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    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 Ca(2+) 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 Ca(2+)-sensor, the Ca(2+) elevation was optically measured under direct photostimulation of the presynaptic terminal. Although this optically evoked Ca(2+) elevation was mostly dependent on the action potential, a significant component remained even in the absence of extracellular Ca(2+). It is suggested that the photo-activation of ChRFR facilitated the release of Ca(2+) from intracellular Ca(2+) 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.


    Ryo Egawa, Shoko Hososhima, Xubin Hou, Hidetaka Katow, Toru Ishizuka, Harukazu Nakamura, Hiromu Yawo. Optogenetic probing and manipulation of the calyx-type presynaptic terminal in the embryonic chick ciliary ganglion. PloS one. 2013;8(3):e59179

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    PMID: 23555628

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