Comprehensive Imaging of Sensory-Evoked Activity of Entire Neurons Within the Awake Developing Brain Using Ultrafast AOD-Based Random-Access Two-Photon Microscopy
Determining how neurons transform synaptic input and encode information in action potential (AP) firing output is required for understanding dendritic integration, neural transforms and encoding. Limitations in the speed of imaging 3D volumes of brain encompassing complex dendritic arbors in vivo us...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2020-06-01
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| Series: | Frontiers in Neural Circuits |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fncir.2020.00033/full |
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| author | Kelly D. R. Sakaki Kaspar Podgorski Tristan A. Dellazizzo Toth Patrick Coleman Kurt Haas |
| author_facet | Kelly D. R. Sakaki Kaspar Podgorski Tristan A. Dellazizzo Toth Patrick Coleman Kurt Haas |
| author_sort | Kelly D. R. Sakaki |
| collection | DOAJ |
| description | Determining how neurons transform synaptic input and encode information in action potential (AP) firing output is required for understanding dendritic integration, neural transforms and encoding. Limitations in the speed of imaging 3D volumes of brain encompassing complex dendritic arbors in vivo using conventional galvanometer mirror-based laser-scanning microscopy has hampered fully capturing fluorescent sensors of activity throughout an individual neuron's entire complement of synaptic inputs and somatic APs. To address this problem, we have developed a two-photon microscope that achieves high-speed scanning by employing inertia-free acousto-optic deflectors (AODs) for laser beam positioning, enabling random-access sampling of hundreds to thousands of points-of-interest restricted to a predetermined neuronal structure, avoiding wasted scanning of surrounding extracellular tissue. This system is capable of comprehensive imaging of the activity of single neurons within the intact and awake vertebrate brain. Here, we demonstrate imaging of tectal neurons within the brains of albino Xenopus laevis tadpoles labeled using single-cell electroporation for expression of a red space-filling fluorophore to determine dendritic arbor morphology, and either the calcium sensor jGCaMP7s or the glutamate sensor iGluSnFR as indicators of neural activity. Using discrete, point-of-interest scanning we achieve sampling rates of 3 Hz for saturation sampling of entire arbors at 2 μm resolution, 6 Hz for sequentially sampling 3 volumes encompassing the dendritic arbor and soma, and 200–250 Hz for scanning individual planes through the dendritic arbor. This system allows investigations of sensory-evoked information input-output relationships of neurons within the intact and awake brain. |
| first_indexed | 2024-12-12T18:41:54Z |
| format | Article |
| id | doaj.art-968b00fd99d0400c9cb41e03cac03398 |
| institution | Directory Open Access Journal |
| issn | 1662-5110 |
| language | English |
| last_indexed | 2024-12-12T18:41:54Z |
| publishDate | 2020-06-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Neural Circuits |
| spelling | doaj.art-968b00fd99d0400c9cb41e03cac033982022-12-22T00:15:37ZengFrontiers Media S.A.Frontiers in Neural Circuits1662-51102020-06-011410.3389/fncir.2020.00033539180Comprehensive Imaging of Sensory-Evoked Activity of Entire Neurons Within the Awake Developing Brain Using Ultrafast AOD-Based Random-Access Two-Photon MicroscopyKelly D. R. Sakaki0Kaspar Podgorski1Tristan A. Dellazizzo Toth2Patrick Coleman3Kurt Haas4Department of Cellular and Physiological Sciences, Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, BC, CanadaJanelia Research, Ashburn, VA, United StatesDepartment of Cellular and Physiological Sciences, Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, BC, CanadaDepartment of Cellular and Physiological Sciences, Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, BC, CanadaDepartment of Cellular and Physiological Sciences, Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, BC, CanadaDetermining how neurons transform synaptic input and encode information in action potential (AP) firing output is required for understanding dendritic integration, neural transforms and encoding. Limitations in the speed of imaging 3D volumes of brain encompassing complex dendritic arbors in vivo using conventional galvanometer mirror-based laser-scanning microscopy has hampered fully capturing fluorescent sensors of activity throughout an individual neuron's entire complement of synaptic inputs and somatic APs. To address this problem, we have developed a two-photon microscope that achieves high-speed scanning by employing inertia-free acousto-optic deflectors (AODs) for laser beam positioning, enabling random-access sampling of hundreds to thousands of points-of-interest restricted to a predetermined neuronal structure, avoiding wasted scanning of surrounding extracellular tissue. This system is capable of comprehensive imaging of the activity of single neurons within the intact and awake vertebrate brain. Here, we demonstrate imaging of tectal neurons within the brains of albino Xenopus laevis tadpoles labeled using single-cell electroporation for expression of a red space-filling fluorophore to determine dendritic arbor morphology, and either the calcium sensor jGCaMP7s or the glutamate sensor iGluSnFR as indicators of neural activity. Using discrete, point-of-interest scanning we achieve sampling rates of 3 Hz for saturation sampling of entire arbors at 2 μm resolution, 6 Hz for sequentially sampling 3 volumes encompassing the dendritic arbor and soma, and 200–250 Hz for scanning individual planes through the dendritic arbor. This system allows investigations of sensory-evoked information input-output relationships of neurons within the intact and awake brain.https://www.frontiersin.org/article/10.3389/fncir.2020.00033/fullcomprehensive imagingrandom-accessmulti-photonacousto-opticsin-vivo calcium imagingencoding |
| spellingShingle | Kelly D. R. Sakaki Kaspar Podgorski Tristan A. Dellazizzo Toth Patrick Coleman Kurt Haas Comprehensive Imaging of Sensory-Evoked Activity of Entire Neurons Within the Awake Developing Brain Using Ultrafast AOD-Based Random-Access Two-Photon Microscopy Frontiers in Neural Circuits comprehensive imaging random-access multi-photon acousto-optics in-vivo calcium imaging encoding |
| title | Comprehensive Imaging of Sensory-Evoked Activity of Entire Neurons Within the Awake Developing Brain Using Ultrafast AOD-Based Random-Access Two-Photon Microscopy |
| title_full | Comprehensive Imaging of Sensory-Evoked Activity of Entire Neurons Within the Awake Developing Brain Using Ultrafast AOD-Based Random-Access Two-Photon Microscopy |
| title_fullStr | Comprehensive Imaging of Sensory-Evoked Activity of Entire Neurons Within the Awake Developing Brain Using Ultrafast AOD-Based Random-Access Two-Photon Microscopy |
| title_full_unstemmed | Comprehensive Imaging of Sensory-Evoked Activity of Entire Neurons Within the Awake Developing Brain Using Ultrafast AOD-Based Random-Access Two-Photon Microscopy |
| title_short | Comprehensive Imaging of Sensory-Evoked Activity of Entire Neurons Within the Awake Developing Brain Using Ultrafast AOD-Based Random-Access Two-Photon Microscopy |
| title_sort | comprehensive imaging of sensory evoked activity of entire neurons within the awake developing brain using ultrafast aod based random access two photon microscopy |
| topic | comprehensive imaging random-access multi-photon acousto-optics in-vivo calcium imaging encoding |
| url | https://www.frontiersin.org/article/10.3389/fncir.2020.00033/full |
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