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Exploration of two-photon photostimulation for neuroscience
Available online at www.sciencedirect.com
Neuroscience Research 65S (2009) S3–S36
Abstracts of the 32nd Annual Meeting of the Japan Neuroscience Society (Neuro 2009) SY1-A1-1 Electrical signaling in dendrites probed by second harmonic generation imaging Mutsuo Nuriya, Masato Yasui Dept. Pharm, Keio University, Tokyo, Japan Fast information processing in neurons is performed using electrical signaling. Morphologies of neurons, especially of dendrites and axons, are complex and such morphological complexities are considered to have large impacts on electrical information processing in neurons. However, physiological investigations on these structures have been challenged by their fine and elaborate structures. To gain quantitative information on electrical signaling in dendrites and axons, we applied second harmonic generation (SHG) imaging to cultured mouse hippocampal neurons. Neurons stained with extracellular FM4-64 give rise to strong SHG signals throughout neurons. SHG signal shows instantaneous and linear response to membrane potential changes, enabling quantitative analyses of membrane potential dynamics in neurons. Analyses of SHG from axons and dendrites provide quantitative information on the dynamic propagation of action potentials to distal parts of neurons. Further applications of SHG imaging to neurons are expected to help investigating electrical nature of neurons. doi:10.1016/j.neures.2009.09.1477
SY1-A1-2 Label-free microscopy Katsumasa Fujita
biological
imaging
by
CARS/Raman
Osaka University, Japan Raman spectroscopy has been utilized to investigate molecular structures and their environments. Since Raman scattering spectra give molecular vibration information of specimen, it allows us to observed and investigate molecules in biological samples without labeling with exogenous chemicals, offering chemical information of biomolecules at cellular micro environments. With the recent development in Raman microscopy, imaging of distributions of biomolecules such as DNA, protein, and lipids is demonstrated without labeling. Coherent anti-Stokes Raman scattering (CARS) has also been utilized to image living specimen since it gives stronger signal than that by Raman scattering and allows us to image samples with a shorter acquisition time. Because CARS is given by multiphoton process with NIR lasers, it can also be used to image thick specimen similarly to multiphoton fluorescence microscopy. In the presentation, basics of Raman and CARS spectroscopy and their applications in biological imaging will be presented. doi:10.1016/j.neures.2009.09.1478
SY1-A1-3 Exploration of two-photon photostimulation for neuroscience Haruo Kasai 1 , Yuya Kanemoto 1 , Masanori Matsuzaki 1 , Graham Ellis-Davis 2 , Atsuya Momotake 4 , Tatsurou Arai 4 , Klaus Hahn 3 , Yi Wu 3 , Motoko Ishikawa 1 , Jun Noguchi 1 , Tatsuya Hayama 1 , Satoshi Watanabe 1 , Susumu Morita 1 1
Lab. Struct. Physiol., University of Tokyo, Tokyo, Japan; 2 Dept. Physiol., Drexel University, Pennsilvenia, USA; 3 Dept. Pharmac., Univ of North Calorina, USA; 4 Dept. Chemist., Tsukuba University, Japan
Two-photon uncaging of caged-glutamate compounds has greatly advanced our understanding of synaptic plasticity in pyramidal neurons. Recently, several new
0168-0102/$ – see front matter
methods for optical stimulation have been developed to facilitate examination of brain function. We will introduce some of the new optical approaches that we use regularly in our laboratory. First, we have developed two-photon-compatible caged GABA, which allows us to study the roles of GABA receptors in dendritic integration. The plasticity of dendritic spines can also be studied using a two-photon-compatible caged-calcium compound (NDBF) or an optogenetic probe (channelrhodopsin2 – ChR2). We can also induce structural plasticity in dendritic spines using another optogenetic probe based on a small G-protein (photoactivatable Rac – PA-Rac), which was developed in Klaus Hahn’s laboratory. doi:10.1016/j.neures.2009.09.1479
SY1-A1-4 Microglia surveillance of the state of synapses Junichi Nabekura 1,2,3 , Hiroaki Wake 1,2 , Yusuke Takatsuru 1,2 , Kei Eto 1 , Hiroyuki Inada 1,3 , Sun Kwang Kim 1,4 1 3
National Institute for physiological Sciecens, Japan; Sokendai, Japan; 4 JSPS, Japan
2
JST CREST, Japan;
