<p class="jove_content"&gt;이 작품은 국립 신경 장애의 연구소와 국립 보건원에서 뇌졸중의 교내 프로그램에 의해 지원되었다. 우리는 또한 메서드와 그림 3의 데이터에 대한 그의 이전 공헌 박사 조지 Mentis 감사드립니다.</p

형광등 dextrans은 해부 학적 도구로 및 이미징의 연결 활동 1-4 위해 사용되었습니다. 필드 외., 2009 년 4 모델 시스템으로 척수에 초점 axonal 책자로 이온 및 전압에 민감한 염료를 적용하기위한 프로토콜을 발표했다. 여기에서-체외 optophysiological 및 형태학의 연구를 위해 컷 복부 뿌리, 지느러미 뿌리 또는 척수의 넓이의 연결로 형광 및 / 또는 이온에 민감한 염료를 적용하기위한 자세한 절차를 설명합니다. 1. 타입 I과 타입 II의 Pipettes 테이퍼 팁과 pipettes를 생성하기 위해 알코올 램프 7 불꽃 위에 폴리에틸렌 튜빙의 두 단편 섹션 (PE90, 클레이 아담스 브랜드)을 벗어서 시작합니다. 한 피펫 (타입 I)은 단단히 대상 루트 또는 넓이를 (:; 내경 0.2-0.4 MM : 0.1-0.3 mm 외경) 저장할 수있는 작은 팁을 함께 (3-7밀리미터) 짧은이어야합니다. 그런 다음 두 번째 피펫 (Type을 끌어-II) 이상 (8~12센티미터) 얇고 축과 아주 좋은 팁 (외경과 : 0.2-0.3 mm, 내경 : 삽입할 수 0.1-0.2 ㎜) 타입 - 난만큼 피펫 그 끝. 두 번째 얇은 피펫은 타입-I 피펫에서 각각 인공 뇌척수 (aCSF)와 염료 솔루션을 대기음하고 소개하는 데 사용됩니다. 2. 의 위치 유형 - 전 피펫 해부 및 척수에게 8,9를 분리하고 로딩 과정 전반에 걸쳐 냉장 aCSF (~ 16 ° C)로 superfused 욕조,에 넣습니다. 타입-I는 다시 오프닝 쉽게 유연한 튜브를 통해 주사기 (1ml U-100 인슐린 주사기, Becton Dickenson 또는 비교)에 연결될 수 있도록 전극 홀더 (H1/12 전극 홀더, Narishige)에 피펫 (PharMed를 배치 BPT, 콜 - Parmer, # AY242002, 10 ㎝) (Fig.1A-B). 3. 염료 신청 <ol><li> 주사기를 사용하여 먼저 그리기유형 - 전 피펫 (Fig.1B, 2A)로 aCSF은 axonal 요로 또는 (그림 2B) 자필 서명하려면 루트로 따라갔다. 플렉시블 튜브 그런 다음 다시 개통에서 제거되어야합니다. 타입-II 피펫은 다른 주사기에 부착된 및 유형 - 전 axonal 요로 또는 루트 개최 피펫에 삽입됩니다. 그래서 잔여 aCSF 방금 axonal 요로 (; 2C-D Fig.1C)를 포함하는 타입-II 피펫, 기음 aCSF에 부착된 주사기를 사용합니다. 그런 타입-I 피펫에서 타입-II 피펫을 철회. axonal 요로이나 신경 좋은 도장을 확인하기 위해 몇 분 동안 aCSF 수준 ( &quot;좋은 씰&quot;에 대한 문제 해결에 제 3 항 참조) 모니터링합니다. </li><li> aCSF의 색소 또는 더블 증류수에 0.2 % 트리톤 X-100를 해산하고 기포를 포함하지 않도록 신경을 쓰는 동안 타입-II 피펫으로 그것을 그립니다. 유형 - 전 피펫으로하고 잔류 aCSF 솔루션 (그림의 2E)로 염료를 포함하는 타입-II 피펫을 넣습니다. 천천히 풀어부드러운 긍정적인 압력 (그림 2F)를 사용 aCSF에 염료. 모든 공기 방울을 소개하거나 팁 안쪽에서 대상 조직의 변위를 발생하지 않도록주의하십시오. 염료 충분한 양의 (염료 용액 3-5 μL) 출시 이후 타입-II 피펫을 철회. </li></ol> 4. 잠복 실험의 종류에 따라 6-20시간 사이 어둠 속에서 조직을 품어. 일단 충분한 시간 충전에 허용되어 부드럽게 영상이나 조직학을위한 조직 준비 떠난 조직에서 전극을 멀리 당기십시오. 문제 해결 <ol><li> 대상 조직의 유형 - 내가 피펫의 일각에 쉽게 입력하거나 한 내부 헐거워되지 않으면 다음 팁 직경은 잘못된 크기입니다. 