This is supported by 973 MOST grant (Grant No. 2011CB504402, 2012CB947602), National Natural Science Foundation of China (Grant No. 91132724, U1332136) and the 'Hundred Talents Project' of Chinese Academy of Science. The protocol was approved by the Committee on the Ethics of Animal Experiments of the USTC (Permit Number: USTCACUC1103013).

1. 수술기구를 구축 참고 :시 및 작업, 드립 마취 솔루션 에틸 3 - 아미노 벤조 에이트 메탄 (MS-222, 또는 Tricaine) 물고기의 입을 통해 1 방울 / sec의 속도로 반 농도 (0.015 %)에서 후 물고기가 살아 남아 있도록하려면 그림 1 및 자작 물방울 시스템을 사용. <ol><li> 도 1a에 도시 된 바와 같이 상자 만들기 (길이 폭 높이 5 cm의 10 cm의 28 cm입니다), 중간 (길이 폭 9cm, 높이 4cm입니다 6 cm입니다) 스폰지를 넣어를 엽니 다 그것에 격차. </li><li> 다음 물고기 입에 삽입됩니다 광중합 결합 (그림 1B)와 바늘의 끝 부분에 부드러운 구형 표면을 확인합니다. 바늘의 표면이 손상되는 물고기의 입을 방지하기 위해 부드러운 지 확인합니다. </li><li> MS-222 (왼쪽 챔버, 길이 6 cm입니다의 절반 농도를 추가, 폭은 10 ℃이다m, 높이 8cm)와 그림 (c)에 도시 한 바와 같이 저수지에 식염수 (오른쪽 챔버, 길이, 높이 8 cm의 폭 10 cm의 4 cm입니다)입니다. 참고 : 신선한 MS-222을 추천합니다. </li><li> 2 분 동안 0.03 % MS-222 물고기를 마취. </li><li> 스펀지의 차이에 물고기를 놓고 가슴 지느러미 뒤에 각각 배설강 기공, 11 옆에 삽입 된 핀, 두 쌍의 물고기를 고정합니다. </li><li> 도 1C에 나타낸 바와 같이 T 형 관 물고기의 입, MS-222와 용수를 연결한다. </li><li> MS-222에 연결된 튜브를 켭니다. </li></ol> 오른쪽 Tectum 2. 역행 라벨 망막 신경절 참고 : 이전에 처음으로, 더 에탄올이 남아 있음을 보장 70~75% 에탄올로 모든 기기를 소독. <ol><li> Rossing : 조금 sclerectome 함께 전체 두개골 위에 피부를 제거 (두개골의 등쪽보기는도 2a 및도 2b에 도시 </stronG&gt;). </li><li> 지우기 : 식염수로 두개골을 씻어 준 후 귀 세정 전구 말립니다. </li><li> 초기 부식 : 10 초 동안 에칭과 두개골을 부식. </li><li> 지우기 : 완전히 면봉으로 에칭을 닦아 후 식염수로 씻어 내고 건조. </li><li> 보호 : 과잉 부식 오른쪽 tectum을 제외한 다른 지역에서 두개골을 보호하기 위해, 광 경화 결합으로이 분야를 커버하고 휴대용 광원과 푸른 빛에 노출을 치료. </li><li> 완전 부식 : 2 분 완료 에칭 다시 오른쪽 tectum의 중심 지역에 에칭을 넣어. 