이 연구는 보조금 F31DC017053-01A1 (Lenell, PI) 및 K23DC014517 (존슨, PI) 청각 장애및 건강의 국립 연구소의 다른 통신 장애에 대한 국립 연구소에서 지원되었다.

이 연구는 뉴욕 대학 의과 대학의 기관 동물 관리 및 사용 위원회에 따라 수행되었습니다.
1. 해부 쥐 후두
<ol><li>제도적으로 승인된 프로토콜에 따라 쥐를 안락사시합니다. 하악에서 마누브리움으로 복부 목을 면도하고 술로 면봉하여 조직 표본의 모피 오염을 방지합니다.</li>
	<li>10배배율을 가진 해부 범위에서 기관체가 노출될 때까지 메스로 중간선 목 절개를 만들어 후두 전체를 소비한다.</li>
	<li>중간선에서 복부 외두 근육을 분리하여 포셉을 사용하여 후두를 노출하고 가위 또는 메스를 해부합니다.</li>
	<li>기관가 를 제3 기관 고리로 분리하고 해부 가위를 사용하여 전체 후두를 소비하기 위해 효이드 뼈에 절개 로스트랄을 만듭니다.</li>
	<li>배율하에서 미세 절도구(핀셋, 핀, 미세시)를 사용하여 후두에서 외두 조직(식도, 갑상선 및 외두 근육)을 제거합니다.</li>
	<li>미세 한 부와 함께, 후위 cricotenoid 근육 사이 중간 라인을 사용 하 여 아리티노이드 사이 후두를 이중으로. 후두의 측면 벽을 핀으로 표시하여 보컬 주름을 노출한 다음 갑상선 연골의 중간선을 통해 미세 시저로 보컬 주름의 전방 분리(도 1)를 통해 양분하게 선회한다. 참고: 이 단계는 선택 사항일 수 있습니다. 후두전체를 유지하기 위해 건너뛸 수 있습니다. 후두의 양절은 동일한 후두의 오른쪽과 왼쪽을 별도로 사용하여 다중 면역 염색 기술을 허용합니다.</li>
	<li>인산완충액(PBS)에서 각각 헤미후두를 헹구고, 동결 시 얼음 결정 형성을 줄이기 위해 작업 와이퍼로 섬세하게 건조시킵니다.</li>
</ol>2. 후두 조직을 수정 및/또는 플래시 동결
참고: 고정은 모든 면역 염색 프로토콜에 적합하지 않을 수 있습니다. 종종 후두 조직은 해부 직후 에새로 얼어 붙습니다. 단계 2.1을 건너뛰고 후두 조직을 고정하지 않고 플래시 동결시합니다.
<ol><li>헤미 후두를 해결하기 위해 70 rpm의 궤도 셰이커에서 실온에서 1 시간 동안 PBS에서 4 % 포름 알데히드로 채워진 원심 분리기 튜브에 조직을 배치합니다. 깨끗한 원심분리기 튜브로 조직을 옮기고 PBS에서 20분 동안 3배 헹구십시오. 그런 다음 깨끗한 원심분리기 튜브로 옮기고 4°C에서 20% 자당/5% 글리세롤 용액(~18h 또는 조직이 가라앉을 때까지)에 침수한다. 주의: 포름알데히드는 위험하며 적절한 개인 보호 장비와 함께 연기 후드에 사용해야합니다.</li>
	<li>모든 헤미 후두를 균일한 위치에 배치하여 최적의 절삭 온도(OCT) 화합물로 채워진 냉동 금형에 넣습니다. hemilarynx의 경우, 극저온의 바닥을 향한 보컬 폴드의 내측 표면과 극저온 개구부의 하부 가장자리에 평행하게 보컬 접힌의 세로 측면을 가진 조직을 배치한다. 전체 후두의 경우, 극저온의 바닥을 향한 후방 리코리테노이드와 극저온 개구부의 하부 가장자리에 평행하게 보컬 접힌 종방향 측면을 가진 조직을 놓습니다. 참고: OCT 화합물 내에서 일관된 후두 방향은 쥐 보컬 폴드의 극저분균에 매우 중요합니다. 헤밀리엔스가 내장되고 얼어 붙으면 방향을 바꾸기 위해 해동하여 여러 해동 동결 주기로 인한 조직 손상의 위험을 소개해야 합니다.</li>
	<li>액체 질소로 둘러싸인 강철 비커로 냉각된 isopentane(2-메틸부탄)를 사용한 플래시 동결 조직. 참고: isopentane는 백색 침전이 비커13의 측면과 바닥에 형성하기 시작할 때 조직 동결을 위한 최적 온도에 도달합니다. 이스펜탄은 액체 질소보다 열 전도도가 높기 때문에 사용되며, 이는 급속한 동결 시 조직 블록의 균열을 방지하는 데 도움이 됩니다. OTC에서 조직을 동결에 대한 자세한 설명은 쿠마르 외.13을 참조하십시오.</li>
	<li>각 금형을 라벨이 붙은 호일로 감싸고 개별 냉동고 가방에 넣어 탈수방지하고 -80°C 냉동고에 보관할 때까지 드라이 아이스에 즉시 보관하십시오.</li>
