シンポジウム
51 光科学者×神経科学者：異なる個性が触発する光神経科学の新展開
51 Optical scientists × neuroscientists: co-creation of new paradigm of opto-neural science
座長：三上 秀治（北海道大学 電子科学研究所）・佐藤 正晃（北海道大学 医学研究員） |
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| 2022年7月1日 9:00～9:18 ラグナガーデンホテル 羽衣：西 第10会場
2S10m-01
次世代神経科学のための高速蛍光顕微鏡技術
High-speed fluorescence microscopy for future neuroscience
*三上 秀治(1)
1. 北海道大学
*Hideharu Mikami(1)
1. Hokkaido University
Keyword: Fluorescence microscopy, High-speed imaging, Light-sheet microscopy
Fluorescence microscopy is an indispensable method in life science as it elucidates morphological details of complex organisms. Specifically, in neuroscience, optical recording of neural activity via calcium or voltage imaging is one of the most fundamental methodologies. Over the last few decades, the spatial resolution of fluorescence microscopy has dramatically improved with the emergence of laser-scanning confocal fluorescence microscopy and recently developed super-resolution techniques, significantly contributing to the evolution of life science. On the other hand, its data acquisition speed (imaging speed) is becoming a bottleneck of neuroscience research in recent years. For example, the emergence of practical genetically encoded voltage indicators enabled us to record millisecond-scale neural activities via fluorescence microscopy directly, but the spatial scale of the imaging is greatly limited as a result of the trade-off between the frame rate and the field of view of current fluorescence microscopes, which is governed by their data acquisition speed. Therefore, we have been developing high-speed fluorescence microscopy techniques that overcome the trade-off to open up new frontiers in neuroscience and other life science fields. I will present on recently developed high-speed fluorescence microscopy techniques and their applications and data analysis using machine learning, including high-speed volumetric imaging at 1,000 volumes/sec and large-scale image-based cytometry. I will also discuss the future of high-speed fluorescence imaging, which will lead to the integration of photonics, informatics, and life sciences. | | 2022年7月1日 9:18～9:48 ラグナガーデンホテル 羽衣：西 第10会場
2S10m-02
Towards cortex-wide volumetric recording of neuroactivity at cellular resolution
*Alipasha Vaziri(1)
1. Rockefeller University
Keyword: calcium imaging , two-photon microscopy, Whole-brain neurorecording , systems neuroscience
Understanding how sensory information is represented, processed and leads to generation of complex behavior is the major goal of systems neuroscience. However, the ability to detect and manipulate such large-scale functional circuits has been hampered by the lack of appropriate tools and methods that allow parallel and spatiotemporally specific manipulation of neuronal population activity while capturing the dynamic activity of the entire network at high spatial and temporal resolutions.
A central focus of our lab is the development and application of new optics-based neurotechnologies for large-scale, high-speed, and single-cell resolution interrogation of neuroactivity across model systems. Through these, we have consistently pushed the limits on speed, volume size, and depth at which neuronal population activity can be optically recorded at cellular resolution. Amongst others have demonstrated whole-brain recording of neuroactivity at cellular resolution in small model systems as well as more recently near-simultaneous recording from over 1 million neurons distributed across both hemispheres and different layers of the mouse cortex at cellular resolution.
I will present on our efforts on neurotechnology development and how the application of some of these optical neurotechnologies could enable solving a qualitatively new range of neurobiological questions that are beyond reach of current methods. Ultimately, our aim is to uncover some of the computational principles underlying representation of sensory information at different levels, its processing across the mammalian brain, and how its interaction with internal states generates behavior. | | 2022年7月1日 9:48～10:12 ラグナガーデンホテル 羽衣：西 第10会場
2S10m-03
Voltage imaging of Purkinje neuron dendrites in awake mice
*Bernd Kuhn(1)
1. OIST Graduate University
Keyword: VOLTAGE IMAGING, DENDRITES, PURKINJE, CEREBELLUM
Previously, we showed that it is possible to simultaneous image dendritic voltage and calcium and record somatic electric activity from Purkinje neurons in awake mice (1). To do this, we fill single Purkinje neurons with the synthetic voltage-sensitive dye ANNINE-6plus (2) by electroporation through a chronic cranial window with access port (3, 4). We imaged with two-photon microscopy and were able to resolve spikelets of the dendritic complex spike triggered by climbing fiber input, and dendritic spikes and hotspots both triggered by parallel fiber input. In response to sensory stimuli, we find depolarizing and hyperpolarizing potentials and we able to show dendritic coincidence detection in Purkinje neurons (5). Now, we study dendritic maps and how they are modulated by inhibitory input. We image presynaptic calcium activity in axons of molecular layer interneurons and postsynaptic potentials in the dendrite of Purkinje neurons. Additionally, we investigate dendritic Purkinje neuron activity during an associative motor learning task. We image dendritic voltage changes repeatedly over days during eye blink conditioning. Thereby, we hope to reveal how dendritic integration changes during learning.