참고 : 이전 물고기의 두개골은 석회화 때문에, 부식이 오래 걸립니다. </li><li> 지우기 : 완전히 면봉으로 에칭을 닦아 후 식염수로 씻어 내고 다시 말린다. </li><li> 두개골 오프닝 : 조심스럽게 오른쪽 tectum (그림 2D를 노출하는 집게와 malacic 두개골 (그림 2C)를 제거 </strong&gt;). </li><li> 지우기 : gelfoam의 조각 구멍에서 점액과 혈액 effusing 닦아 출혈이 멈출 때까지 생리 식염수로 씻어. </li><li> 염료 위치 : 전체 바로 tectum에 염료 (조직 라벨 붙여 넣기)를 넣어 gelfoam로 커버. </li><li> 분리하는 : 같은 동물에서 멸균 규모로 구멍을 커버합니다. </li><li> 규모에 장소 빛 경화 결합을 한 후 40 초 동안 다시 푸른 빛으로 치료 : 바다 표범 어업. </li><li> 리바이벌 : 채권의 표면을 세척하고 식염수로 동물을 부활. </li><li> 간호 사육 : 5 일 동안 28.5 ° C 배아 매체 (ZFIN)에서 물고기를 유지하고 2 회 / 일 공급. 그 염료 실험에서 5 일 동안 tectum에서 숙박을 실패 물고기를 포함하지 마십시오. 참고 : 배아 매체 (EM) 솔루션은 물고기의 생존에 매우 중요하고 온도가 DII 라벨의 핵심 요소입니다. </li></ol> 3. Wholemounting 망막 및 이미징 <ol><li> 망막하기 전에 2 시간 동안 어두운 분야에서 물고기를 놓고 누구lemount. </li><li> 얼음 물과 물고기를 마취. </li><li> 각막의 가장자리에 구멍을 찔린 금성 가위로 각막을 제거하여 아이 컵을 만든다. </li><li> 렌즈를 제거하고 얼음처럼 차가운 1X PBS와 망막의 중심을 세척하십시오. </li><li> 더 안료가 남아 있지 될 때까지 망막과 얼음처럼 차가운 1X PBS과 공막 사이의 간격을 세척합니다. </li><li> 디스크에있는 시신경을 잘라. </li><li> 위로 RGC 층을 유지하고 자체 제작 유리 숟가락 (그림 3a 및 3b)와 망막을 잡아. 드롭퍼는 망막을 전송하는 데 사용될 수있다. </li><li> 위로 RGC 층 (그림 3C)와 슬라이드에 얼음 30 %의 글리세린이 저하 망막을 넣어. </li><li> 글리세린을 분리 한 다음 네 개의 사분면 (그림 3D)으로 망막을 절단하기 전에 유리 슬라이드에 망막을 평평하게. RGC 층으로 전개 망막을 유지하는 것은 위쪽으로 망막을 평평하게하는 데 도움이 될 것입니다. </li><li> t에 얼음 50 %의 글리세린 드립그는 안료의 조각을 씻어 망막. </li><li> 글리세린을 제거하고 망막에 얼음 75 %의 글리세린의 20 μl를 놓습니다. </li><li> 1.8 cm X 1.8 인치의 정사각형 커버 슬립으로 샘플을 커버. </li><li> 매니큐어로 커버 슬립을 봉인. 망막의 즉각적인 영상을 권장합니다. </li><li> 비 인터레이스 이미지 DP72 CCD (각 이미지는 1360 X 1024 픽셀)와 20 배의 대물 렌즈에서 전체 망막 (NA가 0.70 =). 각 필드는 세 가지 초점을 맞추고 있습니다. </li><li> 소프트웨어의 &quot;필드의 깊이를 확장&quot;의 도구를 사용하여 각 필드의 깊이를 확장 할 수 있습니다. </li><li> 바로 Photomerge의 기능을 가진 가상 망막을 조립합니다. </li><li> 망막 신경절의 평균 밀도 (그림 4A)를 분석하는 망막의 각 방향으로 680 X 512 픽셀의 3 개의 필드 (실제 면적이 0.092 mm 2, 264 μm의 350 μm의 경우)를 선택합니다. </li><li> 측정의 기능 영역을 구하는 - 이미지에 다각형 형상을 생성소프트웨어. </li><li> 식으로 망막 신경절의 총 수를 계산 &quot;망막 신경절 번호 ​​= 운명의 X 영역&quot;9. </li></ol>

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We have nothing to disclose.