</ol>3. 단면 평면의 극저부 헤밀리엔스
<ol><li>냉동저온을 -20°C로 설정하여 제조업체 매뉴얼에 의한 근육 조직 단면에 권장되는 온도 범위(15-25°C)의 중간에 있습니다.</li>
	<li>10 μm 두께의 섹션으로 저온 분면 두께를 설정합니다. 참고: 근육 섬유 단면 분석의 경우, 10 μm 두께의 단면은 섬유 타이핑 분석을 위한 라벨이 부착된 근육 섬유의 완전한 염색 및 견고한 이미징 강도를 허용하는 것이 최적입니다14,15,16. 일부 프로토콜신경 근육 대상에 따라 다른 단면도 두께 를 요구할 수 있습니다.</li>
	<li>극저온 챔버로 조직을 옮기고, 저온 시편 디스크(chuck)에 OCT 화합물의 균일한 층을 추가하고, 임베디드 조직 블록을 표본 디스크 위에 놓습니다. 흉선(TA) 근육 섬유 분석을 위한 보컬 접이식의 단면을 얻으려면, 척에 표본을 부착하여 복부 갑상선 연골이 극저온 블레이드를 마주하고 아리티노이드 연골이 표본 디스크에 직면하도록 한다. 참고: 동결시 OCT 화합물이 흰색과 불투명해짐에 따라 이러한 랜드마크가 이 단계에서 표시되지 않습니다. 이러한 가시성 부족은 플래시 동결 단계에서 헤밀리엔스의 방향을 주목하는 것이 중요한 이유입니다.</li>
	<li>갑상선 연골의 복부 부분이 나타날 때까지 표본 머리를 100 μm로 전진시킴으로써 OCT 화합물을 트림합니다.</li>
	<li>그런 다음 갑상선 연골발병부터 라미나 프로프리아, 내측 TA 근육 및 측면 TA 근육이 노출될 때까지 30 μm 섹션을 손질하고 추적합니다. 참고: 후두 랜드마크는 단면각도가 비스듬히 되지 않도록 100μm마다 갑상선 연골이 발병하여 추적하여 주목해야 합니다. 도 2 는 10배배율의 단면 평면에서 두 개의 후두 랜드마크 세트를 나타냅니다.</li>
	<li>대상 TA 근육에 도달하면 10 μm에서 양전하 슬라이드의 섹션을 수집합니다.</li>
	<li>PBS에 섹션을 4°C로 저장하여 염색할 준비가 될 때까지 수분을 유지합니다. 참고: 고정 된 조직은 IHC 표적에 따라 최대 1 주일까지 PBS에 저장 될 수 있지만 고정되지 않은 조직은 즉시 처리되어야합니다.</li>
</ol>4. 경도 평면의 극저부 헤밀리엔스
<ol><li>저온 챔버가 다시 -20°C로 설정되면 단면 두께를 30 μm로 변경합니다. 참고: NMJ 분석의 경우, 30-60 μm 사이의 조직 두께는 신경 단자 또는 모터 엔드플레이트11,12,17의 단편화 없이 후두 근육 내의 몇 가지 완전한 NMJ를 캡처하는 데 사용할 수 있습니다.</li>
	<li>TA 근육의 NMJ 분석을 위한 세로 성보컬 접이식 부분을 얻으려면, 후면이 극저온 블레이드를 향하도록 척에 표본을 부착하고 기관 루멘은 시편 디스크쪽으로 아래로 향합니다.</li>
	<li>갑상선 연골이 나타날 때까지 표본 머리를 100 μm로 전진시켜 OCT 화합물을 다듬습니다.</li>
	<li>TA 근육의 라미나 프로프리아및 내측 및 측면 분열이 드러날 때까지 갑상선 발병으로부터 30 μm의 섹션을 손질하고 추적합니다. 참고: 세로 평면의 후두 랜드마크 5세트는 표적 TA 근육을 향한 조직 깊이 진행을 추적하는 것이 좋습니다. 도 3 은 10배배율로 세로 평면의 후두 랜드마크를 나타낸다.</li>
	<li>표적 TA 근육에 도달하면 30 μm에서 양전하 슬라이드에 대한 섹션을 수집합니다.</li>
	<li>PBS에 섹션을 4°C로 저장하여 염색할 준비가 될 때까지 수분을 유지합니다.</li>
</ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+neuromuscular+junctions+of+laryngeal+and+limb+muscles+in+rats.">Differences in neuromuscular junctions of laryngeal and limb muscles in rats.</a> Laryngoscope. 122 (5), 1093-1098 (2012).</li></ol>

