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Fig. 4 Brain responses to unilateral directional stimulation of neuromasts (A) Stimulation and acquisition setup including two-photon laser scanning microscope (2p LSM), and the stimulation capillary. (B) Maximal z-projection of the reference brain with the region that was imaged boxed in blue. Orange box shows the location of the PLLG ipsilateral to the stimulation side. MON – Medial Octavolateralis Nucleus. A – anterior, L – left, R – right, P – posterior. (C) Activity of individual pLANs in the PLLG ipsilateral to the stimulation side in two larvæ before and after whole-brain scanning. Green and magenta colors show location of responsive cr and rc pLANs, respectively. Traces show z-scored fluorescence of respective pLANs averaged across planes. Note that after whole-brain scanning, the responses of pLANs to stimulation remained similar to the initial responses. (D) Location of all responsive ROIs. Black arrowhead indicates that there were no responsive ROIs in the contralateral PLLG. (E) Bimodal distribution of the phase of the power spectrum frequency component corresponding to the stimulation frequency (see Methods for details). (F) Two example cr- and rc-selective ROIs. (G) Activity of all cr- and rc-selective ROIs detected in all imaged larvæ. The bottom image shows activity, averaged across animals; shaded areas denote SEM across larvæ. (H) Location of all cr- and rc-selective ROIs in the reference brain. Color map is the same as in D. (I) Anatomical regions of the reference brain that contained cr- and rc-selective ROIs consistently across larvæ. (J and K) A maximal projection confocal image of a Gad1b-EGFP (green) ; Sill:Cherry (red) double transgenic fish at 6dpf (dorsal view in J and lateral view in K) showing the relative position of the LAN central lintel (dashed areas). Midbrain-hindbrain boundary is shown by a vertical dashed white line, and PLL ganglion by a white arrow. (L) A magnification of a portion of the LAN central projections (red) and Gad1b(+) neurons (green), showing spatial proximity. (M) Snapshot of a dorsal view of a larval zebrafish reference brain, taken from the mapZebrain Atlas database, depicting all available single-neuron traces. Dashed area indicates the position of the LAN central lintel and white arrow the position of the PLLG. (N) Same reference brain with 3 Gad1(+) neurons overlaid, colored green (ref. 12387), blue (ref. 12352) and red (ref. 12344) in the mapZebrain Atlas. The white dot is an arbitrary spatial reference used to evaluate neuronal projection is three-dimensional renderings (not shown). (O) Magnification of the neurons shown in (N). All 3 Gad1(+) neurons project contralaterally within the hindbrain. (P) Reference brain (gray) overlaid with Gad1(+) neurons. (Q) Reference brain (gray) overlaid with functional cr an rc maps (respectively, green and red). White arrowhead indicates the posterior lateralis ganglion ipsilateral to the mechanical-stimulated side of the fish. Note lack of calcium signal on the contralateral ganglion. (R) Gad1b stack overlaid with functional cr an rc maps (respectively, in green and red, whose overlap is yellow). (S) The number of active voxels overlapping with the Gad1b binary mask. Each fish contributes one point to each category in the x axis, the horizontal line denotes the mean number of voxels across fish and the vertical line denotes the standard deviation. p value = 0.0281 (Mann-Whitney-U-Test). Scale bars are 100 μm in (A) –(I) and 50 μm in (J) –(R). See also Figure S4.