Zebrafish wild-type strain AB was used for all experiments. All animals were maintained according to standard procedures (Westerfield, 1995) and in accordance with legal regulations (EU-Directive 2010/63), the local authorities, the animal welfare representative of the Braunschweig University of Technology, and the Lower Saxony State Office of Consumer Protection and Food Safety (LAVES, Oldenburg, Germany; Az. §4 (02.05) TSchB TU BS).

SSC (Sigma Aldrich) was dissolved at a concentration of 50 mM in distilled water. MK801 (Sigma Aldrich) was dissolved at a concentration of 10 mM in distilled water.

To determine the median lethal concentration of SSC, 20 one- or two-cell embryos were transferred into fresh 30% Danieau medium (290 mM NaCl, 7 mM KCl, 4 mM MgSO₄, 6 mM Ca(NO₃)₂, 50 mM HEPES buffer, pH = 6.8) and used directly or raised until 24, 55, or 72 hours post fertilization (hpf). The medium was changed daily. For SSC treatment, the medium was removed and replaced by different concentrations of SSC in 30% Danieau reaching from 0 to 4 mM. Embryos and larvae were analyzed for 24 h.

Upon SSC treatment, 3 days post fertilization (dpf) larvae were washed 3× with 30% Danieau. Staining was carried out with 10 μg/mL AO (Sigma Aldrich) in 30% Danieau for 1 h in the dark at room temperature. Prior to imaging, larvae were washed 3× with 30% Danieau. Larvae were anesthetized with 0.04% tricaine mesylate (Sigma Aldrich) in 30% Danieau and embedded in 1.5% low melting agarose dissolved in 30% Danieau.

About 3 dpf larvae were exposed to SSC. Mobility of all larvae used for tracking experiments was previously verified. For each experiment, five larvae per well were transferred into fresh 30% Danieau and allowed to habituate 10 min prior to SSC application. SSC was added to a final concentration of 2 mM, and the larvae were allowed to accommodate for 30 s. SSC was either applied alone or in combination with 2 mM MK801. For the control, an equivolume of 30% Danieau was applied. To analyze the effect of SSC, the motor behavior was monitored using the Samsung Galaxy A3 Smartphone camera with a resolution of 13 megapixel at 30 frames per s adapted to a stereo microscope (LEICA S8APO) utilizing the DKA5 Smartphone adapter (recording time: 9 min). Tracking experiments were performed in self-made agarose well plates (1.5% agarose in 30% Danieau heated and solidified and stamped out wells of 16 mm in diameter). To track the movement, movies were analyzed framewise using the Manual Tracking plugin of ImageJ (Schindelin et al., 2012). Measuring points were placed between the eye balls of the analyzed larva. X/Y coordinates were used to analyze and quantify the motor behavior. The traveled distance between frames was calculated by using the difference in X/Y positions between the frames, and the percentage of time spent with movement was determined with calculating the amount of frames, where the distance traveled was >0. Statistical analysis was carried out using a one-tailed unpaired t test.

Inactivation of sensory hair cells of the lateral line (Harris et al., 2003) was carried out by treatment with 400 μM neomycin in 30% Danieau for 1 h and subsequent washing 3× in 30% Danieau. Ablated cells were confirmed by staining with 0.05% DASPEI in 30% Danieau for 15 min.

To test for the effect of SSC on zebrafish motor behavior, we applied 2 mM SSC to 3 dpf larvae and monitored the acute effects. SSC-treated larvae showed an increase in general swimming activity accompanied by circling and convulsive episodes (Figure 1A). We quantified these seizure-like behaviors (Baraban et al., 2005): SSC-treated larvae traveled a significantly longer distance and spent more time with movement as non-treated larvae (Figures 1B,C), hence conforming to “stage I: dramatically increased swim activity.” Also “stage II: rapid ‘whirlpool-like’ circling” and “stage III: brief clonus-like convulsions” were drastically enhanced (Figure 1D). These effects were reversible as demonstrated by the normalized swimming behavior of SSC-treated larvae, which were transferred back to SSC-free medium (Figures 1B,C). To exclude effects of SSC on the peripheral nervous system via the sensory hair cells of the lateral line, we ablated these cells; successful hair cell ablation was confirmed by DASPEI staining [Supplementary Figure S1; (Harris et al., 2003)]. These larvae likewise showed a seizure-like phenotype when 2 mM SSC was applied (Supplementary Video S1) with the corresponding control showed no increased movements (Supplementary Video S2). These findings indicate that the observed seizure-like behaviors are likely caused by an SSC-induced stimulation of the central nervous system. To characterize the observed movements in detail, representative continuous frames were analyzed (Supplementary Figures S2,S3) with an extract of the image sequence of SSC-treated and non-treated larvae being shown in Supplementary Figure S4. Notably are episodes of involuntary erratic movements with aberrant bending of the body axis differentiating the SSC-induced phenotype from the sine-like curved axis during movement of control larvae. Prolonged exposure of larvae to SSC is lethal with the SSC LC₅₀ values varying depending on developmental stages (Supplementary Figure S5). Consistently, increasing levels of cell death in the central nervous system were observable in larvae upon prolonged incubation with SSC (Supplementary Figure S6A). Here, the heads of SSC-treated zebrafish larvae were also found to engorge (Supplementary Figures S6A,B), which might correspond to cerebral edema and increased brain pressure in MoCD patients (Durmaz and Özbakir, 2018).

The NMDA receptor antagonist MK801 was found to partially compensate for excitotoxicity caused by the glutamate analog SSC (Figure 2A) in mammalian cell culture and mice (Kumar et al., 2017). To test for NMDA receptor antagonist efficacy in SSC-treated zebrafish, we applied the non-competitive NMDA receptor antagonist MK801 to 2 and 3 dpf larvae. Upon application to 2 dpf larvae, the afore described seizure-like behavior (Figure 1) was largely compensated as documented by normalized larval movement (Figures 2B,D), reduced clonus-like convulsions, and diminished circling behavior. Therefore, MK801 can partially compensate the SSC-triggered movements. When 3 dpf larvae were co-treated, the compensation of the SSC-induced phenotype was less pronounced and not significant (p = 0.1004, Figure 2C; p = 0.256, Figure 2E), probably because of diminished MK801 penetrance into the brain with ongoing larval differentiation. Nevertheless, these findings in conjunction with the well-known binding and the activation of mammalian NMDA receptors by SSC (Olverman et al., 1984; Kumar et al., 2017) suggest that SSC-mediated hyperactivation of NMDA receptors is conserved in zebrafish.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.