이 작업은 후베이 핵심 연구소 개설 프로젝트(보조금 번호 2021KFY055), 후베이성 자연 과학 재단(보조금 번호 2020CFB240) 및 중앙 대학 기초 연구 기금(보조금 번호 2042020kf0065)의 지원을 받았습니다.

모든 단계는 달리 지시되지 않는 한 실온에서 수행됩니다. 사용된 모든 C57BL/6J 마우스는 우한대학교 동물실험센터에서 입수하였으며, 모든 관련 실험은 우한대학교 동물실험윤리위원회의 승인을 받았다. 생쥐의 고통을 최소화하기 위해 모든 노력을 기울였습니다.
1. 눈의 적출 및 고정
<ol><li>생쥐를 이산화탄소 질식으로 안락사시키고 이빨이 없는 핀셋을 사용하여 안구를 부드럽게 적출합니다.</li>
	<li>인산염 완충 식염수(PBS)로 안구를 한 번 헹굽니다.</li>
	<li>현미경으로 각막에 주사기 바늘로 안구를 뚫습니다(접안렌즈 WF: 10x/22, 연속 배율 대물렌즈: 0.67x-4.5x) 안구로의 고정을 가속화하고 고정 효율을 향상시킵니다.</li>
	<li>4% 파라포름알데히드(PFA)를 함유한 24웰 플레이트로 옮깁니다.</li>
	<li>안구를 4% PFA에 45분 동안 고정한 다음 1x PBS로 옮겨 과잉 PFA를 즉시 제거합니다(3회 세척, 5분/회).</li>
</ol>2. 망막 분리 및 평탄화
<ol><li>안구를 유리 슬라이드로 옮기고 해부 현미경(접안렌즈 WF: 10x/22, 연속 배율 대물렌즈: 0.67x-4.5x) 아래에 놓습니다.</li>
	<li>한 손으로 집게를 잡고 각막의 가장자리를 고정하여 안구 움직임을 방지합니다. 다른 손으로 해부 가위를 잡고 각막 공막 윤부를 통해 약 1mm 깊이의 각막 주위를 자릅니다. 수정체와 유리체를 제거합니다.</li>
	<li>시신경을 중앙에 놓고 해부 가위를 사용하여 4 개의 사분면을 따라 방사상으로 망막을 절단하여 망막 반경의 약 2/3에 도달합니다.</li>
	<li>톱니 집게를 사용하여 공막 가장자리 중 하나를 클램프하십시오. 다른 손으로 톱니가 없는 집게로 공막의 가장자리를 잡고 공막이 톱니가 있는 집게에 의해 고정되는 곳에서 공막 바닥으로 이동합니다. 망막을 조심스럽게 껍질을 벗기십시오.</li>
	<li>피펫(200 μL)을 사용하여 PBS를 빼내어 망막을 플러시하고 적신 다음 작은 흡수성 종이로 과도한 PBS를 제거합니다. 필요한 경우 작은 강모 브러시로 망막을 부드럽게 평평하게 만듭니다.</li>
	<li>차가운 메탄올(-20°C 냉동고에서 예냉)을 망막 내부 표면에 한 방울씩 천천히 피펫팅합니다(망막이 메탄올로 완전히 덮일 수 있는 한 메탄올의 부피는 고정되지 않음). 망막이 하얗게 변하고 끈기가 증가합니다.</li>
	<li>망막을 부드럽게 평평하게하고 작은 강모 브러시로 잔류 색소 막과 유리체와 같은 불순물을 제거합니다. 작은 강모 브러시를 사용하여 망막을 뒤집고 위에서 언급 한 과정을 반복하십시오.</li>
	<li>메탄올-처리된 망막을 24-웰 플레이트의 웰로 옮기고, 메탄올에 담근 상태로 유지한다.</li>
	<li>즉각적인 면역 염색을 위해 -20°C에서 1-2시간 동안 보관하거나 망막을 메탄올에 그대로 두고 장기 보관을 위해 -20°C에서 보관하십시오.</li>
	<li>24-웰 플레이트로부터 메탄올을 제거하고, 망막을 1x PBS로 헹구었다.</li>
</ol>3. RGC의 면역형광 염색
<ol><li>망막을 플라스틱 파스퇴르 피펫으로 24웰 혈구 응집 플레이트로 조심스럽게 옮기고, 5% PBS-TX-BSA (1x PBST 용액 100mL에 용해된 소 혈청 알부민 분말 5g; PBST는 1 x PBS 2,000mL에 Triton X-100 10mL를 첨가하여 형성됨)를 실온에서 4시간 동안 또는 4°C에서 하룻밤 동안 차단합니다. 신경절 쪽을 위로 유지하십시오.</li>
	<li>PBS-TX-BSA를 제거하고, 1:400 희석액을 사용하여 선택된 1차 항체(기니피그 항-RBPMS 항체) 100 μL를 첨가하여 4°C에서 48-96시간 동안 망막을 배양합니다.</li>
	<li>1차 항체를 제거하고, 실온에서 진탕하면서 1x PBS로 망막을 3회(각각 20분) 헹구었다.</li>
	<li>1:400 희석액을 사용하여 해당 2차 항체( 재료 표 참조), 시아닌 Cy3 affiniPure 당나귀 항기니피그 IgG(H+L)와 함께 4°C에서 밤새 부드럽게 흔들면서 망막을 배양합니다.</li>
	<li>1x PBS로 망막을 세 번(각 20분) 헹굽니다.</li>
	<li>신경절 세포층이 위를 향하도록 슬라이드에 망막을 평평하게 놓고 피펫으로 망막 주위에 적절한 양의 형광 소광제를 떨어뜨린 다음 슬라이드를 커버슬립으로 덮습니다(기포가 생기지 않도록 주의). 알림: 적절한 양의 항형광 소광제를 결정하려면 전체 망막을 덮고 있는지 확인하십시오. 그런 다음 망막이 뜨지 않고 슬라이드를 덮고 쉽게 움직입니다.</li>
	<li>슬라이드가 움직이지 않도록 밀봉제로 슬라이드 전체를 밀봉하십시오. 샘플을 -20°C에서 보관하고 빛으로부터 보호하십시오.</li>
	<li>형광/컨포칼 현미경을 사용하여 망막을 이미지화합니다.</li>
</ol>

