ROLE OF DENDRODENDRITIC GAP JUNCTIONS IN SPINAL MICROCIRCUITRY

树突状间隙连接在脊髓微循环中的作用

• 批准号: 7598377
• 负责人: EDUARDO ROSA-MOLINAR
• 金额: $ 2.29万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7598377
• 关键词: 3-Dimensional; Adult; Anesthesia procedures; Anus; Area; Benzocaine; Biological; Biotin; Buffers; Cells; Characteristics; Citrate; Citrates; Classification; Complex; Computer Retrieval of Information on Scientific Projects Database; Computer Simulation; Consultations; Conus genus; Copper; Data Collection; Dendrites; Development; Dextrans; Diamond; Diffusion; Dimethylarsinate; Electron Beam; Electron Microscope; Electrons; Ethanol; Female; Fiber; Fishes; Fixatives; Formvar 1285; Funding; Future; Gap Junctions; Glutaral; Goals; Golgi Apparatus; Grant; Human Resources; Image; Imagery; Immersion Investigative Technique; Implant; Institution; Intention; Interneurons; Label; Laboratories; Lead; Lysine; Measurement; Methods; Microscopic; Microtomy; Modeling; Motor Neurons; Muscle; Neuraxis; Operative Surgical Procedures; Paper; Perfusion; Peroxidase; Peroxidases; Phenylenediamines; Plastics; Preparation; Procedures; Process; Radiation; Research; Research Personnel; Resources; Role; Rosa; Sampling; Series; Solutions; Source; Specimen; Spinal; Spinal Cord; Staging; Staining method; Stains; Surface; Temperature; Thick; Three-Dimensional Imaging; Time; Tissues; Training; Transmission Electron Microscopy; Travel; Trees; United States National Institutes of Health; Viscosity; Work; abstracting; alveolar lamellar body; dextran; fluorescein-dextran; follow-up; high voltage electron microscopy; insight; ionization; male; neural circuit; neural tract; neuronal cell body; paraform; picric acid; polymerization; problem drinker; reconstruction; sample fixation; spine bone structure; teleost; tetramethylrhodamine; three dimensional structure; tissue preparation; tissue processing; tomography; uranyl acetate; vector; voltage

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: The ultrastructural characteristics of identified dendrite bundles in the adult female and male teleost spinal cord of the Western Mosquitofish, Gambusia affinis affinis will be analyzed following retrograde neural tract tracing of anal fin muscles. Dendrite bundles were observed to be arranged in networks that are heavily interconnected by means of gap junctions. The bundles are formed by at least 10 crossing dendrites traveling in different focal planes. In between dendrites, elongated gap junctional complexes are frequently found. Dendrite lamellar bodies, recently described as occurring in relation with gap junctions in the central nervous system, were also observed. The work proposed here will elucidate an alternative mechanism (i.e. gap junctions) of integrating adult-born motor neurons into existing neural circuits. The retrograde labeling, fixations, tissue processing, and staining procedures described in this proposal will be done in the Rosa-Molinar lab, as well as the sectioning. Wadsworth Center's Resource for Visualization of Biological Complexity (RVBC) staff will assist in the operation of the HVEM and training in stereomicroscopy and tomography. Follow-up assistance from, and consultation with, the RVBC staff will be required in making adjustments to optimize the samples and the data collection. Project Description: High voltage electron microscopy (HVEM) and HVEM tomography of selectively retrogradely labeled motor neurons cell bodies, fibers, and terminals as well as interneuron cell bodies, fibers, and terminals will be used to visualize, reconstruct three-dimensionally (3-D), and model dendrodendritic gap junctions as well as filamentous contacts. The retrograde labeling and tissue preparation described in this proposal are routine in my laboratory and this will be completed prior to arriving at the RVBC. Female and male Western