The Experts below are selected from a list of 327 Experts worldwide ranked by ideXlab platform
Koichi Kawakami - One of the best experts on this subject based on the ideXlab platform.
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Real-Time Visualization of Neuronal Activity during Perception
Current Biology, 2013Co-Authors: Akira Muto, Masamichi Ohkura, Gembu Abe, Junichi Nakai, Koichi KawakamiAbstract:To understand how the brain perceives the external world, it is desirable to observe neuronal activity in the brain in real time during perception. The zebrafish is a suitable model animal for fluorescence imaging studies to visualize neuronal activity because its body is transparent through the embryonic and larval stages. Imaging studies have been carried out to monitor neuronal activity in the larval spinal cord and brain using Ca(2+) indicator dyes and DNA-encoded Ca(2+) indicators, such as Cameleon, GFP-aequorin, and GCaMPs. However, temporal and spatial resolution and sensitivity of these tools are still limited, and imaging of brain activity during perception of a natural object has not yet been demonstrated. Here we demonstrate visualization of neuronal activity in the optic tectum of larval zebrafish by genetically expressing the new version of GCaMP. First, we demonstrate Ca(2+) transients in the tectum evoked by a moving spot on a display and identify direction-selective neurons. Second, we show tectal activity during perception of a natural object, a swimming paramecium, revealing a functional visuotopic map. Finally, we image the tectal responses of a free-swimming larval fish to a paramecium and thereby correlate neuronal activity in the brain with prey capture behavior.
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imaging functional neural circuits in zebrafish with a new GCaMP and the gal4ff uas system
Communicative & Integrative Biology, 2011Co-Authors: Akira Muto, Koichi KawakamiAbstract:Measurement of the activity of neuronal ensembles is an essential step to understand how the neuronal network is organized and functioning. Electrical excitation of neurons causes calcium influx via voltage-gated calcium ion channels, which can be monitored by calcium imaging using fluorescent calcium probes. DNA-encoded calcium indicators (DECIs) such as cameleon and GCaMP have been developed to specifically label a subpopulation of neurons. However, in many cases, DECIs that had been developed and tested in vitro did not always show expected performance in vivo. It is necessary to increase its sensitivity and also to adjust its dynamic range to the physiological conditions. In our recent study, we developed an improved version of GCaMP and tested its performance in vivo using transgenic zebrafish. By combining the new GCaMP with targeted gene expression via the Gal4FF-UAS system, we successfully imaged the activity of the spinal motor circuit during spontaneous contractions of zebrafish larvae. Further ...
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genetic visualization with an improved GCaMP calcium indicator reveals spatiotemporal activation of the spinal motor neurons in zebrafish
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Akira Muto, Masamichi Ohkura, Junichi Nakai, Tomoya Kotani, Shinichi Higashijima, Koichi KawakamiAbstract:Animal behaviors are generated by well-coordinated activation of neural circuits. In zebrafish, embryos start to show spontaneous muscle contractions at 17 to 19 h postfertilization. To visualize how motor circuits in the spinal cord are activated during this behavior, we developed GCaMP-HS (GCaMP-hyper sensitive), an improved version of the genetically encoded calcium indicator GCaMP, and created transgenic zebrafish carrying the GCaMP-HS gene downstream of the Gal4-recognition sequence, UAS (upstream activation sequence). Then we performed a gene-trap screen and identified the SAIGFF213A transgenic fish that expressed Gal4FF, a modified version of Gal4, in a subset of spinal neurons including the caudal primary (CaP) motor neurons. We conducted calcium imaging using the SAIGFF213A; UAS:GCaMP-HS double transgenic embryos during the spontaneous contractions. We demonstrated periodic and synchronized activation of a set of ipsilateral motor neurons located on the right and left trunk in accordance with actual muscle movements. The synchronized activation of contralateral motor neurons occurred alternately with a regular interval. Furthermore, a detailed analysis revealed rostral-to-caudal propagation of activation of the ipsilateral motor neuron, which is similar to but much slower than the rostrocaudal delay observed during swimming in later stages. Our study thus demonstrated coordinated activities of the motor neurons during the first behavior in a vertebrate. We propose the GCaMP technology combined with the Gal4FF-UAS system is a powerful tool to study functional neural circuits in zebrafish.
Karel Svoboda - One of the best experts on this subject based on the ideXlab platform.
