To make Tol2-ribeye-ChR2-CFP plasmid, the promoter of zebrafish ribeye a was excised from a ribeye1.8 plasmid with NaeI and EcoRI restriction enzymes, and then cloned into the StuI and EcoRI sites of Tol2-UAS-ChR2-CFP plasmid. For the generation of Tg(Ribeye:ChR2-CFP) fish lines, the mixture of 25 pg DNA and 25 pg transposon mRNA was first injected into one-cell stage embryos, which were raised to adulthood for further screening of fluorescent signalling. F2 generation was used in optogenetic experiments. For optogenetic stimulation during calcium imaging, a 0.5 s pulse of 440-nm laser was applied to ChR2-expressing BC ATs within a circle (20 μm in diameter), while time-lapse images of calcium activities were simultaneously collected with a 900 nm or 488 nm laser via an Olympus FV1000 microscopy. In general, the volume of single BC ATs is about 3.14 × 22 × 3 μm3. On the basis of the scattering of light in larval zebrafish brain's tissues55 (link), it is speculated that around 50–100 BC ATs within a volume of 3.14 × 102 × 10 μm3 (the depth of z axis is about 10 μm) were activated during the optogenetic activation.
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