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: 7721717
• 负责人: EDUARDO ROSA-MOLINAR
• 金额: $ 2.22万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7721717
• 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; 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; Microtome - medical device; Microtomy; Modeling; Motor Neurons; Muscle; Neuraxis; Operative Surgical Procedures; Paper; Perfusion; Peroxidase; Peroxidases; Plastics; Preparation; Procedures; Process; Radiation; Reporting; 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; Visit; Work; abstracting; alveolar lamellar body; day; 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; spurr resin; 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 [MN's] and interneurons [IN's] (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 phenylenediam¿ine 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 (Spurr's 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 MN's, 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. In the previous reporting period, Dr Rosa-Molinar visited the Resource for two days, with blocks of specimens prepared as described. He used the microtome to cut sections three and four micrometers thick, which were post-stained with lead citrate. Many stereopair images were then recorded using the HEVM. This provided the data needed for a grant submission.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 抽象的： 在对臀鳍肌肉进行逆行神经束追踪后，将分析西部食蚊鱼（Gambusia affinis affinis）成年雌性和雄性硬骨鱼脊髓中已识别的树突束的超微结构特征。 观察到树突束排列成网络，通过间隙连接紧密互连。 这些束由至少 10 个在不同焦平面上行进的交叉树突形成。 在树突之间，经常发现细长的间隙连接复合物。 还观察到了树突层状体，最近被描述为与中枢神经系统间隙连接有关。 这里提出的工作将阐明将成年运动神经元整合到现有神经回路中的替代机制（即间隙连接）。 本提案中描述的逆行标记、固定、组织处理和染色程序以及切片将在 Rosa-Molinar 实验室中完成。 沃兹沃斯中心的生物复杂性可视化资源 (RVBC) 工作人员将协助 HVEM 的操作以及立体显微镜和断层扫描方面的培训。 在进行调整以优化样本和数据收集时，需要 RVBC 工作人员的后续帮助和咨询。 项目描述 选择性逆行标记的运动神经元细胞体、纤维和末端以及中间神经元细胞体、纤维和末端的高压电子显微镜 (HVEM) 和 HVEM 断层扫描将用于可视化、三维 (3-D) 重建和树突间隙连接以及丝状接触建模。 本提案中描述的逆行标记和组织制备是我实验室的常规操作，将在到达 RVBC 之前完成。 将雌性和雄性西方蚊鱼用苯佐卡因 (1:5,000) 浸泡麻醉。 麻醉后，葡聚糖、四甲基罗丹明和生物素，3000 MW，赖氨酸可固定（微红宝石），葡聚糖、荧光素和生物素，10,000 MW，阴离子，赖氨酸可固定（迷你祖母绿），葡聚糖、荧光素和生物素，3000 MW，阴离子，赖氨酸可固定（微祖母绿），以及 葡聚糖、荧光素和生物素、10,000 MW、阴离子、赖氨酸可固定（迷你翡翠）饱和滤纸纤维将被植入雌性和雄性西方食蚊鱼的臀鳍肌肉组织中。 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 [IN's] 的高尔基样填充（见图 1）。 8小时后，用苯佐卡因（1:2,000）浸泡，对鱼进行安乐死，并首先用0.2 M二甲胂酸盐-HCl缓冲液（pH 7.4）经动脉圆锥灌注，然后用0.2 M二甲胂酸盐-HCl中的改良卡诺夫斯基固定剂（2.5%戊二醛、2%多聚甲醛、0.2%苦味酸v/v）进行灌注。 室温 (21°C) 下的缓冲液 (pH 7.4)。 灌注完成后，将与椎段7至17相关的脊髓取出并自由解剖，并通过在室温下在用于灌注的相同固定剂中浸没过夜来进行后固定。 标记的运动神经元、纤维和末端的细节将在 VECTASTAIN¿ ABC - 过氧化物酶和底物（DAB、Vector¿ SG 或 Vector¿ VIP）开发后观察到。 然后将脊髓固定在 0.2 M 二甲胂酸盐-HCl 中的 1% OsO4（pH 7.4）中，用 70% 乙醇中的 1% 对苯二胺整体染色 30 分钟，在一系列梯度乙醇中脱水，用 3:1、1:1 和 1:3 无水乙醇和塑料溶液（Spurr 的低粘度）渗透，定向，并嵌入 塑料的横向平面。 块将在 70° C 下聚合 12 小时。 聚合后，将使用金刚石刀在横向和纵向平面上切割厚切片和薄切片。 所有切片均使用 Sorvall MT 5000 超薄切片机进行切割。 厚部分 (1.0 µm) 将按顺序安装在 2.0 x 1.0 mm 涂有 Formvar 的重槽铜网上。 网格将用酒精醋酸双氧铀和柠檬酸三铅进行后染色。 上述标记和组织处理程序使我们能够可视化未染色的 1.0 µm 切片（见图 2）。 对称的丝状接触发生在标记的 MN 的相邻树突 (D) 之间、它们的胞体 (S) 之间以及胞体和树突之间。 这些网格将被运送到 RVBC，在那里使用 AEI EM-7 Mk II 高压电子显微镜对其进行观察和拍照。 我想让 RVBC 工作人员评估网格并提供有关优化标本制备、切片和染色的指导。 我还希望 RVBC 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 测量将与树突样品的系统采样和连续电子显微镜重建相结合，以获得准确的表面积估计。 这将仅由从事该拟议项目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方面。 为什么我们需要使用 HVEM？ 硬骨鱼脊髓中的运动神经元和中间神经元有许多复杂的过程，并且研究很少。 它们对于宽视场显微镜研究来说非常精细，但对于传统透射电子显微镜来说过于复杂和广泛传播。 对逆行填充的脊髓运动神经元和中间神经元的厚切片进行高压电子显微镜 (HVEM) 立体观察可以提供其突起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 米厚的切片。 高压电子显微镜（HVEM）的使用将使我们能够利用HVEM电子束的更高穿透力。 厚切片还将通过立体显微镜为我们提供有关三维结构的信息。 证明使用 HVEM 合理性的最后一个重要方面是辐射损伤。 在生物样本中，例如我们将要处理的脊髓组织，较低的加速电压可能会导致电离损伤。 通过增加加速电压来减少损坏程度。 这项研究获得的结果不仅将为运动神经元和中间神经元以及间隙连接的关系提供新的见解，而且还清楚地表明了厚样本的 HVEM 立体观察对于树突状间隙连接的详细形态学分析的有用性。 最后，我们的最终目标是开发脊髓运动神经元和中间神经元的树突间隙连接的真实计算模型。 我们还需要开发方法来估计具有这些树突间隙连接的脊髓运动神经元和中间神经元的树突树的表面积。 在上一个报告期，罗莎-莫利纳尔博士参观了资源中心两天，并按照所述准备了大块标本。 他用切片机切成三到四微米厚的切片，并用柠檬酸铅进行后染色。 然后使用 HEVM 记录许多立体对图像。 这提供了拨款申请所需的数据。