망막 신경절의 역행 라벨은 포유류 동물의 망막 신경절의 생존을 연구하는 것이 중요하지만, 제브라 피쉬에서 사용되지 않았습니다. 다른 방법은, 그가 7 염색 및 항체 염색 (8), 망막 신경절의 수를 계산 골드 표준, 표시된 모든 망막 신경절과 유전자 변형 라인하지 않는 것은 아직 12, 13을 구성되지 않았습니다. 이 비디오에서는, 우리는 성인 zebrafish의 망막에 라벨 망막 신...

성인 제브라 피쉬가 계산하는 시신경 손상 1 일 이후 축삭을 재생 할 수있는 강력한 능력, 적절한 방법이으로 전체 망막 신경절은 망막 신경절의 생존과 재생 2을 평가하는 것이 필수적입니다. 포유 동물과 금붕어 3 역행 라벨 망막 신경절의 방법에 따라 - 5, 우리는 성인 제브라 피쉬의 tectum에서 망막 신경절 레이블을하는 방법을 건설했다. 성인 제브라 피쉬의 경우, 두 가지 중요한 기술적 인 문제는 주목해야한다 : 성인 제브라 피쉬의 두개골에는 6 개의 아주 작은; 그들은 물 환경에서 살고있다. 여기, 우리는 5 시추와 관련된 위험을 최소화 에칭액 두개골을 취급합니다. 다음, 우리는 수술 후 동물의 생존을 향상 광중합 결합으로 구멍을 밀봉. 이전에는 몇 가지 다른 기술은 간접 방법으로 망막 신경절의 수를 계산하기 위해 채택되었다. HE가 망막 부분에서 염색하면 RGCL 7 세포의 모든 종류의 라벨. 전체 R 항체 라벨링이러한 섬-1과 etina는 또한 무 축삭 세포 8 레이블을 지정할 수 있습니다. 시신경의 그루터기에서 역행 레이블이 망막에있는 망막 신경절 라벨을 할 수 있지만 그것은 시신경에 추가 부상의 원인이되기 때문에, 그것은 호감 모델에 채택 할 수 없습니다. tectum에서 역행 라벨을 활용, 우리는 시신경의 호감에서 망막 신경절의 생존과 재생을 연구했다. 결과는 거의 모든 망막 신경절이 살아 망막 신경절의 90 % 이상이 호감 모델 9의 첫 번째 주에 tectum에 재생 보여줍니다. 모두 성공적으로 망막 신경절 레이블을하기 위해, DII 페이스트는 여러 가지 다른 상업용 염료 (10)와 비교 후 선택되었다. 첫째, 특히 생체 조직 라벨링을 위해 설계되었습니다. 둘째, 물을 확산 할 수있는 친 유성 염료이다. 또한,이 형광은 망막 신경절 생존 연구를위한 훌륭한 후보하게 오랫동안 지속 할 수 있습니다.

<table border="0" cellpadding="0" cellspacing="0" width="608">
	<tbody>
		<tr height="37">
			<td height="37" style="height:37px;width:152px;">MS222</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:152px;">E10521</td>
			<td style="width:152px;">USA</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:152px;">DiI</td>
			<td style="width:152px;">Invitrogen</td>
			<td style="width:152px;">N22880</td>
			<td style="width:152px;">USA</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:152px;">lightcuring bond</td>
			<td style="width:152px;">Heraeus Kulzer&#160;</td>
			<td style="width:152px;">Durafill bond</td>
			<td style="width:152px;">Germany</td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;width:152px;">Gluma Etch</td>
			<td style="width:152px;">Heraeus Kulzer&#160;</td>
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retrograde labeling of retinal ganglion cells in adult zebrafish with fluorescent dyes

