Simultaneous voltage and calcium imaging and mRNA extraction from single neurons in-vivo
Publicly Offered Research
| Project Area | Census-based biomechanism of circuit construction and transition for adaptive brain functions |
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| Project/Area Number |
22H05517
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| Research Category |
Grant-in-Aid for Transformative Research Areas (A)
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| Allocation Type | Single-year Grants |
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| Review Section |
Transformative Research Areas, Section (III)
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| Research Institution | Okinawa Institute of Science and Technology Graduate University |
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Principal Investigator |
ROOME Christopher 沖縄科学技術大学院大学, 光学ニューロイメージングユニット, スタッフサイエンティスト (60955392)
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| Project Period (FY) |
2022-06-16 – 2024-03-31
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| Project Status |
Granted (Fiscal Year 2023)
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| Budget Amount *help |
¥7,670,000 (Direct Cost: ¥5,900,000、Indirect Cost: ¥1,770,000)
Fiscal Year 2023: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000)
Fiscal Year 2022: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000)
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| Keywords | dendrites / voltage imaging / protein expression / calcium imaging / dendritic integration / neuronal computation / two-photon imaging / cerebellum / Neuron signaling / mRNA analysis |
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| Outline of Research at the Start |
For a deeper understanding of neuronal function, this research will record multidimensionally from a single neuron in the awake behaving animal. The functional activity of neurons will then be connected to their unique protein expression pattern.
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| Outline of Annual Research Achievements |
Dendritic spikes are thought to play an essential role in synaptic integration in vivo, yet precisely how they convey information remains unknown. Dendritic computations may be performed by modulating the propagation of dendritic spikes through specific dendritic branches. Propagation of dendritic spikes could be determined by the activity of local synaptic inputs to generate ‘dendritic computational units’ specific for each dendritic spike. As such, these dendritic computational units, i.e., the spatiotemporal propagation of dendritic spikes, could decode specific synaptic input patterns that, for example, encode a sensory stimulus.
High resolution spatiotemporal voltage maps are necessary to investigate how dendritic spikes propagate in vivo. Using two-photon voltage imaging, we recorded dendritic voltage spikes in awake mice with high spatiotemporal resolution (Roome & Kuhn 2018). Single cerebellar Purkinje neurons were double labelled with voltage sensor, ‘ANNINE-6plus’ and genetically encoded calcium sensor GCaMP6f, using chronic cranial window with access port (Roome & Kuhn 2014). Dendrites were imaged by two-photon microscopy in linescan mode at 2kHz. Using rapid linescan imaging it was possible to detect dendritic spikes across several dendritic processes simultaneously, to determine how dendritic spikes propagate through the Purkinje neuron dendrites.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
We recorded rapid (1ms) dendritic spikes in the distal spiny dendritic branchlets of Purkinje neurons. Dendritic complex spikes (DCS) typically comprising a ‘burst’ of 2-5 dendritic spikelets. These dendritic spikes are ‘calcium spikes’ triggered by climbing fiber synaptic input, and so could be confirmed by their corresponding calcium signals. Climbing fiber synaptic inputs occurred spontaneously at a characteristic rate of 1Hz and could also be ‘evoked’ by sensory stimulation (air-puff directed towards the eye). To investigate how local synaptic inputs modulate dendritic spike propagation we used pharmacology, applied via micropipette, to selectively block metabotropic receptors (mGluR1). The preliminary data was presented at an international conference on dendrites (GRC dendrites) in March 2022. In preparation for protein expression analysis aspect of this project we have also received protocols from our collaborators, for performing single neuron mRNA extraction, in vivo.
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| Strategy for Future Research Activity |
The next step of the research is to combine functional imaging techniques with protein expression analysis so that a relationship between functional signaling and protein expression patterns can be established.
Now that the preliminary functional imaging data is collected and being analyzed, we focus on optimizing mRNA extraction techniques and/or developing techniques for determining protein expression within the Purkinje neuron dendrites.
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Report
(1 results)
Research Products
(2 results)