저자는 공개 할 것이 없습니다.

신경 근육 분석을 위한 쥐 보컬 주름을 준비하는 것은 각종 도전으로 제시할 수 있습니다. 후두 근육은 작고 연골에 둘러싸여 있어 표적 근육을 직접 추출하기가 어려워질 뿐만 아니라 후두 해부학 적 랜드 마크의 깊이에서 동물 들 사이에서 높은 가변성을 발견했습니다. 근육의 경우, 전체 보컬 접이식 섹션은 NMJ 분석 프로토콜(표 1)에 대한 종로 평면에서 63-126 단면(섹션당 35 μm)보다...

쥐 후두는 발달, 노화, 질병 및 약리학 에이전트에 구조적이고 기능적인 신경 근육 후두 적응을 조사하는 잘 확립 된 모델입니다1,2,3,4,5. 조직학적 방법의 일관성은 후두 연골 내에서 캡슐화 된 근육의 후두 크기, 모양 및 지형과 관련된 도전뿐만 아니라 근육 준비 및 분석에 관련된 여러 가지 복잡성이 있기 때문에이 작업 라인에 중요합니다1,6,7,8,9,10,11 . 쥐 내성 후두 근육의 작은 크기로 인해 체계적인 포함, 동결 및 냉동 절개는 일관되고 정확한 결과를 달성하는 데 중요합니다. 예를 들어, 관상 평면에서 쥐 보컬 접을 분리할 때, 내래 후두 근육 4개 중 4개의 신경근육 접합부(NMJs)는 조직 깊이11의 1.8mm 미만 내에 위치한다. 따라서, 동결 절제 동안 후두 근육 해부학의 정밀한 모니터링은 정확하게 관심의 섹션을 식별하고 조직의 과부를 방지하는 것이 필수적이다. 대상 근육의 과분은 NMJs11의 수와 지형을 부정확하게 식별할 수 있거나 랜드마크 방향 혼동으로 인해 대상 근육이 버려지면 시료 크기가 전반적으로 감소될 수 있다12. 후두 근육과 각각의 적응에 대한 새로운 모델이 개발됨에 따라 연구 전반에 걸쳐 결과가 정확하고 신뢰할 수 있으며 재현가능한지 확인하는 표준 수술 절차가 필수적입니다.
이 문서의 목적은 최적의 세로 및 단면 분석을 위해 쥐 보컬 폴드의 세부 준비입니다. 우리의 실험실에서 정기적으로 사용되는 상세한 방법은 냉동 절제 동안 대상 근육 랜드 마크를 식별하기 위해 설명된다. 유사한 방법은 여러 실험실에서 사용되지만 초보 조사관이 구현할 때 신뢰할 수 있고 정확한 복제를 보장하기 위해 문헌보다 더 자세한 정보가 여기에 제공됩니다. 이 튜토리얼의 목표는 실험실 및 조사 전반에 걸쳐 일관성을 개선하기 위해 쥐 보컬 접이의 면역 조직화학 (IHC) 평가를위한 표준 방법론을 제공하는 것입니다.

