title: Effective synaptome analysis of itch-mediating neurons in the spinal cord: A novel immunohistochemical methodology using high-voltage electron microscopy.
abstract: Transmission electron microscopy (TEM) is used for three-dimensional (3-D) analysis of synaptic connections in neuroscience research. However, 3-D reconstruction of the synapses using serial ultrathin sections is a powerful but tedious approach requiring advanced technical skills. High-voltage electron microscopy (HVEM) allows examination of thicker sections of biological specimens due to the increased penetration of the more accelerated electrons, which is useful to analyze the 3-D structure of biological specimens. However, it is still difficult to visualize the neural networks and synaptic connections in 3-D using HVEM because of insufficient and non uniform heavy metal staining in the membranous structures in semi-thin sections. Here, we present the successful chemical 3-D neuroanatomy of the rat spinal dorsal horn at the ultrastructural level as a first step for effective synaptome analysis by applying a high-contrast en bloc staining method to immune-HVEM tomography. Our new approach made it possible to examine many itch-mediating synaptic connections and neural networks in the spinal cord simultaneously using HVEM tomography. This novel 3-D electron microscopy is very useful for the analysis of synaptic structure and the chemical neuroanatomy at the 3-D ultrastructural level.
authors.affiliation: Ushimado Marine Institute, Graduate School of Natural Science and Technology, Okayama University, Ushimado, Setouchi, Okayama 701-4303, Japan.
authors.forename: Keita
authors.initials: K
authors.lastname: Satoh
authors.affiliation: Department of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Takanami
authors.affiliation: National Institute for Physiological Sciences, Nishigonaka, Myodaiji, Okazaki 444-8585, Japan.
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Murata
authors.affiliation: Department of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan.
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Kawata
authors.affiliation: Ushimado Marine Institute, Graduate School of Natural Science and Technology, Okayama University, Ushimado, Setouchi, Okayama 701-4303, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Sakamoto
authors.affiliation: Ushimado Marine Institute, Graduate School of Natural Science and Technology, Okayama University, Ushimado, Setouchi, Okayama 701-4303, Japan. Electronic address: hsakamo@okayama-u.ac.jp.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Sakamoto
mesh_terms: D000818:Animals; D007150:Immunohistochemistry; D008297:Male; D046529:Microscopy, Electron, Transmission / Q000379:methods; D020671:Posterior Horn Cells / Q000378:metabolism / Q000648:ultrastructure*; D011537:Pruritus / Q000378:metabolism / Q000473:pathology*; D017208:Rats, Wistar; D013569:Synapses / Q000502:physiology*
keywords: High-voltage electron microscopy; Itch sensation; Spinal cord; Synapse; Three-dimensional analysis; Tomography