Resting microglia dynamically extend and retract their processes as if actively surveying the microenvironment. Using in vivo two-photon imaging of fluorescent labeled neurons and microglia we demonstrate that the resting microglial processes make brief (5 min) and direct contacts with neuronal synapses at a frequency of about once per hour. The frequency of contacts is activity-dependent. Following transient cerebral ischemia, the duration of these microglia-synapse contacts are markedly prolonged (1–2 hours), and are frequently followed by the disappearance of the presynaptic bouton. Furthermore, the striking finding that some synapses in the ischemic areas disappear following prolonged microglial contact suggests microglia contribute to the subsequent increased turnover of synaptic connections. Further understanding of the mechanisms involved in the microglial detection of the functional state of synapses, and of their role in remodeling neuronal circuits disrupted by ischemia, may lead to novel therapies for treating brain injury that target microglia. doi:10.1016/j.neures.2009.09.1480
SY1-A1-5 Functional micro-architecture of visual cortex Kenichi Ohki Harvard Medical School, USA We labeled thousands of neurons of the visual cortex with a calcium-sensitive indicator in vivo. We then imaged the activity of neuronal populations at single-cell resolution with two-photon microscopy. In rat primary visual cortex, neurons had robust orientation selectivity but there was no discernible local structure; neighboring neurons often responded to different orientations. In cat visual cortex, functional maps were organized at a fine scale. Neurons with opposite preferences for stimulus direction were segregated with extraordinary spatial precision in three dimensions, with columnar borders one to two cells wide. These results indicate that cortical maps can be built with single-cell precision. In monkey V1, we examined the finescale arrangement of color-selective cells in V1. By imaging the activity over 1000 neurons, we could reveal that color-selective cells are organized into functionally defined clusters. We found that most highly orientation selective cells were located at interblob regions. Most neurons in color blobs showed broad or no orientation selectivity. This finding strongly supports that there are parallel representations of orientation and color in monkey V1. doi:10.1016/j.neures.2009.09.1481
Neuroscience Research 65S (2009) S3–S36
Abstracts of the 32nd Annual Meeting of the Japan Neuroscience Society (Neuro 2009) SY1-A1-1 Electrical signaling in dendrites probed by second harmonic generation imaging Mutsuo Nuriya, Masato Yasui Dept. Pharm, Keio University, Tokyo, Japan Fast information processing in neurons is performed using electrical signaling. Morphologies of neurons, especially of dendrites and axons, are complex and such morphological complexities are considered to have large impacts on electrical information processing in neurons. However, physiological investigations on these structures have been challenged by their fine and elaborate structures. To gain quantitative information on electrical signaling in dendrites and axons, we applied second harmonic generation (SHG) imaging to cultured mouse hippocampal neurons. Neurons stained with extracellular FM4-64 give rise to strong SHG signals throughout neurons. SHG signal shows instantaneous and linear response to membrane potential changes, enabling quantitative analyses of membrane potential dynamics in neurons. Analyses of SHG from axons and dendrites provide quantitative information on the dynamic propagation of action potentials to distal parts of neurons. Further applications of SHG imaging to neurons are expected to help investigating electrical nature of neurons. doi:10.1016/j.neures.2009.09.1477
SY1-A1-2 Label-free microscopy Katsumasa Fujita
biological
imaging
by
CARS/Raman
Osaka University, Japan Raman spectroscopy has been utilized to investigate molecular structures and their environments. Since Raman scattering spectra give molecular vibration information of specimen, it allows us to observed and investigate molecules in biological samples without labeling with exogenous chemicals, offering chemical information of biomolecules at cellular micro environments. With the recent development in Raman microscopy, imaging of distributions of biomolecules such as DNA, protein, and lipids is demonstrated without labeling. Coherent anti-Stokes Raman scattering (CARS) has also been utilized to image living specimen since it gives stronger signal than that by Raman scattering and allows us to image samples with a shorter acquisition time. Because CARS is given by multiphoton process with NIR lasers, it can also be used to image thick specimen similarly to multiphoton fluorescence microscopy. In the presentation, basics of Raman and CARS spectroscopy and their applications in biological imaging will be presented. doi:10.1016/j.neures.2009.09.1478