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characterization and subcellular targeting of GCaMP type genetically encoded calcium indicators
PLOS ONE, 2008Co-Authors: Junichi Nakai, Tianyi Mao, Daniel H Oconnor, Volker Scheuss, Karel SvobodaAbstract:Genetically-encoded calcium indicators (GECIs) hold the promise of monitoring [Ca(2+)] in selected populations of neurons and in specific cellular compartments. Relating GECI fluorescence to neuronal activity requires quantitative characterization. We have characterized a promising new genetically-encoded calcium indicator-GCaMP2-in mammalian pyramidal neurons. Fluorescence changes in response to single action potentials (17+/-10% DeltaF/F [mean+/-SD]) could be detected in some, but not all, neurons. Trains of high-frequency action potentials yielded robust responses (302+/-50% for trains of 40 action potentials at 83 Hz). Responses were similar in acute brain slices from in utero electroporated mice, indicating that long-term expression did not interfere with GCaMP2 function. Membrane-targeted versions of GCaMP2 did not yield larger signals than their non-targeted counterparts. We further targeted GCaMP2 to dendritic spines to monitor Ca(2+) accumulations evoked by activation of synaptic NMDA receptors. We observed robust DeltaF/F responses (range: 37%-264%) to single spine uncaging stimuli that were correlated with NMDA receptor currents measured through a somatic patch pipette. One major drawback of GCaMP2 was its low baseline fluorescence. Our results show that GCaMP2 is improved from the previous versions of GCaMP and may be suited to detect bursts of high-frequency action potentials and synaptic currents in vivo.
Akira Muto - One of the best experts on this subject based on the ideXlab platform.
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Real-Time Visualization of Neuronal Activity during Perception
Current Biology, 2013Co-Authors: Akira Muto, Masamichi Ohkura, Gembu Abe, Junichi Nakai, Koichi KawakamiAbstract:To understand how the brain perceives the external world, it is desirable to observe neuronal activity in the brain in real time during perception. The zebrafish is a suitable model animal for fluorescence imaging studies to visualize neuronal activity because its body is transparent through the embryonic and larval stages. Imaging studies have been carried out to monitor neuronal activity in the larval spinal cord and brain using Ca(2+) indicator dyes and DNA-encoded Ca(2+) indicators, such as Cameleon, GFP-aequorin, and GCaMPs. However, temporal and spatial resolution and sensitivity of these tools are still limited, and imaging of brain activity during perception of a natural object has not yet been demonstrated. Here we demonstrate visualization of neuronal activity in the optic tectum of larval zebrafish by genetically expressing the new version of GCaMP. First, we demonstrate Ca(2+) transients in the tectum evoked by a moving spot on a display and identify direction-selective neurons. Second, we show tectal activity during perception of a natural object, a swimming paramecium, revealing a functional visuotopic map. Finally, we image the tectal responses of a free-swimming larval fish to a paramecium and thereby correlate neuronal activity in the brain with prey capture behavior.
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imaging functional neural circuits in zebrafish with a new GCaMP and the gal4ff uas system
Communicative & Integrative Biology, 2011Co-Authors: Akira Muto, Koichi KawakamiAbstract:Measurement of the activity of neuronal ensembles is an essential step to understand how the neuronal network is organized and functioning. Electrical excitation of neurons causes calcium influx via voltage-gated calcium ion channels, which can be monitored by calcium imaging using fluorescent calcium probes. DNA-encoded calcium indicators (DECIs) such as cameleon and GCaMP have been developed to specifically label a subpopulation of neurons. However, in many cases, DECIs that had been developed and tested in vitro did not always show expected performance in vivo. It is necessary to increase its sensitivity and also to adjust its dynamic range to the physiological conditions. In our recent study, we developed an improved version of GCaMP and tested its performance in vivo using transgenic zebrafish. By combining the new GCaMP with targeted gene expression via the Gal4FF-UAS system, we successfully imaged the activity of the spinal motor circuit during spontaneous contractions of zebrafish larvae. Further ...
