ROLE OF DENDRODENDRITIC GAP JUNCTIONS IN SPINAL MICROCIRCUITRY

这个子项目是众多研究子项目之一

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.