Poster Presentation The International Congress of Neuroendocrinology 2014

Visualization of a three-dimensional ultrastructure of neuropeptide in the axon terminals by 3view-scanning electron microscopy (#261)

Keiko Takanami 1 , Hirotaka Sakamoto 2 , Naoyuki Miyazaki 3 , Kazuyoshi Murata 3 , Keita Satoh 2 , Tatsuya Sakamoto 2 , Mitsuhiro Kawata 1
  1. Anatomy and Neurobiology, Kyoto Prefectural University of Medicine, Kyoto, KYOTO, Japan
  2. Ushimado Marine Institute, Graduate School of Natural Science and Technology, Okayama University, Setouchi, OKAYAMA, Japan
  3. Brain Structure Information, Supportive Center for Brain Research, National Institute for Physiological Sciences, Okazaki, AICHI, Japan

Gastrin-releasing peptide (GRP) is widely distributed in the central nervous system, and is involved in a variety of physiological processes and behaviors, including the circadian rhythm, food intake, anxiety/fear responses, and male sexual function.  Recently, the spinal GRP/GRP-receptor system has been identified as an itch-specific mediator in the somatosensory system.  We demonstrated that the expression of GRP was predominant in the small-sized neurons of the primary afferents, and GRP-immunoreactive axons terminated the superficial layers of the spinal dorsal horn in rats.  Furthermore, ultrastructure of GRP-immunoreactive axon terminals was analyzed by various kinds of electron microscopy.  The analysis of high-voltage electron microscopy showed that GRP-immunoreactive axon terminals formed a series of the varicosities.  The analysis of transmission electron microscopy showed the GRP-immunoreactive presynaptic terminals contained many clear microvesicles and large dense-cored vesicles.  Serial block-face scanning electron microscopy (SBF-SEM) that combines SEM and ultramicrotome makes serial ultrathin sections automatically.  Until now was technically impossible, but the reconstruction of a three-dimensional ultrastructure has become possible owing to this electron microscopy.We have developed a technology that applies immunohistochemistry to label the target molecule in this microscope.  SBF-SEM analysis showed that not one but some dendritic spines contacted a single varicosity containing GRP-immunoreactive dens-cored vesicles.  Thus, by combination of several kind of electron microscopy, visualization of a three-dimensional ultrastructure of neuropeptide in the axon terminals has become possible.