<table>
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			<td>Cryostat blades</td>
			<td>C.L. Sturkey D554X50</td>
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			<td>Thermo Scientific</td>
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			<td>Medline</td>
			<td>23-666-062</td>
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			<td>Thermo Scientific</td>
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			<td>Immunostain Moisture Chamber</td>
			<td>Ted Pella Inc</td>
			<td>NC9425474</td>
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			<td>Needle holders</td>
			<td>Assi</td>
			<td>ASSI.B148</td>
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			<td>Non-Woven Sponges, 4 Ply</td>
			<td>Quick Medical</td>
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			<td>Paraformaldehyde, 16% w/v aq. soln., methanol free</td>
			<td>Alfa Aesar</td>
			<td>50-00-0</td>
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			<td>Premium Microcentrifuge Tubes</td>
			<td>Fisherbrand</td>
			<td>5408129</td>
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			<td>Specimen Storage Bags</td>
			<td>Fisherbrand</td>
			<td>19240093</td>
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			<td>Stainless Steel Graduated Measure 32 oz/100 mL</td>
			<td>Polar Ware</td>
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			<td>Superfrost Plus Microscope Slides</td>
			<td>Fisherbrand</td>
			<td>12-550-15</td>
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			<td>Task wiper</td>
			<td>Kimberly-Clark Professional&#8482; 34155</td>
			<td>06666A</td>
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			<td>Timer</td>
			<td>Fisherbrand</td>
			<td>2261840</td>
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			<td>Vannas Pattern Scissors</td>
			<td>Assi</td>
			<td>ASSI.SAS15RV</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">NOTE: For all supplies, these are examples of equipment to purchase. The exact model is not necessary to complete our methods.</td>
		</tr>
	</tbody>
</table>

preparation of the rat vocal fold for neuromuscular analyses

이 튜토리얼의 목적은 조직화학 신경 근육 연구에 대한 쥐 보컬 접기의 준비를 설명하는 것입니다. 이 프로토콜은 쥐 후두 해부, 플래시 동결 및 보컬 주름의 저온 절제에 대한 절차를 간략하게 설명합니다. 이 연구는 세로 및 단면 평면 모두에서 저온 절 보컬 주름을 하는 방법을 설명합니다. 이 프로토콜의 참신은 내성 후두 근육의 정확한 식별을 보장하고 조직 손실의 기회를 감소 극저온 절제 하는 동안 후두 추적. 수치는 두 평면에서 점진적 극저온절을 보여줍니다. 29마리의 쥐 헤미 후두는 갑상선 연골의 출현에서 전체 보컬 접이를 포함하는 첫 번째 섹션의 출현까지 극저고 를 추적했다. 전체 보컬 폴드는 두 평면의 모든 동물을 위해 시각화되었습니다. 갑상선 연골의 출현에서 두 평면에서 전체 보컬 폴드의 출현까지 의 거리에서 높은 가변성이 있었다. 무게는 후두 랜드 마크의 깊이와 상관 관계가 없었다, 개별 가변성 및 조직 준비와 관련된 다른 요인은 단면 동안 랜드 마크의 출현에 높은 가변성에 대한 책임이있을 수 있음을 시사. 이 연구는 방법론을 자세히 설명하고 조직 화학 신경 근육 조사를위한 쥐 보컬 접이를 준비하기위한 형태학적 데이터를 제시합니다. 높은 개별 가변성 때문에, 후두 랜드 마크는 조직과 조직 손실을 과분하는 것을 방지하기 위하여 극저온 절제 도중 밀접하게 추적되어야 합니다. 쥐 후두 내의 랜드 마크의 적절 한 조직 준비 및 인식을 포함 하 여 일관 된 방법론의 사용, 연구 전반에 걸쳐 일관 된 결과 지원 하 고 후두 신경 근육 메커니즘을 조사 하는 모델로 쥐 보컬 접을 사용 하 여 관심 있는 새로운 연구원을 원조.

대표적인 결과는 후두 신경 근육 시스템에 음성 운동의 효력의 지속적인 조사의 일부였습니다. 29 남성 피셔 344/갈색 노르웨이 쥐 (12 9 개월, 17 24 개월)는 CO2 흡입으로 무게와 안락사 되었다 양측 thoracotomy.
절차는 NMJs와 측면 및 내측 TA 근육의 섬유 크기를 표시하는 설명 된 프로토콜을 따랐다. 후두 랜드 마크 사이의 거리는 후두 근육과 주변 연골을 사용하여 경도 및 ?...