-
genetic visualization with an improved GCaMP calcium indicator reveals spatiotemporal activation of the spinal motor neurons in zebrafish
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Akira Muto, Masamichi Ohkura, Junichi Nakai, Tomoya Kotani, Shinichi Higashijima, Koichi KawakamiAbstract:Animal behaviors are generated by well-coordinated activation of neural circuits. In zebrafish, embryos start to show spontaneous muscle contractions at 17 to 19 h postfertilization. To visualize how motor circuits in the spinal cord are activated during this behavior, we developed GCaMP-HS (GCaMP-hyper sensitive), an improved version of the genetically encoded calcium indicator GCaMP, and created transgenic zebrafish carrying the GCaMP-HS gene downstream of the Gal4-recognition sequence, UAS (upstream activation sequence). Then we performed a gene-trap screen and identified the SAIGFF213A transgenic fish that expressed Gal4FF, a modified version of Gal4, in a subset of spinal neurons including the caudal primary (CaP) motor neurons. We conducted calcium imaging using the SAIGFF213A; UAS:GCaMP-HS double transgenic embryos during the spontaneous contractions. We demonstrated periodic and synchronized activation of a set of ipsilateral motor neurons located on the right and left trunk in accordance with actual muscle movements. The synchronized activation of contralateral motor neurons occurred alternately with a regular interval. Furthermore, a detailed analysis revealed rostral-to-caudal propagation of activation of the ipsilateral motor neuron, which is similar to but much slower than the rostrocaudal delay observed during swimming in later stages. Our study thus demonstrated coordinated activities of the motor neurons during the first behavior in a vertebrate. We propose the GCaMP technology combined with the Gal4FF-UAS system is a powerful tool to study functional neural circuits in zebrafish.
Junichi Nakai - One of the best experts on this subject based on the ideXlab platform.
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Real-Time Visualization of Neuronal Activity during Perception
Current Biology, 2013Co-Authors: Akira Muto, Masamichi Ohkura, Gembu Abe, Junichi Nakai, Koichi KawakamiAbstract:To understand how the brain perceives the external world, it is desirable to observe neuronal activity in the brain in real time during perception. The zebrafish is a suitable model animal for fluorescence imaging studies to visualize neuronal activity because its body is transparent through the embryonic and larval stages. Imaging studies have been carried out to monitor neuronal activity in the larval spinal cord and brain using Ca(2+) indicator dyes and DNA-encoded Ca(2+) indicators, such as Cameleon, GFP-aequorin, and GCaMPs. However, temporal and spatial resolution and sensitivity of these tools are still limited, and imaging of brain activity during perception of a natural object has not yet been demonstrated. Here we demonstrate visualization of neuronal activity in the optic tectum of larval zebrafish by genetically expressing the new version of GCaMP. First, we demonstrate Ca(2+) transients in the tectum evoked by a moving spot on a display and identify direction-selective neurons. Second, we show tectal activity during perception of a natural object, a swimming paramecium, revealing a functional visuotopic map. Finally, we image the tectal responses of a free-swimming larval fish to a paramecium and thereby correlate neuronal activity in the brain with prey capture behavior.
-
genetic visualization with an improved GCaMP calcium indicator reveals spatiotemporal activation of the spinal motor neurons in zebrafish
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Akira Muto, Masamichi Ohkura, Junichi Nakai, Tomoya Kotani, Shinichi Higashijima, Koichi KawakamiAbstract:Animal behaviors are generated by well-coordinated activation of neural circuits. In zebrafish, embryos start to show spontaneous muscle contractions at 17 to 19 h postfertilization. To visualize how motor circuits in the spinal cord are activated during this behavior, we developed GCaMP-HS (GCaMP-hyper sensitive), an improved version of the genetically encoded calcium indicator GCaMP, and created transgenic zebrafish carrying the GCaMP-HS gene downstream of the Gal4-recognition sequence, UAS (upstream activation sequence). Then we performed a gene-trap screen and identified the SAIGFF213A transgenic fish that expressed Gal4FF, a modified version of Gal4, in a subset of spinal neurons including the caudal primary (CaP) motor neurons. We conducted calcium imaging using the SAIGFF213A; UAS:GCaMP-HS double transgenic embryos during the spontaneous contractions. We demonstrated periodic and synchronized activation of a set of ipsilateral motor neurons located on the right and left trunk in accordance with actual muscle movements. The synchronized activation of contralateral motor neurons occurred alternately with a regular interval. Furthermore, a detailed analysis revealed rostral-to-caudal propagation of activation of the ipsilateral motor neuron, which is similar to but much slower than the rostrocaudal delay observed during swimming in later stages. Our study thus demonstrated coordinated activities of the motor neurons during the first behavior in a vertebrate. We propose the GCaMP technology combined with the Gal4FF-UAS system is a powerful tool to study functional neural circuits in zebrafish.