망막 신경절 세포 연구를 위한 마우스 망막 분리 및 메탄올 치료

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저자는 이 연구의 객관성에 영향을 미칠 수 있는 소속, 회원 자격, 자금 또는 재정 보유에 대해 알지 못합니다.

고정은 망막을 보존하기 위한 필수 단계이며, 이는 형태학을 기반으로 한 후속 RGC 조사에 영향을 미칠 수 있습니다. 성공적인 고정은 망막을 고정 매체에 노출시키는 순간 망막의 구조와 상태를 빠르게 포착하며, 이는 추가 분석에 중요합니다. 포름 알데히드는 조직 및 세포 고정 및 보존을위한 가장 일반적인 고정제 중 하나로 간주되어 왔지만, 포름 알데히드만으로는 일부 조사에서 최적의 화학...

망막 신경절 세포(Retinal ganglion cell, RGC)는 망막에 있는 유일한 투영 뉴런으로, 외부 시각 정보를 통합하여 뇌에 전달한다1. 녹내장 및 외상성 시신경병증과 같은 많은 신경퇴행성 질환은 비가역적 손상과 RGC의 손실을 특징으로 한다 2,3. RGC의 형태학적 및 정량적 변화를 분석하는 것은 신경퇴행성 질환이 어떻게 발생하고 진행되는지를 결정하는 중요한 단계입니다 4,5.
간접 면역형광 분석은 단백질 분포 및 세포 계수를 모니터링하는 데 널리 사용되는 방법입니다. 실험실에서 망막의 생리학적 및 병리학적 상태를 평가하기 위해 RGC에 대한 실험적 연구에서 전체 마운트 망막 면역염색법이 일반적으로 사용됩니다6. 전체 망막에서 RGC 정량화에 사용되는 가장 일반적인 마커는 Brn3a, 다중 스플라이싱을 갖는 RNA 결합 단백질(RBPMS) 등을 포함합니다 7,8. RGC의 양과 분포를 특성화하려면 고품질의 전체 마운트 망막 면역염색이 필요합니다. 일반적으로, 면역염색 프로토콜에서, 망막은 항체에서 배양되기 전에 화학적 고정제에 담근다. 이상적인 고정제는 세포의 모양, 항체에 대한 에피토프의 접근성 또는 친화도, 또는 조직의 선형 치수를 변화시키지 않아야 한다 9,10.
망막의 복잡한 구조로 인해 망막의 취약성 및 접힘과 같은 문제뿐만 아니라 세포 수축 및 불분명한 핵을 포함한 몇 가지 일반적인 어려움이 전체 마운트 망막 패치를 만들 때 발생하기 쉬우며 이는 실험 연구에 부정적인 영향을 미칩니다. 또한, 모든 망막이 즉시 면역염색되는 것은 아니며, 특히 귀중한 기원의 값비싼 가격을 가진 형질전환 마우스의 망막의 경우 추가 사용을 위해 여분의 망막 샘플을 보존해야 합니다.
적절한 고정액은 조직을 신속하게 고정하고, 조직 자가분해를 피하고, 조직 세포의 정상적인 형태와 구조를 보존하고, 단백질 및 기타 물질의 항원성을 유지할 수 있다10. 현재, 포름알데히드계 고정술은 분리된 망막, 반절제된 안구, 안구 전체를 포함한 다양한 조직에서 널리 사용되고 있다10. 조직 수축과 세포의 형태학적 변화는 포름알데히드에 담근 후 직면하게 되는 두 가지 중요한 문제이다11. 또한, 망막 및 표적 세포의 본래 특성의 보유를 최대화하기 위해 변형된 고정 제형이 점점 더 등장하고 있다 9,10. 다른 망막 고정 치료는 망막 구조, 단백질 면역원성, 형광 여기 및 감쇠 소멸 주기에 다르게 영향을 미칠 수 있습니다12,13. 데이비슨 용액으로 고정된 망막은 포르말린으로 고정된 망막에 비해 형태학적으로 더 온전하지만, 데이비슨 용액은 소교세포 마커-이온화 칼슘 결합 어댑터 분자 112와 같은 일부 항체와의 호환성이 떨어진다. 망막의 깨지기 쉬운 특성을 고려할 때 연구자들은 망막 무결성과 표적 세포의 특성 및 형태가 장기 보관 후에 변할지 여부를 자연스럽게 궁금해할 것입니다. 그러나 수개월 보관 후 망막 및 RGC 세포 형태에 대한 고정 용액의 가능한 효과는 거의 보고되지 않았습니다. 망막 고정의 최적화는 RGC의 평가 및 보존에 중요합니다.
우리는 전체 마운트 쥐 망막 염색에 사용하는 신뢰할 수 있고 기술적으로 간단한 방법에 대한 자세한 설명을 제공합니다. 우리의 방법은 망막 조직의 장기 보관의 필요성과 형광단 형성 또는 분해의 특정 측면을 고려하여 RGC 조사를 위한 망막의 적절한 준비 및 보관을 강조합니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-well cell culture cluster</td>
			<td>Costar</td>
			<td></td>
			<td>Eyeball fixation</td>
		</tr>
		<tr>
			<td>24-well hemagglutination plate</td>