Watch this Scientific Journal Video about Preparation of the Rat Vocal Fold for Neuromuscular Analyses at JoVE.com

Preparation of the Rat Vocal Fold for Neuromuscular Analyses

이 프로토콜은 조직 화학 신경 근육 연구에 대 한 쥐 보컬 주름을 준비 하는 데 사용 하는 방법을 설명 합니다.

신경 근육 분석을위한 쥐 보컬 폴드의 준비

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures ...

preparation-of-the-rat-vocal-fold-for-neuromuscular-analyses

Research

JoVE Journal

Neuroscience

2.6K 조회수.  New York University School of Medicine. 이 프로토콜은 조직 화학 신경 근육 연구에 대 한 쥐 보컬 주름을 준비 하는 데 사용 하는 방법을 설명 합니다.

비디오: Preparation of the Rat Vocal Fold for Neuromuscular Analyses

Morphometric Analyses of Retinal Sections

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)&#x2013;induced excitotoxicity1, retinal ischemia-reperfusion injury2 and glaucoma3. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity4. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure3,5,6. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.
The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.
 	This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience &mdash; MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

망막 섹션 Morphometric 분석

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

연구

신경과학

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 ...

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 ...

Microstate and Omega Complexity Analyses of the Resting-state Electroencephalography

Microstate and omega complexity are two reference-free electroencephalography (EEG) measures that can represent the temporal and spatial complexities of EEG data and have been widely used to investigate the neural mechanisms in some brain disorders. The goal of this article is to describe the protocol underlying EEG microstate and omega complexity analyses step by step. The main advantage of these two measures is that they could eliminate the reference-dependent problem inherent to traditional spectrum analysis. In addition, microstate analysis makes good use of high time resolution of resting-state EEG, and the four obtained microstate classes could match the corresponding resting-state networks respectively. The omega complexity characterizes the spatial complexity of the whole brain or specific brain regions, which has obvious advantage compared with traditional complexity measures focusing on the signal complexity in a single channel. These two EEG measures could complement each other to investigate the brain complexity from the temporal and spatial domain respectively.

This article describes the protocol underlying electroencephalography (EEG) microstate analysis and omega complexity analysis, which are two reference-free EEG measures and highly valuable to explore the neural mechanisms of brain disorders.

미세 및 휴식 상태 Electroencephalography의 오메가 복잡도 분석

Microstate and omega complexity are two reference-free electroencephalography (EEG) measures that can represent the temporal and spatial complexities of ...

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ex vivo preparation of the levator auris longus (LAL) is used because it is a thin muscle that provides easy visualization of the neuromuscular junction for microelectrode impalement at the motor endplate. This method allows for the recording of spontaneous miniature endplate potentials and currents (mEPPs and mEPCs), nerve-evoked endplate potentials and currents (EPPs and EPCs), as well as the membrane properties of the motor endplate. Results obtained from this method include the quantal content (QC), number of vesicle release sites (n), probability of vesicle release (prel), synaptic facilitation and depression, as well as the muscle membrane time constant (&#964;m) and input resistance. Application of this technique to mouse models of human disease can highlight key pathologies in disease states and help identify novel treatment strategies. By fully voltage-clamping a single synapse, this method provides one of the most detailed analyses of synaptic transmission currently available.

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

The protocol described in this paper uses the mouse levator auris longus (LAL) muscle to record spontaneous and nerve-evoked postsynaptic potentials (current-clamp) and currents (voltage-clamp) at the neuromuscular junction. Use of this technique can provide key insights into mechanisms of synaptic transmission under normal and disease conditions.

Levator Auris Longus 포유류 신경 근육 학 전송 전압 클램프 조건에서의 시험을 위한 준비

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...

Collection of Frozen Rodent Brain Regions for Downstream Analyses

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5&#8211;1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 &#176;C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.

다운스트림 분석을 위한 냉동 설치류 뇌 영역 모음

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

면역형광및 전체산 신경근육 접합의 형태 분석에 대한 단일 골격 근육 섬유의 해부

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative.
This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.