로 역행 라벨 망막 신경절 세포 (망막 신경절)이 사이트를 죽음에서 망막 신경절의 인 somata를 분리 할 수​​ 있습니다, 그것은 망막 신경절의 생존과 재생 실험에서 망막 신경절을 계산위한 황금 표준이되었습니다. 많은 연구 시신경 손상 후 망막 신경절 생존 조회 포유류 동물에서 수행되었다. 몇 가지 다른 방법은 망막 신경절 세포 레이어 (RGCL)의 세포 수를 셀 수 있지만 그러나, 성인 zebrafish에있는 망막 신경절의 역행 라벨은 아직보고되지 않았습니다. 성인 zebrafish의 두개골의 작은 크기와 두개골에 드릴링 후에 사망 고위험 감안하면 산 - 에칭에 도움으로 두개골을 열고 크게 생존율을 향상시킬 수있는 광 경화성 본드로 구멍을 밀봉. 5 일 동안 염료를 흡수 한 후, 거의 모든 망막 신경절이 표시되어 있습니다. 이 방법은 시신경을 절개 할 필요가 없다, 그것은 성인 zebrafish에있는 시신경의 호감 후 망막 신경절 생존의 연구에 바꾸어 놓을 수없는 것입니다. 여기, 우리는 소개이 방법을 단계별로하고 대표적인 결과를 제공합니다.

도 4B-D 쇼 같이, DII + 세포의 수는 RGCL 소재 DAPI + 세포의 3 분의 2이다. 정상적인 망막에서 전체 망막 (그림 4E)의 몽타주 이미지 DII + 망막 신경절이 중앙 지역에있는 망막 신경절이 그들의 목표에 다시 생성되지 않은으로 전체 망막에 있지만, 재생 된 망막 (그림 4 층)에 분포되어 있음을 보여줍니다 첫 주에, 그들은 표시 할 수 없습니다....

Watch this Scientific Journal Video about Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes at JoVE.com

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

형광 염료와 성인 Zebrafish의 망막 신경절 세포의 퇴행성 라벨링

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

retrograde-labeling-retinal-ganglion-cells-adult-zebrafish-with

Research

JoVE Journal

Neuroscience

9.4K 조회수.  University of Science and Technology of China. We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

비디오: Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

Zebrafish의 Hindbrain에 라벨 부착 및 이미징 셀

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

연구

신경과학

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 ...

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

전압에 민감한 염료와 크랩 Stomatogastric 신경절에서 뉴런의 광학 이미징

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

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 ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

역행 형광 라벨이 확인 된 신경 세포의 표적 세포 외 단일 단위 기록을 허용<em&gt; 생체 내</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. Generally, the dyes are applied as solid crystals or by local pressure injection using glass pipettes. However, this can result in dilution of the dye and reduced labeling intensity, particularly when several hours are required for dye diffusion. Here we demonstrate a simple and low-cost technique for introducing fluorescent and ion-sensitive dyes into neurons using a polyethylene suction pipette filled with the dye solution. This method offers a reliable way for maintaining a high concentration of the dye in contact with axons throughout the loading procedure.

We describe a simple and low cost technique for introducing high concentration of fluorescent and calcium-sensitive dyes into neurons or any neuronal tract using a polyethylene suction pipette.

신경, 책자, 그리고 형광 염료와 척수 뿌리 역행로드

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

교양 성인 지느러미 루트 신경절의 신경 세포와 축삭 재생의 유전 연구

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

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 ...

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, photoreceptor degeneration triggers M&#252;ller glial cells to re-enter the cell cycle and produce transient-amplifying progenitors. These progenitors continue to proliferate as they migrate to the damaged area, where they ultimately give rise to new photoreceptors. Currently, there are two widely-used LIRD paradigms, each of which results in varying degrees of photoreceptor loss and corresponding differences in the regeneration response. As more genetic and pharmacological tools are available to test the role of individual genes of interest during regeneration, there is a need to develop a robust LIRD paradigm. Here we describe a LIRD protocol that results in widespread and consistent loss of both rod and cone photoreceptors in which we have combined the use of two previously established LIRD techniques. Furthermore, this protocol can be extended for use in pigmented animals, which eliminates the need to maintain transgenic lines of interest on the albino background for LIRD studies.

Multiple light damage protocols have been described to damage photoreceptors and consequently induce a retinal regeneration response in adult zebrafish. This protocol describes an improved method that can be used in pigmented animals and that damages the vast majority of rod and cone photoreceptors across the entire retina.

성인 Zebrafish의 망막 재생 연구에있는 사용을위한 비발 한 가벼운 손상 패러다임

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, ...

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 ...