴림-1 . BRP는 또한, 같은 HRP CSP의 및 SYT를 다른 마커의 폭 넓은 응용 프로그램이 프로토콜에 표시되는 매크로는 구 개 형태 NMJ 기능을 평가할 수 있습니다. NMJ 지역, NMJ 주변, 튼스의 수, NMJ 길이를 NMJ 긴 지점 길이, 섬의 수, 지점의 수 및 분기점 NMJ 단말의 활성 영역의 수의 수.

Watch this Scientific Journal Video about Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology at JoVE.com

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

두 이미지 분석 알고리즘, &quot;초파리 NMJ Morphometrics&quot;및 &quot;초파리 NMJ Bouton은 Morphometrics&quot;가 자동 초파리 신경근 접합부 (NMJ)의 구 개 형태 적 특징을 정량화하기 위해 만들어졌다.

높은 처리량 및 다중 파라 메트릭 정량화를위한 ​​두 알고리즘<em&gt; 초파리</em&gt; 신경 근육 접합 형태학

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The ...

two-algorithms-for-high-throughput-multi-parametric-quantification

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JoVE Journal

Neuroscience

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

9.6K 조회수.  Radboud University Medical Center.  두 이미지 분석 알고리즘, "초파리 NMJ Morphometrics"및 "초파리 NMJ Bouton은 Morphometrics"가 자동 초파리 신경근 접합부 (NMJ)의 구 개 형태 적 특징을 정량화하기 위해 만들어졌다. 

비디오: Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

의 활성 영역의 해부 및 이미징 Drosophila 신경근육학 융티온

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

연구

신경과학

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

개발 마우스 망막의 생체내 Electroporation에

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

연속 신경 근육 분기점에서 싱글 Schwann 세포를 윤곽을 그리다하는 사진 표백

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

전신 탈염 신경 근육 정션 분석을위한 트랜스 대 복부 근육의 해부

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

검출<em&gt; 현장에서</em상기&gt; 단백질 - 단백질 복합체<em&gt; 초파리</em근접 내고 분석을 사용하기&gt; 애벌레 신경 근육 접합부

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

FIM Imaging and FIMtrack: Two New Tools Allowing High-throughput and Cost Effective Locomotion Analysis

The analysis of neuronal network function requires a reliable measurement of behavioral traits. Since the behavior of freely moving animals is variable to a certain degree, many animals have to be analyzed, to obtain statistically significant data. This in turn requires a computer assisted automated quantification of locomotion patterns. To obtain high contrast images of almost translucent and small moving objects, a novel imaging technique based on frustrated total internal reflection called FIM was developed. In this setup, animals are only illuminated with infrared light at the very specific position of contact with the underlying crawling surface. This methodology results in very high contrast images. Subsequently, these high contrast images are processed using established contour tracking algorithms. Based on this, we developed the FIMTrack software, which serves to extract a number of features needed to quantitatively describe a large variety of locomotion characteristics. During the development of this software package, we focused our efforts on an open source architecture allowing the easy addition of further modules. The program operates platform independent and is accompanied by an intuitive GUI guiding the user through data analysis. All locomotion parameter values are given in form of csv files allowing further data analyses. In addition, a Results Viewer integrated into the tracking software provides the opportunity to interactively review and adjust the output, as might be needed during stimulus integration. The power of FIM and FIMTrack is demonstrated by studying the locomotion of Drosophila larvae.

FIM is a novel, cost effective imaging system designed to track small moving objects such as C. elegans, planaria or Drosophila larvae. The accompanying FIMTrack program is designed to deliver fast and efficient data analysis. Together, these tools allow high-throughput analysis of behavioral traits.

FIM 이미징 및 FIMtrack : 높은 처리량 및 비용 효율적인 로코 분석을 허용하는 두 가지 새로운 도구

The analysis of neuronal network function requires a reliable measurement of behavioral traits. Since the behavior of freely moving animals is variable to ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

높은 처리량 분석은 개인의 알을 낳는 환경 설정을 검사합니다<em&gt; 노랑 초파리</em

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

상기 표현형의 특성을 : 정량이 중요한 이유<em&gt; 초파리</em&gt; 애벌레 신경 근육 학 분기점

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Quantification of Drosophila Grooming Behavior

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a grooming assay protocol and novel chamber design that is cost-efficient and scalable for either small or large-scale studies of Drosophila grooming. Flies are dusted all over their body with Brilliant Yellow dye and given time to remove the dye from their bodies within the chamber. Flies are then deposited in a set volume of ethanol to solubilize the dye. The relative spectral absorbance of dye-ethanol samples for groomed versus ungroomed animals are measured and recorded. The protocol yields quantitative data of dye accumulation for individual flies, which can be easily averaged and compared across samples. This allows experimental designs to easily evaluate grooming ability for mutant animal studies or circuit manipulations. This efficient procedure is both versatile and scalable. We show work-flow of the protocol and comparative data between WT animals and mutant animals for the Drosophila type I Dopamine Receptor (DopR).

This protocol describes a scalable individual grooming assay technique in Drosophila that yields robust, quantitative data to measure grooming behavior. The method is based on comparing the difference in dye accumulation on the bodies of un-groomed versus groomed animals over a set period of time.

Drosophila 그루밍 행동의 부량

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a ...

높은 처리량 및 다중 파라 메트릭 정량화를위한 ​​두 알고리즘

높은 처리량 및 다중 파라 메트릭 정량화를위한 두 알고리즘