[Introduction] In various nervous systems, a presynaptic neuron once branches extensively and projects its axons to many postsynaptic neurons in early developmental stages. The number of branches is reduced with development and the connections between presynaptic and postsynaptic neurons become refined. The ciliary ganglion of chick embryo has been one of model systems of synaptogenesis. As early as in embryonic day 8 (E8), postsynaptic ciliary neuron is multiply innervated by the presynaptic axons. Before E14 a postsynaptic ciliary neuron receives only one calyx-type presynaptic terminal of a midbrain neuron in the Edinger-Westphal nucleus. It has been hypothesized that the refinement of neuronal connections is dependent on the activity of presynaptic and/or postsynaptic neurons. Here, we invented new system using in ovo electroporation and shell-less culture and investigated if the activity of presynaptic neurons is necessary and sufficient for the synapse refinement in the ciliary ganglion using optogenetics. [Method] CAGGS plasmids containing a tandem construct of chimeric variants of channelrhodopsins (ChRs), such as ChRWR, ChRGR and ChRFR(C167A), and yellow florescence protein (Venus), were introduced in the midbrain of E2 embryo and incorporated in the neuroblasts using in ovo electroporation method (Egawa et al., 2013). Some chick embryos that expressed channelrhodopsin were cultured using shell-less culture method. These embryos were LED-flashed between E8 and E14, and examined under confocal microscopy at E14. The unflashed embryos were used as control. The synaptic current was measured from the postsynaptic ciliary neuron at E14 under conventional whole-cell patch clamp. [Results & Discussion] We found the expression of venus-conjugated ChRs in neurons of the Edinger-Westphal nucleus, axons in the oculomotor nerve and calyx-type presynaptic terminals in the ciliary ganglion of E8-14 embryo. When the ChR-expressing presynaptic terminal was flashed by LED, the light-dependent synaptic currents were evoked in the postsynaptic neuron. Although the ciliary ganglion was somewhat distorted in shape under shell-less culture during E2-14, the calyx-type presynaptic terminals were almost normally developed in appearence. It is suggested that the activity of a presynaptic axon/terminal should be optogenetically manipulated during embryogenesis in the shell-less culture to reveal the relationship between the presynaptic activity and the synapse refinement.