이런 경우 뽑아 다른 피펫을 사용합니다. </li><li> 타입-I 피펫의 aCSF에 염료를 추가하기 전에 충분한 aCSF는 그것에 도달할 수 있도록 남아 있는지 확인타입-II 피펫 팁 (그림 2D). 염료가 추가되면 그렇지 않으면, 에어 갭이 존재합니다. 이런 경우 염료를 추가하고 피펫에서 axonal 요로를 제거한 다음 유형-II 피펫에 의해 도달할 수있는 충분한 aCSF으로 다시 그려하지 않습니다. </li><li> 타입-I 피펫 증가에 aCSF 수준이 그것이 타입-II 피펫으로 aspirated되자면 조직과 인감이 좋지 않다는 것을 의미합니다. 다른 전극을 사용, 그렇지 않으면 염료가 라벨 과정에서 희석됩니다. </li><li> 염료를 주입하면서 공기 방울이 유형 - 전 피펫으로 도입되면, 풍선에 타입-II 피펫 팁 주변을 발전 부드럽게 연결된 주사기를 사용하여 약한 부정적인 압력을 적용하여 거품을 배수. </li></ol> 5. 대표 결과 방법의 응용을 설명하기 위해서, 우리는 동시에로드 motoneurons, 감각 afferents 세 가지 dext와 척수 interneurons형광 염료 (그림 3A) 복합 조사 했어요. 다른 이름으로 그림 그림. 신경 세포의 3B, 세 강좌로 분류되며, 관능 afferents (적색), 복부 삭조 (녹색)과 motoneurons (파란색)으로 돌출 interneurons. <img alt="그림 1" src="/files/ftp_upload/4008/4008fig1.jpg" /> 1 그림. 개략도 피펫을 보여주는 타입 I과 타입 II 조립. () 타입 - 나는 그것이 개통이 쉽게 유연한 엘라스토머 튜브 (B)에 연결하고있는로 타입 II 피펫은 (C) 삽입될 수 돌아와요 있도록 피펫 (하늘색)는 전극 홀더에 배치됩니다. <img alt="그림 2" src="/files/ftp_upload/4008/4008fig2.jpg" /> 그림 2. 도식은 형광 염료와 motoneurons의 로딩 과정을 보여주고 있습니다. () 타입-I의 피펫은 자필 서명 복부 루트 근처에 위치합니다. (B) 흡입이 루트 다음에 피펫으로 aCSF을 그릴 수있는 피펫에 적용됩니다. (C-D) 종류 - 내가 피펫의 백엔드에서 유연한 엘라스토머 튜브를 분리하고 타입-II 피펫으로 흡입을 적용하여 aCSF의 양을 줄일 수 있습니다. (E) 타입-I 피펫의 나머지 aCSF에 녹아있는 염료를 포함하는 타입-II 피펫의 팁을 넣습니다. (EF) 천천히 염료와 유형 - 전 피펫을 채울 후 타입-II 피펫을 제거합니다. <img alt="그림 3" src="/files/ftp_upload/4008/4008fig3.jpg" /> 그림 3. 고립된 마우스 척수로 작성 절차의 응용. () 도식이 보여주는 세 가지 유형 - 전 척수의 연결에서 다른 클래스를 작성하는 데 사용 pipettes.에게 Motoneurons (파란색, 캐스케이드 블루 dextran)와 성례의 감각 afferents (적색, 텍사스 - 빨강 dextran)은 각각 복부와 지느러미 뿌리를 통해 backfilled되었다. 플루오레신 dextran은 누구의 axons 복부 삭조 (VF)를 통해 승천 척추 interneurons를 (녹색)로드하는 데 사용되었습니다. 각 dextran 염료의 1 밀리그램 (10,000 MW, 분자 프로브)6 μL 증류수는 0.2 %를 포함에 녹아되었다 트리톤 X-100 (B). 라벨 감각 afferents 몇 contralateral 전망과 주로 ipsilateral 것을 A. 노트에 설명된대로 뉴런은 백 라벨이 붙지만하는 척수의 성례의 코드에서 섹션 공촛점 이미지입니다. 표시된 interneurons는 채우기의 측면 (화살표 머리)에 contralateral 반면 표시된 motoneurons이 가득한 복부 루트에 ipsilateral 있습니다. 200 μm의 보정 바.

