title: In Vivo Two-Photon Imaging of Dendritic Spines in Marmoset Neocortex
abstract: Two-photon microscopy in combination with a technique involving the artificial expression of fluorescent protein has enabled the direct observation of dendritic spines in living brains. However, the application of this method to primate brains has been hindered by the lack of appropriate labeling techniques for visualizing dendritic spines. Here, we developed an adeno-associated virus vector-based fluorescent protein expression system for visualizing dendritic spines in vivo in the marmoset neocortex. For the clear visualization of each spine, the expression of reporter fluorescent protein should be both sparse and strong. To fulfill these requirements, we amplified fluorescent signals using the tetracycline transactivator (tTA)-tetracycline-responsive element system and by titrating down the amount of Thy1S promoter-driven tTA for sparse expression. By this method, we were able to visualize dendritic spines in the marmoset cortex by two-photon microscopy in vivo and analyze the turnover of spines in the prefrontal cortex. Our results demonstrated that short spines in the marmoset cortex tend to change more frequently than long spines. The comparison of in vivo samples with fixed samples showed that we did not detect all existing spines by our method. Although we found glial cell proliferation, the damage of tissues caused by window construction was relatively small, judging from the comparison of spine length between samples with or without window construction. Our new labeling technique for two-photon imaging to visualize in vivo dendritic spines of the marmoset neocortex can be applicable to examining circuit reorganization and synaptic plasticity in primates.
authors.affiliation: Laboratory for Molecular Analysis of Higher Brain Function, Brain Science Institute, RIKEN, Wako, Saitama 351-0198, Japan ; Division of Brain Biology, National Institute for Basic Biology , Aichi 444-8585, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Sadakane
authors.affiliation: Laboratory for Molecular Analysis of Higher Brain Function, Brain Science Institute, RIKEN, Wako, Saitama 351-0198, Japan ; Division of Brain Biology, National Institute for Basic Biology , Aichi 444-8585, Japan.
authors.forename: Akiya
authors.initials: A
authors.lastname: Watakabe
authors.affiliation: Laboratory for Molecular Analysis of Higher Brain Function, Brain Science Institute, RIKEN, Wako, Saitama 351-0198, Japan ; Division of Brain Biology, National Institute for Basic Biology , Aichi 444-8585, Japan.
authors.forename: Masanari
authors.initials: M
authors.lastname: Ohtsuka
authors.affiliation: Laboratory for Molecular Analysis of Higher Brain Function, Brain Science Institute, RIKEN, Wako, Saitama 351-0198, Japan ; Division of Brain Biology, National Institute for Basic Biology , Aichi 444-8585, Japan.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Takaji
authors.affiliation: Department of Ultrastructural Research, National Center of Neurology and Psychiatry, National Institute of Neuroscience , Tokyo 187-8502, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Sasaki
authors.affiliation: Division of Behavioral Development, National Institute for Physiological Science , Aichi 444-8585, Japan.
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Kasai
authors.affiliation: Division of Behavioral Development, National Institute for Physiological Science , Aichi 444-8585, Japan.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Isa
authors.affiliation: Division of Homeostatic Development, National Institute for Physiological Science , Aichi 444-8585, Japan.
authors.forename: Go
authors.initials: G
authors.lastname: Kato
authors.affiliation: Division of Homeostatic Development, National Institute for Physiological Science , Aichi 444-8585, Japan.
authors.forename: Junichi
authors.initials: J
authors.lastname: Nabekura
authors.affiliation: Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical University , Tochigi 329-0498, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Mizukami
authors.affiliation: Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical University , Tochigi 329-0498, Japan.
authors.forename: Keiya
authors.initials: K
authors.lastname: Ozawa
authors.affiliation: Department of Biophysical Genetics, Graduate School of Medical Sciences, Kanazawa University , Ishikawa 920-8640, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kawasaki
authors.affiliation: Laboratory for Molecular Analysis of Higher Brain Function, Brain Science Institute, RIKEN, Wako, Saitama 351-0198, Japan ; Division of Brain Biology, National Institute for Basic Biology , Aichi 444-8585, Japan.
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Yamamori
keywords: dendrite; marmoset; neocortex; primate; spine