			<td>Labedit Company</td>
			<td></td>
			<td>Incubation antibody</td>
		</tr>
		<tr>
			<td>Adhesion microscope slides</td>
			<td>Citotest</td>
			<td></td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Anti-fluorescent quenching mountant</td>
			<td>Servicebio</td>
			<td>G1401</td>
			<td>Slow down fluorescence quenching</td>
		</tr>
		<tr>
			<td>BSA (bovine serum albumin)</td>
			<td>Servicebio</td>
			<td>GC305010</td>
			<td>Blocking reagent</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>OLYMPUS</td>
			<td></td>
			<td>Apply 40x objective lens</td>
		</tr>
		<tr>
			<td>Curved scissors</td>
			<td>Jiangsu Kanghua Medical Equipment Co., Ltd.</td>
			<td></td>
			<td>Dissecting tools</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>RWD Life science Co.,LTD&#160;</td>
			<td>77001S</td>
			<td>Dissecting tools</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Jiangsu Kanghua Medical Equipment Co., Ltd.</td>
			<td></td>
			<td>Dissecting tools</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd.</td>
			<td>20210624</td>
			<td>GC&#8805;99.5%</td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>SecheVite</td>
			<td></td>
			<td>Sealing agent</td>
		</tr>
		<tr>
			<td>Needles&#160;</td>
			<td>Shanghai Kindly Enterprises Development Group Co., Ltd.</td>
			<td></td>
			<td>Accelerate the fixation</td>
		</tr>
		<tr>
			<td>Paraformaldehyde solution</td>
			<td>Servicebio</td>
			<td>G1101</td>
			<td>Eyeball fixation</td>
		</tr>
		<tr>
			<td>PBS (phosphate buffered saline pH 7.4)</td>
			<td>Servicebio</td>
			<td>G0002</td>
			<td>Rinse the eyeball&#160;</td>
		</tr>
		<tr>
			<td>Primary antibody: guinea pig anti-RNA-binding protein with multiple splicing (RBPMS)</td>
			<td>PhosphoSolutions</td>
			<td>Cat. #1832-RBPMS</td>
			<td>For immunofluorescence. Used at 1:400</td>
		</tr>
		<tr>
			<td>Secondary antibody: Cy3 affiniPure donkey anti-guinea pig IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>706-165-148</td>
			<td>For immunofluorescence. Used at 1:400</td>
		</tr>
		<tr>
			<td>Straight scissors</td>
			<td>Jiangsu Kanghua Medical Equipment Co., Ltd.</td>
			<td></td>
			<td>Dissecting tools</td>
		</tr>
	</tbody>
</table>

