The proper execution of the auditory-evoked startle response relies on hair cells to sense the auditory stimulus, Mauthner cells to integrate the signals, and motor neurons and musculature to execute the startle behavior.

a Representative short-latency C-bend (SLC) and long-latency C-bend (LLC) startle response of a control zebrafish larva at 6 dpf in comparison to a typical response of a PCB153 treated larva. b Vibro-acoustic startle latency at highest stimulus intensity (43 dB) in vehicle control (n = 448), NDL PCB153 1000 nM (n = 115), NDL PCB153 100 nM (n = 226), NDL PCB153 30 nM (n = 117), NDL PCB153 10 nM (n = 112), NDL PCB138 (n = 68), NDL PCB118 (n = 99), NDL PCB52 (n = 97), and DL PCB126 (n = 86) exposed larvae. c Bias of SLC and LLC and d response rate of different exposure groups at different vibro-acoustic intensities. All data points are biologically independent samples from at least three independent experiments and mean ± SEM shown in the plots of (c, d). Binomial GLM was used for statistical analysis in (c, d). Significant differences to DMSO controls (p < 0.01) are indicated by open symbols.

a Diagram indicating L3 neuromast. b Hair cell innervating neurons at neuromast L3 shown using Tg(cntn1b:EGFP-CAAX) and volume of hair cell innervating neurons of DMSO (n = 30) and PCB153 (n = 29) treated larvae. Scale bar = 20 µm. c Whole-mount immunostaining of kinocilia and hair cell innervating neurons (anti-acetylated tubulin antibody; green) and counterstain of stereocilia bundles and muscle tissue (actin; magenta) of DMSO (n = 26) and PCB153 (n = 24) treated larvae. The hair cell number was determined based on the number of kinocilia. Scale bar = 10 µm. d Uptake of FM1-43 dye as indication of functional hair cells of DMSO (n = 32) and PCB153 (n = 28) treated larvae. CTCF = Corrected total cell fluorescence. Scale bar = 10 µm. All data points are biologically independent samples from three independent experiments and mean ± SD are shown in the plots. Unpaired two-tailed t-test with Welch’s correction was used for statistical analysis in (b, c), a two-way ANOVA accounting for trial and treatment in (d). Asterisks indicate significant differences to controls (***p < 0.001).

a Diagram indicating localization of Mauthner cells in the hindbrain and brain tissue immunostaining with anti-neurofilament 3A10 antibody labeling reticulospinal neurons in control and PCB153 treated larvae at 6 dpf (ventral view). Mauthner (arrow) cells were present in all brain tissues (DMSO: n = 23, PCB153: n = 21, biologically independent samples). Scale bar = 50 µm. b Diagram of electrical stimulation performed to assess the functionality of Mauthner cells. Both DMSO (n = 44) and PCB153 (n = 45) treated larvae show similar c latency to electrical stimulation and d response frequency, indicating that the Mauthner cells are present and functional, e while the C-bend angle in PCB153 exposed larvae was increased (Welch’s t-test, **p = 0.0091). All data points are biologically independent samples (one value representing the mean of seven consecutive electric field pulses) from three independent experiments and mean ± SD are shown in the plots. Mann–Whitney test was used for statistical analysis in (c), binomial GLM in (d), and unpaired two-tailed t-test with Welch’s correction in (e). Dashed lines represent median and quartiles and asterisks indicate significant differences to controls.

a Neurotransmitter levels in 6 dpf larvae exposed to 1000 nM PCB153 in comparison to control (n = 4, pool of 18 larvae per sample). Unpaired t-test GABA ***p = 0.00007, choline-chloride ***p = 0.0001, L-glutamine ***p = 0.0010, 3-methoxytyramine hydrochloride **p = 0.0089, L-valine *p = 0.0202. b Startle bias shift induced by 30-min exposure to serotonin, dopamine, and GABA neurotransmitter modulators in 6 dpf control and 100 nM PCB153 exposed larvae. Control (n = 172), 5-HTP (n = 80), L-DOPA (n = 94), haloperidol (Control n = 178, PCB153 n = 81), and bicuculline (Control n = 160, PCB153 n = 173). All data points are biologically independent samples from at least three independent experiments and mean ± SEM are shown in the plots. Significant differences to respective controls (GLM, p < 0.01) are indicated by open symbols. c Representative maximum intensity z-projection from confocal stack after photoconversions of freely swimming 6 dpf CaMPARI larvae. Scale bar = 100 µm. Ratio of red to green fluorescence intensity in the rostral hindbrain (indicated by dashed line in image) in DMSO (n = 14) and 1000 nM PCB153 (n = 14) exposed swimming larvae in 4 ˚C (cold) medium. Asterisks indicate significant differences to controls (two-way ANOVA, *p = 0.0277, ***p = 0.0004). All data points are biologically independent samples from two independent experiments (trials) and the horizontal lines are indicating the mean.