In immobilized tadpoles, the head skin was cut along the midline using superfine scissors to minimize the stretching and the damage to the peripheral LL organs. The hindbrain dorsal roof was opened and some ependymal cells inside the neurocoele were removed to expose neuronal somata using a tungsten dissection needle (47 (link)). Tadpoles were left further in 5 µM Fluo-4 AM (Thermo Fisher Scientific, UK) saline solution for ∼20 min in darkness. After resting the tadpole for ∼20 min, fluorescence images were captured at 10 Hz using a ×10 water immersion lens with a Neo5.5 CMOS camera and the Andor Solis software (Oxford Instruments, UK), around the time when the anterior lateral line was activated by suction. Regions of interest (ROIs) were chosen based on visible increases of fluorescence intensity following suction during video replay. A large blank area void of tissue was chosen to determine background illumination, which was subtracted from all ROI fluorescence intensity measurements. Fluorescence intensities were given as change of intensity in percentages compared with the baseline, i.e., in the absence of sensory stimulation or motor activity at the same ROIs. Any fluorescence increase lower than 5% within 1 s after suction stimulus was classified as lack of response.
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