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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中央赠款提供的资源 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此，在列出的机构中可能会被压制 对于中心，这不一定是调查员的工厂。 抽象的： 佐治亚州西部蚊子的成年和雄性脊髓中的甲壳虫的超级特征将在肛门最终最终最终鳍上的神经区域进行某些时间互联通过在不同的焦点平面上进行的租赁树突形成。 这里支撑的工作将阐明将成年运动神经元整合到现有神经IT的替代机制（即差距连接）。 Rosa-Molinar实验室中描述的逆行标签，裂缝加工和WADSWORTH中心可视化生物复杂性（RVBC）的资源将有助于HVEM的运作和训练，并进行训练与RVBC员工的协助和咨询，以优化样本和数据收集。 项目描述：高压电子显微镜（HVEM）和选择性标记的运动神经元纤维的HVEM层析成像，终端以及中间神经元细胞体，纤维和终端遗嘱将用于可视化，重建三位数（3- d），3- d），，3- d），，和模型树突状缝隙连接以及丝状接触。 该提案中描述的逆行labledale命运是我的实验室常规，这次是到达RVBC。 女性和蚊子将被浸入苯佐卡因（1：5,000）和生物素，3000 MW，赖氨酸固定（Micro-Ruby），葡萄糖，荧光素和生物素，10,000 MW，10,000 MW，偏anionic，乳酸，赖氨酸固定（Mini-Emerald，Mini-Emerald），将其麻醉（1：5,000）。荧光素和生物素，3000兆瓦，阴离子，赖氨酸，可固定（微型矿物质）和葡萄糖，10,000兆瓦，离子，赖氨酸，赖氨酸，赖氨酸，赖氨酸（Mini-Emerald）饱和滤纸纤维材料遗嘱遗嘱遗嘱遗嘱遗嘱是遗嘱中的，是一位西方的西部雌性，是一些女性的西部幼体，是一些女性的最终法令。 。 7.4）然后在0.2 m cacodylate-HCl缓冲液（pH 7.4）中改性的卡诺夫斯基固定剂（2.5％戊二醛2％甲醛0.2％苦味甲醛V/V） c）压缩灌注后，与椎骨段7相关的脊髓在室温下在室温下隔夜在灌注的相同固定剂中隔夜过夜。 ABC - 过氧酶和底物（DAB，向量sg或载体VIP）发育。在30分钟内，在一系列的ethez中脱水，具有3：1、1：1：1：1：3的绝对和塑料低粘度的溶液，定向，并嵌入塑料中的横向平面中。 » c在厚度和薄后，厚度为12小时。铀酰和柠檬酸三铅。 标记和组织加工程序描述使我们可视化未染色的1.0€ M部分（见图2）。 将使用AM-7 MK II高压电子显微镜查看网格。训练。该项目在不久的将来的这一方面。 为什么我们需要使用HVEM：脊髓中的运动神经元和中间神经元有许多竞争的过程，并且差异很差。传统的电子显微镜» M厚电压电子显微镜（HVEM）将使用Tom em的塔，还将提供有关三维结构的信息。在生物学的唱片中，较低的加速电压会导致电离损伤。显示了厚标本的HVemes观察到详细的形态。 最后，我们的目标是用于脊柱运动神经元和神经元的树突状间隙连接的去效率计算模型。