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<p class="jove_content"&gt; 우리는 공개 할게 없다.</p

<p class="jove_content"&gt; 여기 뉴런, 신경 및 척수의 책자로 염료를 도입을위한 간단하고 비용 효율적인 프로토콜을 설명합니다. 이 방법은 로딩 과정의 기간 동안 고도의 농축 염료 용액에 확인된 해부 학적 경로를 노출하는 것입니다. 약간의 배경과 대상 사이트의 역행 라벨이 결과 microinjections, 욕조 응용 프로그램과 electroporation 기술에 비해. 그러나 방법은 axons의 넓이는 격리 수 신경계의 사이트로 제한됩니다. 염료...

<table border="1"&gt;<tbody&gt;<tr&gt;<td colspan="2"<strong&gt; 자재 명칭</strong</td&gt;<td<strong&gt; 회사 소개</strong</td&gt;<td<strong&gt; 카탈로그 번호</strong</td&gt;<td<strong&gt; 코멘트</strong</td&gt;</tr&gt;<tr&gt;<td rowspan="2"&gt; 튜브</td&gt;<td&gt; PE90 (IDxOD : 0.034 &quot;x0.050&quot;; 벽면 Thikness : 0.008 &quot;)</td&gt;<td&gt; 클레이​​ 아담스 브랜드 Intermedic</td&gt;<td&gt; 427,421</td&gt;<td&gt; 타입 I &amp; II 타입 pipettes위한</td&gt;</tr&gt;<tr&gt;<td&gt; NSF-51 (IDxOD : 1/16x1/8, 벽면 Thikness : 1 / 32)</td&gt;<td&gt; PharMed BPT, 콜 - Parmer</td&gt;<td&gt; AY242002</td&gt;<td&gt; 플렉시블 튜빙의 경우</td&gt;</tr&gt;<tr&gt;<td&gt; 주사기</td&gt;<td&gt; 1ml 인슐린 주사기 U-100</td&gt;<td&gt; Becton Dickenson</td&gt;<td&gt; 329,650</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; 니들</td&gt;<td&gt; 19G 1 개-1 / 2 &quot;메탈 허브 니들</td&gt;<td&gt; MONOJECT</td&gt;<td&gt; 200,136</td&gt;<td&gt; 주사기로 타입 II 피펫 연결하기</td&gt;</tr&gt;<tr&gt;<td&gt;</td&gt;<td&gt; 알콜 램프</td&gt;<td&gt;</td&gt;<td&gt;</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; 꼭지</td&gt;<td남성 Luer 슬립 어댑터&gt; 세 방향 꼭지</td&gt;<td&gt; 박스터 헬스케어 주식 회사</td&gt;<td&gt; 2C6241</td&gt;<td&gt; 주사기와 함께 사용</td&gt;</tr&gt;<tr&gt;<td rowspan="3"&gt; 홀더</td&gt;<td&gt; H-1 전극 홀더</td&gt;<td&gt; Narishige</td&gt;<td&gt; H-1/12</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; 자석 스탠드</td&gt;<td&gt; Narishige</td&gt;<td&gt; GJ-8</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; Micromanipulator</td&gt;<td&gt; Narishige</td&gt;<td&gt; M-3333</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; 족집게</td&gt;<td&gt; S &amp; T 뒤몽 스위스 00632-11</td&gt;<td&gt; 뒤몽</td&gt;<td&gt; JF-5 TC</td&gt;<td&gt;</td&gt;</tr&gt;</tbody&gt;</table&gt;<p class="jove_content"&gt; *들은 제안 자료입니다. 어떤 호환 제품으로 교체할 수 있습니다</p

retrograde loading of nerves, tracts, and spinal roots with fluorescent dyes

뉴런의 역행 라벨은 또한 뉴런 3-6 칼슘 및 전압에 민감한 염료를로드하는 데 사용되었습니다 표준 해부 방법 1,2입니다. 일반적으로 염료는 고체 크리스털이나 유리 pipettes를 사용하여 로컬 압력 주입에 의해 적용됩니다. 그러나 이것은 몇 시간이 염료 확산이 필요합니다 특히 때 염료 및 감소 라벨 강도의 희석이 발생할 수 있습니다. 여기 염료 용액으로 가득 폴리에틸렌 흡입 피펫을 사용하여 뉴런으로 형광등 및 이온에 민감한 염료를 도입을위한 간단하고 저비 용의 기술을 보여줍니다. 이 방법은 로딩 과정 전반에 걸쳐 axons과 접촉 염료의 높은 농도를 유지하는 안정적인 방법을 제공합니다.

Watch this Scientific Journal Video about Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes at JoVE.com

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

우리는 뉴런이나 폴리에틸렌 흡입 피펫을 사용하여 모든의 연결을 요로에 형광등과 칼슘에 민감한 염료의 높은 농도를 도입을위한 간단하고 저렴한 비용으로 기술을 설명합니다.

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. ...

retrograde-loading-nerves-tracts-spinal-roots-with-fluorescent

Research

JoVE Journal

Neuroscience

12.2K 조회수.  National Institute of Neurological Disorders and Stroke, National Institutes of Health. 우리는 뉴런이나 폴리에틸렌 흡입 피펫을 사용하여 모든의 연결을 요로에 형광등과 칼슘에 민감한 염료의 높은 농도를 도입을위한 간단하고 저렴한 비용으로 기술을 설명합니다.

