title: Reconstitution of Giant Mammalian Synapses in Culture for Molecular Functional and Imaging Studies.
abstract: Giant presynaptic terminal brain slice preparations have allowed intracellular recording of electrical signals and molecular loading, elucidating cellular and molecular mechanisms underlying neurotransmission and modulation. However, molecular genetic manipulation or optical imaging in these preparations is hampered by factors, such as tissue longevity and background fluorescence. To overcome these difficulties, we developed a giant presynaptic terminal culture preparation, which allows genetic manipulation and enables optical measurements of synaptic vesicle dynamics, simultaneously with presynaptic electrical signal recordings. This giant synapse reconstructed from dissociated mouse brainstem neurons resembles the development of native calyceal giant synapses in several respects. Thus, this novel preparation constitutes a powerful tool for studying molecular mechanisms of neurotransmission, neuromodulation, and neuronal development.
  
  
  CONCLUSIONS
  We have developed a novel culture preparation of giant mammalian synapses. These presynaptic terminals make it possible to perform optical imaging simultaneously with presynaptic electrophysiological recording. We demonstrate that this enables one to dissect endocytic and acidification times of synaptic vesicles. In addition, developmental elimination and functional maturation in this cultured preparation provide a useful model for studying presynaptic development. Because this giant synapse preparation allows molecular genetic manipulations, it constitutes a powerful new tool for studying molecular mechanisms of neurotransmission, neuromodulation, and neuronal development.
authors.affiliation: Cellular and Molecular Synaptic Function Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, 904-0495, Japan, and ddimitrov@oist.jp ttakahas@oist.jp.
authors.forename: Dimitar
authors.identifier: http://orcid.org/0000-0002-5703-491X
authors.initials: D
authors.lastname: Dimitrov
authors.affiliation: Cellular and Molecular Synaptic Function Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, 904-0495, Japan, and.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Takagi
authors.affiliation: Cellular and Molecular Synaptic Function Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, 904-0495, Japan, and.
authors.forename: Laurent
authors.initials: L
authors.lastname: Guillaud
authors.affiliation: Doshisha University Graduate School of Brain Science, Kyoto 610-0394, Japan.
authors.forename: Naoto
authors.initials: N
authors.lastname: Saitoh
authors.affiliation: Cellular and Molecular Synaptic Function Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, 904-0495, Japan, and.
authors.forename: Kohgaku
authors.initials: K
authors.lastname: Eguchi
authors.affiliation: Cellular and Molecular Synaptic Function Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, 904-0495, Japan, and ddimitrov@oist.jp ttakahas@oist.jp.
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Takahashi
mesh_terms: D000200:Action Potentials / Q000502:physiology*; D000818:Animals; D000831:Animals, Newborn; D001921:Brain / Q000166:cytology* / Q000502:physiology; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D051379:Mice; D008853:Microscopy / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods; D013569:Synapses / Q000502:physiology* / Q000648:ultrastructure*
keywords: giant synapse; membrane capacitance measurements; neuronal cell culture; shRNA knockdown; synaptic maturation; vesicle imaging