1. Roome CJ & Kuhn B (2018) Simultaneous dendritic voltage and calcium imaging and somatic recording from Purkinje neurons in awake mice. Nat Commun 9(1):3388.
2. Kuhn B & Roome CJ (2019) Primer to Voltage Imaging With ANNINE Dyes and Two-Photon Microscopy. Front Cell Neurosci 13:321.
3. Roome CJ & Kuhn B (2014) Chronic cranial window with access port for repeated cellular manipulations, drug application, and electrophysiology. Front Cell Neurosci 8:379.
4. Roome CJ & Kuhn B (2019) Voltage imaging with ANNINE dyes and two-photon microscopy. Multiphoton Microscopy, Springer Nature Neuromethods, ed Hartveit E (Springer Nature (Neuromethods)).
5. Roome CJ & Kuhn B (2020) Dendritic coincidence detection in Purkinje neurons of awake mice. Elife 9. | | 2022年7月1日 10:12～10:36 ラグナガーデンホテル 羽衣：西 第10会場
2S10m-04
センチメートル視野内の全細胞を観るトランススケールスコープ
Trans-scale scope for dynamic observation of all cells in a centimeter field of view
*市村 垂生(1,2)、永井 健治(1,3)、橋本 均(1,4)
1. 大阪大学先導的学際研究機構、2. JSTさきがけ、3. 大阪大学産業科学研究所、4. 大阪大学薬学研究科
*Taro Ichimura(1,2), Takeharu Nagai(1,3), Hitoshi Hashimoto(1,4)
1. OTRI, Osaka University, Osaka, Japan, 2. JST PRESTO, 3. SANKEN, Osaka University, Osaka, Japan, 4. Grad Sch Pharmacy, Osaka University, Osaka, Japan
Keyword: microscopy, fluorescence imaging, multicellular system
Optical imaging plays the vital role in the studies of multicellular systems including neuroscience. In order to find important cells that trigger a state transition of a multicellular system, it is desirable for an optical imaging tool to have both a wide field-of-view (FOV) to see the state of the entire system in the millimeter/centimeter scale and high spatial resolution to see the dynamics of all the element cells in the micrometer scale. We have been developing a “trans-scale scope” to cover the scale range from the micrometer to centimeter beyond the limitation of conventional microscopy, which we named AMATERAS. In the presentation, we will introduce the first version of AMATERAS which has realized fluorescence imaging at sub-cellular spatial resolution in an over-one-centimeter FOV. We demonstrated its ability of time-lapse imaging of multicellular system with capturing the dynamics of 105-106 cells in the system in a single FOV. By utilizing automatic analytical tools, one can find exceptional cells responsible for the fate of millions of cells. We applied the technology to imaging of many biological systems including mouse brain, and verified that our method is very powerful in multicellular biology. | | 2022年7月1日 10:36～11:00 ラグナガーデンホテル 羽衣：西 第10会場
2S10m-05
神経細胞樹状突起スパインの光遺伝学的操作
Optogenetic manipulation of dendritic spines in neurons
*村越 秀治(1)
1. 生理学研究所
*Hideji Murakoshi(1)
1. National Institute for Physiological Sciences
Keyword: Photoactivatable protein, 2-photon excitation, Synaptic plasticity
Optogenetic approaches to studying neuronal function have proven their usefulness in neurosciences. However, optogenetic tools capable of inducing synaptic plasticity at the level of single synapses have been lacking. Here, we engineered a photoactivatable (pa)CaMKII by fusing a light-sensitive domain, LOV2, to CaMKIIα. Blue light or two-photon excitation reversibly activated paCaMKII. Activation in single spines was sufficient to induce structural long-term potentiation (sLTP) in vitro and in vivo. paCaMKII activation was also sufficient for the recruitment of AMPA receptors and functional LTP in single spines. We applied paCaMKII for understanding the mechanisms of metaplasticity at the single-synapse level. When neurons are exposed to signals that induce aberrant neuronal excitation, they increase the threshold for the induction of long-term potentiation (LTP), known as metaplasticity. However, the metaplastic regulation of structural LTP (sLTP) remains unclear. Here, we investigate glutamate uncaging/photoactivatable (pa)CaMKII-dependent sLTP induction in hippocampal CA1 neurons after chronic neuronal excitation by GABAA receptor antagonists. We found that the neuronal excitation decreases the glutamate uncaging-evoked Ca2+ influx mediated by GluN2B-containing NMDA receptors and suppresses sLTP induction. In addition, single-spine optogenetic stimulation using paCaMKII indicates the suppression of CaMKII signaling. While the inhibition of Ca2+ influx is protein synthesis-independent, the paCaMKII-induced sLTP suppression depends on it. Our findings demonstrate that chronic neuronal excitation suppresses sLTP in two independent ways (i.e., dual inhibition of Ca2+ influx and CaMKII signaling). This dual inhibition mechanism may contribute to robust neuronal protection in excitable environments. Thus, paCaMKII for dissecting the function of CaMKII activation and for manipulating synaptic plasticity is expected to have many applications in neuroscience and other fields. | | | |
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