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

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye 1; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K+). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1Ai-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1Aiv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining).

Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy 2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.

FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.

Styryl 염료의 Photoconversion를 사용하여 시냅틱 소포 풀 유학

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

연구

신경과학

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 ...

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 ...

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.

Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

중증 척수 손상 후 섬유소 및 성장 인자 칵테일 신경 줄기 세포의 생존 및 분화 촉진

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

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

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 survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

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 ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

마우스 좌골 신경에 생체 전기 생리 측정에서

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of F&#246;rster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

A method for imaging changes in membrane potential using genetically encoded voltage indicators is described.

유전자 인코딩 된 형광 전압 센서의 두 가지 유형의 잠재적 이미징 막

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating the respiratory diaphragm. In spinal cord slices, phrenic motor neurons cannot be identified from other motor neurons on morphological or biochemical criteria. We provide the description of procedures for visualizing phrenic motor neuron cell bodies in mice, following intrapleural injections of cholera toxin subunit beta (CTB) conjugated to a fluorophore. This fluorescent neuroanatomical tracer has the ability to be caught up at the diaphragm neuromuscular junction, be carried retrogradely along the phrenic axons and reach the phrenic cell bodies. Two methodological approaches of intrapleural CTB delivery are compared: transdiaphragmatic versus transthoracic injections. Both approaches are successful and result in similar number of CTB-labeled phrenic motor neurons. In conclusion, these techniques can be applied to visualize or quantify the phrenic motor neurons in various experimental studies such as those focused on the diaphragm-phrenic circuitry.

Here, we describe a protocol for identifying phrenic motor neurons in mice after intrapleural delivery of fluorophore conjugated cholera toxin subunit beta. Two techniques are compared to inject the pleural cavity: transdiaphragmatic versus transthoracic approaches.

마우스에 인할지 모터 신경의 퇴행 성 신경 해부학 추적

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic ...

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) on a treadmill is widely used as a rehabilitation tool in the SCI population with many benefits and improvements to daily life. We utilize this method of activity-based task-specific training (ABT) in rodents after SCI to both elucidate the mechanisms behind such improvements and to enhance and improve upon existing clinical rehabilitation protocols. Our current goal is to determine the mechanisms underlying ABT-induced improvements in urinary, bowel, and sexual function in SCI rats after a moderate to severe level of contusion. After securing each individual animal in a custom-made adjustable vest, they are secured to a versatile body weight support mechanism, lowered to a modified three-lane treadmill and assisted in step-training for 58 minutes, once a day for 10 weeks. This setup allows for the training of both quadrupedal and forelimb-only animals, alongside two different non-trained groups. Quadrupedal-trained animals with body weight support are aided by a technician present to assist in stepping with proper hind limb placement as necessary, while forelimb-only trained animals are raised at the caudal end to ensure no hind limb contact with the treadmill and no weight-bearing. One non-trained SCI group of animals is placed in a harness and rests next to the treadmill, while the other control SCI group remains in its home cage in the training room nearby. This paradigm allows for the training of multiple SCI animals at once, thus making it more time-efficient in addition to ensuring that our pre-clinical animal model mimics the clinical representation as close as possible, particularly with respect to the body weight support with manual assistance.

This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol.

척수와 디딜 방 아에 활동 기반 교육 부상 Wistar 쥐

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) ...

Dissection of Pelvic Autonomic Ganglia and Associated Nerves in Male and Female Rats

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons innervating pelvic organs of rodents; the functionally equivalent structure in humans is the inferior hypogastric plexus. The major pelvic ganglia also provide the route by which lumbar and sacral sensory axons reach the pelvic organs. These complex, mixed ganglia can prove challenging to identify and dissect for further experimental study of normal autonomic mechanisms or to establish preclinical models of disease, injury or visceral pain. Here we describe a protocol to access and visualize these ganglia and their associated nerve tracts. We provide this protocol with schematics for both male and female rats, as the ganglion size and landmarks for identification differ between sexes. The protocol describes removal of the ganglion for in vitro studies, but this method can be integrated into a surgical recovery protocol for experimental interventions (e.g., nerve crush, nerve resection) or for mapping neuronal circuits (e.g., by microinjection of neural tracers). We also demonstrate the primary structures of the ganglion and its associated nerves immediately following dissection and following immunohistochemical staining.

The major pelvic ganglia contain parasympathetic and sympathetic neurons that innervate pelvic organs. Here we describe a dissection method and provide schematics for identification of these ganglia and their associated nerves. These methods can be applied to experimental manipulation of these ganglia in vivo or removal post-mortem for further study.