SY1-A1-3 Exploration of two-photon photostimulation for neuroscience Haruo Kasai 1 , Yuya Kanemoto 1 , Masanori Matsuzaki 1 , Graham Ellis-Davis 2 , Atsuya Momotake 4 , Tatsurou Arai 4 , Klaus Hahn 3 , Yi Wu 3 , Motoko Ishikawa 1 , Jun Noguchi 1 , Tatsuya Hayama 1 , Satoshi Watanabe 1 , Susumu Morita 1 1
Lab. Struct. Physiol., University of Tokyo, Tokyo, Japan; 2 Dept. Physiol., Drexel University, Pennsilvenia, USA; 3 Dept. Pharmac., Univ of North Calorina, USA; 4 Dept. Chemist., Tsukuba University, Japan
Two-photon uncaging of caged-glutamate compounds has greatly advanced our understanding of synaptic plasticity in pyramidal neurons. Recently, several new
0168-0102/$ – see front matter
methods for optical stimulation have been developed to facilitate examination of brain function. We will introduce some of the new optical approaches that we use regularly in our laboratory. First, we have developed two-photon-compatible caged GABA, which allows us to study the roles of GABA receptors in dendritic integration. The plasticity of dendritic spines can also be studied using a two-photon-compatible caged-calcium compound (NDBF) or an optogenetic probe (channelrhodopsin2 – ChR2). We can also induce structural plasticity in dendritic spines using another optogenetic probe based on a small G-protein (photoactivatable Rac – PA-Rac), which was developed in Klaus Hahn’s laboratory. doi:10.1016/j.neures.2009.09.1479
SY1-A1-4 Microglia surveillance of the state of synapses Junichi Nabekura 1,2,3 , Hiroaki Wake 1,2 , Yusuke Takatsuru 1,2 , Kei Eto 1 , Hiroyuki Inada 1,3 , Sun Kwang Kim 1,4 1 3
National Institute for physiological Sciecens, Japan; Sokendai, Japan; 4 JSPS, Japan
2
JST CREST, Japan;
Resting microglia dynamically extend and retract their processes as if actively surveying the microenvironment. Using in vivo two-photon imaging of fluorescent labeled neurons and microglia we demonstrate that the resting microglial processes make brief (5 min) and direct contacts with neuronal synapses at a frequency of about once per hour. The frequency of contacts is activity-dependent. Following transient cerebral ischemia, the duration of these microglia-synapse contacts are markedly prolonged (1–2 hours), and are frequently followed by the disappearance of the presynaptic bouton. Furthermore, the striking finding that some synapses in the ischemic areas disappear following prolonged microglial contact suggests microglia contribute to the subsequent increased turnover of synaptic connections. Further understanding of the mechanisms involved in the microglial detection of the functional state of synapses, and of their role in remodeling neuronal circuits disrupted by ischemia, may lead to novel therapies for treating brain injury that target microglia. doi:10.1016/j.neures.2009.09.1480
SY1-A1-5 Functional micro-architecture of visual cortex Kenichi Ohki Harvard Medical School, USA We labeled thousands of neurons of the visual cortex with a calcium-sensitive indicator in vivo. We then imaged the activity of neuronal populations at single-cell resolution with two-photon microscopy. In rat primary visual cortex, neurons had robust orientation selectivity but there was no discernible local structure; neighboring neurons often responded to different orientations. In cat visual cortex, functional maps were organized at a fine scale. Neurons with opposite preferences for stimulus direction were segregated with extraordinary spatial precision in three dimensions, with columnar borders one to two cells wide. These results indicate that cortical maps can be built with single-cell precision. In monkey V1, we examined the finescale arrangement of color-selective cells in V1. By imaging the activity over 1000 neurons, we could reveal that color-selective cells are organized into functionally defined clusters. We found that most highly orientation selective cells were located at interblob regions. Most neurons in color blobs showed broad or no orientation selectivity. This finding strongly supports that there are parallel representations of orientation and color in monkey V1. doi:10.1016/j.neures.2009.09.1481