methanol-based whole-mount preparation for the investigation of retinal ganglion cells

망막의 투영 뉴런인 망막 신경절 세포(RGC)는 외부 시각 정보를 뇌로 전파합니다. RGC의 병리학 적 변화는 수많은 망막 퇴행성 질환과 밀접한 관련이 있습니다. 전체 마운트 망막 면역염색은 망막의 발달 및 병리학적 상태를 평가하기 위해 RGC에 대한 실험적 연구에서 자주 사용됩니다. 어떤 상황에서는 형질전환 마우스의 샘플과 같은 일부 귀중한 망막 샘플을 RGC의 형태나 수에 영향을 미치지 않고 장기간 보관해야 할 수도 있습니다. 신뢰할 수 있고 재현 가능한 실험 결과를 위해서는 효과적인 보존 배지를 사용하는 것이 필수적입니다. 여기에서 우리는 망막 전체 장착 준비 및 장기 보관을 위한 보조 고정 배지로서의 메탄올의 효과를 설명합니다. 간단히 말해서, 분리 과정에서 차가운 메탄올(-20°C)을 망막 표면에 피펫팅하여 조직을 고정하고 투과성을 촉진한 다음 망막을 면역염색하기 전에 차가운 메탄올(-20°C)에 보관할 수 있습니다. 이 프로토콜은 RGC 조사에 유용하고 실용적인 망막 분리 워크플로 및 조직 샘플 보관 프로토콜을 설명합니다.