-
characterization and subcellular targeting of GCaMP type genetically encoded calcium indicators
PLOS ONE, 2008Co-Authors: Junichi Nakai, Tianyi Mao, Daniel H Oconnor, Volker Scheuss, Karel SvobodaAbstract:Genetically-encoded calcium indicators (GECIs) hold the promise of monitoring [Ca(2+)] in selected populations of neurons and in specific cellular compartments. Relating GECI fluorescence to neuronal activity requires quantitative characterization. We have characterized a promising new genetically-encoded calcium indicator-GCaMP2-in mammalian pyramidal neurons. Fluorescence changes in response to single action potentials (17+/-10% DeltaF/F [mean+/-SD]) could be detected in some, but not all, neurons. Trains of high-frequency action potentials yielded robust responses (302+/-50% for trains of 40 action potentials at 83 Hz). Responses were similar in acute brain slices from in utero electroporated mice, indicating that long-term expression did not interfere with GCaMP2 function. Membrane-targeted versions of GCaMP2 did not yield larger signals than their non-targeted counterparts. We further targeted GCaMP2 to dendritic spines to monitor Ca(2+) accumulations evoked by activation of synaptic NMDA receptors. We observed robust DeltaF/F responses (range: 37%-264%) to single spine uncaging stimuli that were correlated with NMDA receptor currents measured through a somatic patch pipette. One major drawback of GCaMP2 was its low baseline fluorescence. Our results show that GCaMP2 is improved from the previous versions of GCaMP and may be suited to detect bursts of high-frequency action potentials and synaptic currents in vivo.
Tianyi Mao - One of the best experts on this subject based on the ideXlab platform.
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imaging neural activity in worms flies and mice with improved GCaMP calcium indicators
Nature Methods, 2009Co-Authors: Lin Tian, Jasper Akerboom, Andrew S Hires, Tianyi Mao, Daniel Huber, Eugenia M Chiappe, Sreekanth H Chalasani, Leopoldo Petreanu, Sean A MckinneyAbstract:An improved version of the GCaMP genetically encoded calcium indicator, called GCaMP3, has higher calcium affinity and increased baseline fluorescence, dynamic range and stability. GCaMP3 performs better than existing genetically encoded calcium indicators in several assays and organisms, including in vivo imaging of neuronal signaling in worms, flies and mice. Genetically encoded calcium indicators (GECIs) can be used to image activity in defined neuronal populations. However, current GECIs produce inferior signals compared to synthetic indicators and recording electrodes, precluding detection of low firing rates. We developed a single-wavelength GCaMP2-based GECI (GCaMP3), with increased baseline fluorescence (3-fold), increased dynamic range (3-fold) and higher affinity for calcium (1.3-fold). We detected GCaMP3 fluorescence changes triggered by single action potentials in pyramidal cell dendrites, with signal-to-noise ratio and photostability substantially better than those of GCaMP2, D3cpVenus and TN-XXL. In Caenorhabditis elegans chemosensory neurons and the Drosophila melanogaster antennal lobe, sensory stimulation–evoked fluorescence responses were significantly enhanced with GCaMP3 (4–6-fold). In somatosensory and motor cortical neurons in the intact mouse, GCaMP3 detected calcium transients with amplitudes linearly dependent on action potential number. Long-term imaging in the motor cortex of behaving mice revealed large fluorescence changes in imaged neurons over months.
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characterization and subcellular targeting of GCaMP type genetically encoded calcium indicators
PLOS ONE, 2008Co-Authors: Junichi Nakai, Tianyi Mao, Daniel H Oconnor, Volker Scheuss, Karel SvobodaAbstract:Genetically-encoded calcium indicators (GECIs) hold the promise of monitoring [Ca(2+)] in selected populations of neurons and in specific cellular compartments. Relating GECI fluorescence to neuronal activity requires quantitative characterization. We have characterized a promising new genetically-encoded calcium indicator-GCaMP2-in mammalian pyramidal neurons. Fluorescence changes in response to single action potentials (17+/-10% DeltaF/F [mean+/-SD]) could be detected in some, but not all, neurons. Trains of high-frequency action potentials yielded robust responses (302+/-50% for trains of 40 action potentials at 83 Hz). Responses were similar in acute brain slices from in utero electroporated mice, indicating that long-term expression did not interfere with GCaMP2 function. Membrane-targeted versions of GCaMP2 did not yield larger signals than their non-targeted counterparts. We further targeted GCaMP2 to dendritic spines to monitor Ca(2+) accumulations evoked by activation of synaptic NMDA receptors. We observed robust DeltaF/F responses (range: 37%-264%) to single spine uncaging stimuli that were correlated with NMDA receptor currents measured through a somatic patch pipette. One major drawback of GCaMP2 was its low baseline fluorescence. Our results show that GCaMP2 is improved from the previous versions of GCaMP and may be suited to detect bursts of high-frequency action potentials and synaptic currents in vivo.
