Fluorescent Ca2+ Imaging of Ciona Larvae

The fixed Ciona larvae on a glass-based dish were observed by fluorescence microscopy with a 3CCD camera. A long-term Ca²⁺ transient was captured with pSP-CiVAChT:GCaMP6s or pSP-CiVAChT-H2B:GCaMP6s transduced Ciona embryos (N = 10, Figure 2; Figure 3., Supplementary Table S1). The same results can be obtained with either pSP-CiVAChT:GCaMP6s or pSP-CiVAChT-H2B:GCaMP6s (data not shown). An inverted microscope (Nikon Eclipse, IX71) with a 10×, 20×, 60× oil immersion objective lens (LUCPlanFLN) was used for fluorescence imaging with a U-MWBV2 mirror unit (Olympus, Shinjuku, Japan). SOLA LED light (Lumencor, Beaverton, OR) was used as a light source; fluorescence images were acquired with a 3CCD camera (C7800-20, Hamamatsu Photonics, Hamamatsu, Japan) and processed by the AQUACOSMOS software (Hamamatsu Photonics). Room temperature was maintained at 20°C. As a result, Ca²⁺ transients for both MN2L and MN2R, located at the Ciona motor ganglion region (Figure 1C), were continuously imaged from St.22 (mid tailbud II) to St.34 (late tail absorption) (N = 10, Supplementary Table S1). Ciona developmental staging (Hotta et al., 2007 (link)) was used to estimate the developmental stage from morphology and time after fertilization.