title: Optogenetic probing and manipulation of the calyx-type presynaptic terminal in the embryonic chick ciliary ganglion.
abstract: 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.
authors.affiliation: Department of Developmental Biology and Neuroscience, Tohoku University Graduate School of Life Sciences, Sendai, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Egawa
authors.forename: Shoko
authors.initials: S
authors.lastname: Hososhima
authors.forename: Xubin
authors.initials: X
authors.lastname: Hou
authors.forename: Hidetaka
authors.initials: H
authors.lastname: Katow
authors.forename: Toru
authors.initials: T
authors.lastname: Ishizuka
authors.forename: Harukazu
authors.initials: H
authors.lastname: Nakamura
authors.forename: Hiromu
authors.initials: H
authors.lastname: Yawo
mesh_terms: D000200:Action Potentials / Q000502:physiology / Q000528:radiation effects; D000818:Animals; D001369:Axons / Q000502:physiology / Q000528:radiation effects; D002118:Calcium / Q000378:metabolism; D002467:Cell Nucleus / Q000502:physiology / Q000528:radiation effects; D002642:Chick Embryo; D018274:Electroporation; D005726:Ganglia, Parasympathetic / Q000502:physiology* / Q000528:radiation effects; D015870:Gene Expression / Q000528:radiation effects; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D017136:Ion Transport / Q000528:radiation effects; D008027:Light; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D062308:Optogenetics / Q000379:methods*; D017729:Presynaptic Terminals / Q000502:physiology* / Q000528:radiation effects; D013569:Synapses / Q000502:physiology* / Q000528:radiation effects; D009435:Synaptic Transmission / Q000528:radiation effects*; D014162:Transfection
chemical_list: D009419:Nerve Tissue Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D002118:Calcium
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