| 研究実績の概要 |
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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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
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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