Observation chamber

Individual 5-dpf embryos were distributed into a well of a 96-well plate, in 100 μl of E3 embryo medium, and allowed to habituate for 1 hr prior to drug treatment. Following 1 hr of drug treatment, the behavior of the larvae was tracked for another hour in the DanioVison Observation Chamber and the behavior was analyzed using EthoVision XT 8.5 software (Noldus Information Technology). For the swimming activity data shown in Figure 1H, data were pooled across several plates to better examine data distributions. In these experiments data were normalized to the grand average of each individual plate to minimize plate-to-plate variation.

Groups of 30 zebrafish larvae at 6 dpf were exposed to each of the five types of magnetic nanocomposites at concentrations of 10 and 100 µg/mL for 96 h. At 10 dpf, animals underwent behavioral testing, including open-field, startle response, and color preference tests, using a DanioVision Observation Chamber and the results were analyzed with EthoVision^® XT 14 software (Noldus Information Technology, Wageningen, The Netherlands). Open-field videos were recorded for 5 min, and then the distance traveled, average speed, and freezing time of the animals were analyzed. After that, the startle response time was evaluated (the animal’s reaction time to a single white light flash of 0.1 s). The distance traveled, speed, and elongation during the 5 s before and after the flash of light were measured. Another group of treated animals were studied for the color preference test. For this test, the wells of a 6-well plate were divided into two regions, one green and the other transparent, while the behavior of the larvae in terms of time and frequency of entry into each region was monitored.

We assessed the response and habituation to acoustic startle stimuli in wild type and mutant larvae at 5 dpf in the presence and absence of the dopamine D2/D3 receptor antagonist amisulpride and non-selective dopamine receptor agonist apomorphine. Behavioral assays were conducted between 9 a.m. and 4 p.m. Larvae were placed in individual wells of a 48 multi-well plate. A control (0.05% DMSO) or treatment (0.01 mg/L amisulpride or 0.2 mg/L apomorphine in 0.05% DMSO) dose was added to each well in a final volume of 300 μL. Larvae were acclimatized for 1 h before testing. Then, plates were placed in a DanioVision Observation Chamber containing a dedicated tapping device (Noldus Information Technology, Wageningen, Netherlands). After 5 min of acclimation, larvae were subjected to 20 sound/vibration stimuli over 40 s (2 s intervals between each stimulus). For all experiments, distance traveled was recorded using EthoVision XT software (Noldus Information Technology, Wageningen, Netherlands), and data were outputted in 1 s time bins and analyzed as previously described (García-González et al., 2021 (link)).