Optogenetic Probing and Manipulation of the Calyx-Type Presynaptic Terminal in the Embryonic Chick Ciliary Ganglion

Ryo Egawa, Shoko Hososhima, Xubin Hou, Hidetaka Katow, Toru Ishizuka, Harukazu Nakamura, Hiromu Yawo

    研究成果: Article査読

    9 被引用数 (Scopus)

    抄録

    The calyx-type synapse of chick ciliary ganglion (CG) has been intensively studied for decades as a model system for the synaptic development, morphology and physiology. Despite recent advances in optogenetics probing and/or manipulation of the elementary steps of the transmitter release such as membrane depolarization and Ca2+ elevation, the current gene-manipulating methods are not suitable for targeting specifically the calyx-type presynaptic terminals. Here, we evaluated a method for manipulating the molecular and functional organization of the presynaptic terminals of this model synapse. We transfected progenitors of the Edinger-Westphal (EW) nucleus neurons with an EGFP expression vector by in ovo electroporation at embryonic day 2 (E2) and examined the CG at E8-14. We found that dozens of the calyx-type presynaptic terminals and axons were selectively labeled with EGFP fluorescence. When a Brainbow construct containing the membrane-tethered fluorescent proteins m-CFP, m-YFP and m-RFP, was introduced together with a Cre expression construct, the color coding of each presynaptic axon facilitated discrimination among inter-tangled projections, particularly during the developmental re-organization period of synaptic connections. With the simultaneous expression of one of the chimeric variants of channelrhodopsins, channelrhodopsin-fast receiver (ChRFR), and R-GECO1, a red-shifted fluorescent Ca2+-sensor, the Ca2+ elevation was optically measured under direct photostimulation of the presynaptic terminal. Although this optically evoked Ca2+ elevation was mostly dependent on the action potential, a significant component remained even in the absence of extracellular Ca2+. It is suggested that the photo-activation of ChRFR facilitated the release of Ca2+ from intracellular Ca2+ stores directly or indirectly. The above system, by facilitating the molecular study of the calyx-type presynaptic terminal, would provide an experimental platform for unveiling the molecular mechanisms underlying the morphology, physiology and development of synapses.

    本文言語English
    論文番号e59179
    ジャーナルPloS one
    8
    3
    DOI
    出版ステータスPublished - 2013 3 21

    ASJC Scopus subject areas

    • Biochemistry, Genetics and Molecular Biology(all)
    • Agricultural and Biological Sciences(all)
    • General

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