Article Figures & Data
Figures
Visual Abstract
- Figure 1.
Experimental setup and procedure. Before auditory stimulation, fish were allowed to acclimatize to the enclosure overnight (left) before auditory stimulation began (middle). After auditory stimulation, fish were sacrificed at eight different time points (t = 0, 10, 70, 100, 130, 160, 190, and 250 min). The 0 minute time group served as a baseline control without auditory stimulation.
- Figure 2.
Illustration of the location within the experimental tank where the broad frequency range stimulus was measured (on the right). In the center of the figure, the corresponding waveforms of the auditory stimulation are displayed, with measurements taken at various distances from the tank's border: center (depicted in magenta), 10 cm from the border (depicted in cyan), and 1 cm from the border (depicted in green). Additionally, the fast Fourier transform (FFT) of the recorded stimuli in these different positions is presented on the right.
- Figure 3.
Summary of tracing in the central division of the TSc. A, Schematic drawing of the zebrafish brain from the sagittal view highlights the ascending auditory regions and the coronal plane locations (B–F) along the anterior–posterior axis. B–F, Schematic drawings of the coronal sections showing the distribution of labeling (red circles, cell bodies; red lines, fibers) and the corresponding photographs: Neurobiotin-labeled cells and fibers in DON (B1), in SON (C1), in AON (D1); in the LL and RF (D2) and the injection site in the TSc (E1). Please note that the fibers visible in the TSvl do not terminate in this region and are projections from TSc to other regions; E2, The location of NI, delineated based on ChAT staining in which no Neurobiotin was found. F, Labeling in the AT is limited to dAT and is absent in the vAT region. Inset in F1 shows a magnification in the dAT in which labeled cell bodies were present, as indicated by the white arrows. F2, Labeling in the CP. Scale bars represent 200 µm in the schematic drawings and 100 µm in the staining sections. DON, dorsal octaval nucleus; SON, secondary octaval nucleus; AON, anterior octaval nucleus; LL, lateral lemniscus; RF, reticular formation; CP, central posterior thalamic nucleus; AT, anterior tuberal nucleus; TSc, the central nucleus of the torus semicircularis; NI, nucleus isthmi; CP, central posterior thalamic nucleus; CCe, corpus cerebelli; EG, eminentia granularis; VIII, octaval nerve; TeO, tectum opticum; TSvl, ventrolateral nucleus of torus semicircularis; RV, rhombencephalic ventricle; vAT, ventral part of the anterior tuberal nucleus; DP, dorsal posterior thalamic nucleus; LH, lateral hypothalamic nucleus; DP, dorsal zone of periventricular nucleus.
- Figure 4.
Maximum projections of confocal micrographs depicting the main toral connectivity. Schematic drawings of coronal brain sections and photographs of connected nuclei. Neurobiotin-labeling was found in the SON (A1), the DON (A2), the AON (B1), the RF (B2), fibers of the LL (C2), the dAT (D1), and CP (D2). Further depicted is the injection site in the central nucleus of TSc (C1) with labeled fibers and cells. CCe, corpus cerebelli; EG, eminentia granularis; VIII, octaval nerve; TeO, tectum opticum; TSvl, ventrolateral nucleus of torus semicircularis; RV, rhombencephalic ventricle; vAT, ventral part of the anterior tuberal nucleus; DP, dorsal posterior thalamic nucleus; LH, lateral hypothalamic nucleus; DP, dorsal zone of periventricular nucleus. Scale bars are 200 µm in the schematics and 50 µm in the confocal projections.
- Figure 5.
A, Schematic drawing of a coronal section with a typical placement of a cell counting zone (blue rectangle) within the central nucleus of the TSc. B, Example photograph of a pS6 staining and the positioned counting zone within the TSc. C, Zoom to the cell counting zone within TSc. pS6-ir cells are stained in magenta (full arrowheads) and are easily distinguishable from the nonactivated cells (empty arrowheads). Cell nuclei are stained with DAPI (blue). DIL, diffuse nucleus of the anterior lobe; Nin, nucleus intrapeduncularis; TTB, tractus tectobulbaris; NLV, nucleus lateralis valvulae; TSvl, ventrolateral nucleus of torus semicircularis (TSvl); TL, torus longitudinalis; TeO, tectum opticum; PGZ, periventricular gray zone of optic tectum; RV, rhombencephalic ventricle; Ctec, commissura tecti; Val, lateral division of valvula cerebelli; Vam, medial division of valvula cerebelli.
- Figure 6.