Mosquitofish will be anesthetized by immersion with benzocaine (1:5,000). Following anesthesia, dextran, tetramethylrhodamine and biotin, 3000 MW, lysine fixable (micro-ruby), dextran, fluorescein and biotin, 10,000 MW, anionic, lysine fixable (mini-emerald), dextran, fluorescein and biotin, 3000 MW, anionic, lysine fixable (micro-emerald), and dextran, fluorescein and biotin, 10,000 MW, anionic, lysine fixable (mini-emerald) saturated filter paper fibers will be implanted into the anal fin musculature of female and male Western Mosquitofish. Preliminary studies have shown that an 8 h diffusion time was sufficient to obtain Golgi-like filling of spinal motor neurons [MNs] and interneurons [INs] (see Figure 1). After 8 h the fish will be euthanized by immersion with benzocaine (1:2,000) and perfused transcardially through the conus arteriosus first with 0.2 M cacodylate-HCl buffer (pH 7.4) followed by modified Karnovsky's fixative (2.5% glutaraldehyde 2% paraformaldehyde 0.2% picric acid v/v) in 0.2 M cacodylate-HCl buffer (pH 7.4) at room temperature (21¿C). After completion of the perfusion, the spinal cord associated with vertebral segments 7 through 17 will be removed dissected free and post-fixed by immersion overnight at room temperature in the same fixative used for the perfusion. Details of labeled motor neurons, fibers, and terminals will be observed after VECTASTAIN¿ ABC - Peroxidase and substrate (DAB, Vector¿ SG or Vector¿ VIP) development. The spinal cord will then be post fixed in 1% OsO4 in 0.2 M cacodylate-HCl (pH 7.4), en bloc stained with 1% p phenylenediamine in 70% ethanol for 30 minutes, dehydrated in a graded series of ethanol, infiltrated with 3:1, 1:1, and 1:3 solution of absolute ethanol and plastic (Spurrs Low Viscosity), oriented, and embedded in a transverse plane in plastic. Blocks will be polymerized at 70¿ C for 12 hr. After polymerization, thick and thin sections will be cut using a diamond knife in a transverse and longitudinal plane. All sections will be cut using a Sorvall MT 5000 Ultramicrotome. Thick sections (1.0 ¿m) will be mounted on 2.0 x 1.0 mm Formvar-coated heavy slot copper grids in serial order. Grids will be post-stained with alcoholic uranyl acetate and triple lead citrate. The labeling and tissue processing procedures describe above have allowed us visualize unstained 1.0 ¿m sections (see Figure 2). Symmetrical filamentous contacts occur between adjacent dendrites (D) of the labeled MNs, between their somata (S) and between soma and dendrite. The grids will be transported to the RVBC where they will be viewed and photographed using a AEI EM-7 Mk II High-Voltage Electron Microscope. I would like to have the RVBC staff evaluate the grids and provide pointers on the optimization of the specimen preparation, sectioning, and staining. I would also like for the RVBC staff to provide me training on the operation of the HVEM and stereomicroscopy. Finally, in the initial stages of the project, HVEM measurements will be combined with systematic sampling and serial electron microscopic reconstruction of samples from the dendrites, in order to yield accurate surface area estimates. This will be done solely by the Biological Imaging Group personnel working on this proposed project. It is our intention to use tomography to accelerate this aspect of the project in the near future. Why do we need to use the HVEM: Motor neurons and interneurons in the teleost spinal cord have many complicated processes and have been very poorly studied. They are extremely fine for wide-field microscopic study and are too complicated and widely spread for traditional transmission electron microscopy. High-voltage electron microscopic (HVEM) stereo observation of thick sections of retrogradely-filled spinal motor neurons and interneurons can provide detailed three-dimensional (3-D) images