해부 후 망막은 편평한 네잎 클로버처럼 보일 것입니다. 이 연구에서는 위에서 설명한 프로토콜을 사용하여 메탄올을 첨가한 후 망막이 하얗게 변했습니다(그림 1). 한편, 망막은 부드러움에서 유연하고 평평한 것으로 바뀌었습니다. 다음으로, RGC는 anti-RBPMS8로 라벨링되었습니다. 컨포칼 현미경(접안렌즈: 10x, 대물렌즈: 40x)을 사용하여 전체 마운트 망막(n = ...

Watch this Scientific Journal Video about Methanol-Based Whole-Mount Preparation for the Investigation of Retinal Ganglion Cells at JoVE.com

저자 스포트라이트: 망막 신경절 세포 연구를 위한 메탄올 기반 전체 장착 준비  

메탄올은 망막 전체 마운트 제제 및 장기 보관을 위한 보조 고정 배지로 사용할 수 있으며, 이는 망막 신경절 세포의 조사에 유용합니다.

망막 신경절 세포 조사를 위한 메탄올 기반 전체 마운트 준비

Retinal ganglion cells (RGCs), which are the projection neurons of the retina, propagate external visual information to the brain. Pathological changes in ...

이 연구는 RGC를 조사하는 데 유용하고 실용적인 수정된 망막 분리 워크플로 및 조직 샘플 저장 프로토콜을 제공하는 것을 목표로 합니다. 망막은 깨지기 쉽고 접혀 있어 전체 마운트 망막 패치를 만들 때 실험적 연구를 제한합니다. 또한 모든 망막은 즉각적인 면역 염색이 필요하지 않습니다.때때로, 여분의 망막 샘플은 추가 사용을 위해 보존이 필요합니다. 우리는 RGC 조사를 위한 망막의 적절한 준비와 장기 보관의 필요성을 강조하는 전체 마운트 망막 염색의 신뢰할 수 있고 기술적으로 간단한 방법에 대한 자세한 설명을 제공합니다. 우리는 퇴행성 시신경 병증의 기초 연구를 계속할 것입니다.또한, 시신경 손상 및 사고 재생의 분자 메커니즘과 신경퇴행성 질환의 진단 및 치료를 위한 잠재적인 임상 표적을 탐구할 것입니다.

methanol-based-whole-mount-preparation-for-investigation-retinal

Research

JoVE Journal

Neuroscience

Methanol-Based Whole-Mount Preparation for the Investigation of Retinal Ganglion Cells

2.9K 조회수.  Renmin Hospital of Wuhan University. 이 연구는 RGC를 조사하는 데 유용하고 실용적인 수정된 망막 분리 워크플로 및 조직 샘플 저장 프로토콜을 제공하는 것을 목표로 합니다. 망막은 깨지기 쉽고 접혀 있어 전체 마운트 망막 패치를 만들 때 실험적 연구를 제한합니다. 또한 모든 망막은 즉각적인 면역 염색이 필요하지 않습니다.때때로, 여분의 망막 샘플은 추가 사용을 위해 보존이 필요합니다. 우리는 RGC ?...

비디오: 저자 스포트라이트: 망막 신경절 세포 연구를 위한 메탄올 기반 전체 장착 준비  

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

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

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

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

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

연구

신경과학

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

망막 안료 상피의 전체 마운트를위한 마우스 아이의 해부

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

마우스 망막 신경절 세포 Transfection로 생체내에 Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

성인 전체 마운트 준비 Drosophila</em장 섬유 염료 주입을위한> 복부 신경 코드

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

마우스 망막 신경절 세포의 시냅스와 인공 컨덕턴스와 동적 클램프 구현

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

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 ...

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 ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

간단한 한 단계 성인의 전체 마운트 준비에 대한 해부 프로토콜<em&gt; 초파리</em&gt; 두뇌

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

망막 신경절 세포의 정의 된 서브 세트의 단일 세포 RNA-Seq

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

광학 일관성 단층 촬영: 마우스 망막 신경 절 세포에서 Vivo에서 화상 진 찰

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...