The time-dependent pattern of up- and downregulation of pS6 phosphorylation across the three regions of interest. A, Central nucleus of the TSc. B, Dorsal portion of the anterior tuberal nucleus (dAT). C, NI. Bar plots show mean ± SEM with dots representing the individual values. Time groups in which the peak level was reached are in red. The unstimulated baseline is in gray and the other bars in blue. Asterisks mark significant differences compared with the baseline (*p < 0.05; **p < 0.01). The right side of the figure depicts representative example photos of pS6-ir cells (magenta) of the 130 min and baseline time groups in the three brain regions with DAPI (blue) and the ChAT (green, used only to delineate NI).
- Figure 7.
pS6-ir cell densities per mm2 in the 100 min (red) and baseline groups (gray) across regions of interest: dAT and vAT, central nucleus of the TSc and NI. Bar plots show mean ± SEM, with dots representing the individual values (**p < 0.01; ***p <0.001).
Tables
- Table 1.
pS6-ir cell densities across the time groups in the TSc, dAT, and NI (mean ± SEM, rounded numbers)
Time groups TSc dAT NI 0 1,174.1 ± 263 1,970.8 ± 272 5,875 ± 572 10 1,785.1 ± 280 2,162.5 ± 285 6,808.3 ± 959 70 2,895.9 ± 215 2,825 ± 299 7,258.3 ± 642 100 3,043.7 ± 355 4,989.6 ± 606 6,025 ± 706 130 3,346.8 ± 319 4,264.6 ± 544 6,416.7 ± 593 160 2,694.9 ± 472 3,536.8 ± 531 6,708.3 ± 788 190 1,723.5 ± 196 2,674.3 ± 353 6,750 ± 557 250 1,706.8 ± 296 2,104.2 ± 459 5,933.3 ± 745 - Table 2.
Results of the post hoc Dunn’s tests for TSc and dAT with significant p values in bold
Comparison between time groups TSc dAT 0 vs 10 Z = 1.093; p = 0.274 Z = 0.124; p = 0.902 0 vs 70 Z = 3.134; p = 0.002 Z = 1.454; p = 0.146 0 vs 100 Z = −3.175; p = 0.001* Z = −3.464; p = 0.001* 0 vs 130 Z = 3.629; p < 0.001* Z = 2.928; p = 0.003 0 vs 160 Z = 2.577; p = 0.010 Z = 2.217; p = 0.027 0 vs 190 Z = 0.825; p = 0.409 Z = 1.000; p = 0.317 0 vs 250 Z = 0.907; p = 0.364 Z = 0.031; p = 0.975 10 vs 70 Z = −2.041; p = 0.041 Z = −1.330; p = 0.184 10 vs 100 Z = −2.083; p = 0.037 Z = −3.341; p = 0.001* 10 vs 130 Z = −2.536; p = 0.011 Z = −2.804; p = 0.005 10 vs 160 Z = −1.485; p = 0.138 Z = −2.093; p = 0.036 10 vs 190 Z = 0.268; p = 0.789 Z = −0.876; p = 0.381 10 vs 250 Z = 0.186; p = 0.853 Z = 0.093; p = 0.926 70 vs 100 Z = −0.041; p = 0.967 Z = −2.011; p = 0.044 70 vs 130 Z = 0.495; p = 0.621 Z = 1.474; p = 0.140 70 vs 160 Z = −0.557; p = 0.578 Z = 0.763; p = 0.445 70 vs 190 Z = −2.309; p = 0.021 Z = −0.454; p = 0.650 70 vs 250 Z = −2.227; p = 0.026 Z = −1.423; p = 0.155 100 vs 130 Z = 0.454; p = 0.650 Z = −0.536; p = 0.591 100 vs 160 Z = −0.598; p = 0.550 Z = −1.248; p = 0.212 100 vs 190 Z = −2.351; p = 0.019 Z = −2.464; p = 0.014 100 vs 250 Z = −2.268; p = 0.023 Z = −3.433; p = 0.001* 130 vs 160 Z = 1.052; p = 0.293 Z = 0.711; p = 0.477 130 vs 190 Z = 2.804; p = 0.005 Z = 1.928; p = 0.054 130 vs 250 Z = 2.722; p = 0.006 Z = 2.897; p = 0.004 160 vs 190 Z = 1.753; p = 0.080 Z = 1.217; p = 0.224 160 vs 250 Z = 1.670; p = 0.095 Z = 2.186; p = 0.029 190 vs 250 Z = −0.082; p = 0.934 Z = 0.969; p = 0.332 ↵* Significant also after a Bonferroni’s correction for multiple comparisons.
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