of their processes and gap junctions. For this research we are proposing to use 1.0 ¿m thick sections. The use of the high voltage electron microscope (HVEM) will allow us to use the higher penetrating power of the electron beam of the HVEM. Thick sections will also provide us information about three-dimensional structures through stereomicroscopy. A final but important aspect justifying use of the HVEM is that of radiation damage. In biological specimens, such as the spinal cord tissue that we will be processing, lower accelerating voltages can cause ionization damage. The degree of the damage is reduced by increasing the accelerating voltage. The results obtained from this research will not only provide new insight into the relationship of motor neurons and interneurons and gap junctions but also clearly show the usefulness of HVEM stereo observation of thick specimens for detailed morphological analysis of dendrodendritic gap junctions. Finally, our ultimate goal is to develop realistic computational models of dendrodendritic gap junctions of spinal motor neurons and interneurons. We are also in need of developing methods to estimate the surface area of the dendritic trees of the spinal motor neurons and interneurons that posses these dendrodendritic gap junctions.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 抽象的： 在肛门鳍肌肉的逆行神经段追踪后，将分析已鉴定出的树突束的超微结构特征。观察到树突束被排列在通过间隙连接互联的网络中。束由至少10个以不同焦平面行驶的十个十个树突形成。在树突之间，经常发现伸长的间隙连接络合物。还观察到了中枢神经系统中最近描述为与缝隙连接相关的树突层状体。 这里提出的工作将阐明将成人运动神经元整合到现有神经元中的替代机制（即间隙连接）。 该提案中描述的逆行标签，固定，组织加工和染色程序将在Rosa-Molinar实验室以及分段中进行。 Wadsworth中心的生物复杂性可视化资源（RVBC）工作人员将有助于HVEM的操作和立体显微镜和层析成像的培训。在进行调整以优化样品和数据收集时，需要与RVBC工作人员进行后续协助和咨询。 Project Description: High voltage electron microscopy (HVEM) and HVEM tomography of selectively retrogradely labeled motor neurons cell bodies, fibers, and terminals as well as interneuron cell bodies, fibers, and terminals will be used to visualize, reconstruct three-dimensionally (3-D), and model dendrodendritic gap junctions as well as filamentous contacts. 该提案中描述的逆行标记和组织制剂是常规的，在我的实验室中，这将在到达RVBC之前完成。 女性和雄性西方蚊子将被浸入苯佐卡因（1：5,000）的麻醉。 Following anesthesia, dextran, tetramethylrhodamine and biotin, 3000 MW, lysine fixable (micro-ruby), dextran, fluorescein and biotin, 10,000 MW, anionic, lysine fixable (mini-emerald), dextran, fluorescein and biotin, 10,000 MW, anionic, lysine fixable (mini-emerald) saturated filter纸纤维将植入女性和雄性西部蚊子的肛门鳍肌肉中。初步研究表明，8小时的扩散时间足以获得脊髓运动神经元[Mn S]和中间神经元[S]中的高尔基体样填充（见图1）。 8小时后，将通过浸入苯佐卡因（1：2,000）（1：2,000）的浸入式，并先通过圆锥动脉蛋白酶灌注，首先用0.2 m cacodylate-HCl缓冲液（pH 7.4）进行灌注（pH 7.4）在室温下（21¿C）。灌注完成后，将在室温下以与灌注相同的固定固定的室温，将与椎骨7至17相关的脊髓脱离并在室温下浸入过夜。在Vectastain€abc-过氧化物酶和底物（DAB，Vector？SG或Vector？VIP）开发后，将观察到标记的运动神经元，纤维和末端的细节。然后，脊髓将在0.2 m cacodylate-HCl（pH 7.4）中固定在1％OSO4中，并用1％P苯二胺在70％乙醇中染色30分钟，在一系列的一系列乙醇中脱水，并在3：1：1：1：1：1：1：1：1：1：1：1：1：1：1：1：3的溶液中脱水（Sprient），并溶解了Ablys and Obled and proding and Obery S的溶液（Sprient）。塑料中的横平。块将在70°C下聚合12小时。聚合后，将使用横向和纵向平面中的钻石小针切成厚和薄的部分。所有部分将使用Sorvall MT 5000超级机体切割。厚的部分（1.0？m）将以串行顺序安装在2.0 x 1.0 mm FormVar涂层的重槽铜网格上。网格将用含酒精的乙酸铀酰和三铅柠檬酸含量染色。 上面描述的标签和组织处理程序使我们能够可视化未染色的1.0段（见图2）。对称的丝状接触发生在标记的Mn S的相邻树突（D）之间，其somata（s）之间以及索马和树突之间。 网格将被运输到RVBC，并使用AEI EM-7 MK II高压电子显微镜查看和拍照。我想让RVBC的工作人员评估网格，并提供有关样品准备，切片和染色的优化的指针。我还希望RVBC的工作人员为我提供有关HVEM和立体显微镜操作的培训。最后，在项目的初始阶段，HVEM测量将与来自树突的样品进行系统的采样和串行电子显微镜重建，以产生准确的表面积估计。这将仅由在此拟议项目中工作的生物成像小组人员完成。我们打算在不久的将来使用断层扫描来加速该项目的这一方面。 为什么我们需要使用HVEM：硬骨脊髓中的运动神经元和中间神经元具有许多复杂的过程，并且研究得很糟糕。它们对于宽场显微镜研究非常好，并且对于传统的传播电子显微镜而言太复杂，广泛传播。高压电子显微镜（HVEM）立体观察逆行填充的脊柱运动神经元和中间神经元的厚部分可以提供其过程和间隙连接的详细三维（3-D）图像。对于这项研究，我们建议使用1.0`厚的部分。高压电子显微镜（HVEM）的使用将使我们能够使用HVEM电子束的较高穿透力。较厚的部分还将通过立体显微镜为我们提供有关三维结构的信息。最终但重要的方面证明使用HVEM是辐射损伤。在我们将要处理的生物标本（例如脊髓组织）中，较低的加速电压会导致电离损伤。通过增加加速度电压来降低损伤程度。从这项研究获得的结果不仅将提供有关运动神经元和中间神经元和间隙连接的关系的新见解，而且还清楚地表明了HVEM立体观察对厚标本的有用性，以详细介绍了树突状差异的详细形态分析。 最后，我们的最终目标是开发脊柱运动神经元和神经元的树突状间隙连接的现实计算模型。我们还需要开发方法来估计脊柱运动神经元和中间神经元的树突树的表面积，这些神经元和中间神经元构成了这些树突状间隙连接。