Drugs prescribed for Phelan-McDermid syndrome differentially impact sensory behaviors in shank3 zebrafish models.

Ethics, fish maintenance and husbandry

Zebrafish were housed and maintained at 28°C in system-water on a 14:10 hour circadian light:dark cycle in the zebrafish core facility at the University of Miami where they were fed twice a day using a combination of dry fish food and brine shrimp. Adult and larval zebrafish used in this study were handled in accordance with NIH guidelines and experiments were approved by the University of Miami Institutional Care and Use Committee protocol #’s 15-128 (approval date 9/22/2015) and 18-128 (approval date 9/27/2018). To limit harm to the animals and ensure experimental reproducibility, after natural spawnings, unfertilized eggs were removed and embryos were maintained in 10 cm dishes with ~50 larvae per dish until behavioral observations. Embryos were raised with the same 14:10 light cycle as their parents. Zebrafish lines used in this study were; ABTL wildtype (WT), shank3abN-/- (Kozol et al., 2021) and shank3abC-/- (James et al., 2019).

This study is reported in line with the Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines (Kozol & Dallman, 2023).

Behavioral assays

Sample

All exact sample sizes can be found in the figure legends. Sample sizes were derived from a previous study based on the same VMR behavioral endpoint (Kozol et al., 2021).

High-throughput behavioral screens

Experimental plans were developed and refined during weekly meetings but there was no protocol registered prior to initiation of experiments. The DanioVision system^tm (Noldus, Wageningen, NTD) with the DanioVision observation chamber (DVOC-0040) was used to record videos of larval behaviors during experiments using the following settings: 25 fps, 1280 × 960 resolution using a Basler acA1300-60 gm camera fitted with a 12 mm Megapixel lens. White light for the visual motor response assay was set at 12% intensity on the high-power setting. Larvae were pipeted into an ANSI-SBS-compatible 96 well microtiter plate at a density of one larva per well, at a depth of 10 mm. Six-day-old larvae were acclimated to the observation chamber at 28 °C in the dark for at least 1 hr. Larval sex is unknown at this stage. Larvae were monitored during behavioral recordings, to ensure no signs of distress were exhibited during light cycles. DanioVision EthoVision XT software version 11.5 (Noldus) was used to set up data collection and for preliminary analyses. Visual motor response (VMR) experiments consisted of four cycles of alternating lights-on (five min.)/lights-off (five min.) for a total of 40 minutes. All behavioral experiments were conducted between 11 am and 3 pm, with 2-5 independent trials. Larvae were randomly assigned across each 96-well plate, blinded to experimenters, then were genotyped following behavioral experiments using restriction digest assays previously described (James et al., 2019; Kozol et al., 2021), allowing larvae to be binned by genotype for subsequent analyses. Following experiments, larvae were humanely euthanized using MS222.

Drug screening

Zebrafish were exposed to drugs dissolved in 0.1% DMSO system water (water from the system that houses the adult fish) 24 hours prior to running VMR assays. A range of risperidone, MPEP, CBZ and LiCl concentrations were derived from previously published papers (Tucker et al., 2006; Bruni et al., 2016; Hoffman et al., 2016), then dose-response curves were generated to determine an effective dose in relation to the VMR response of WT zebrafish. Concentrations used for comparing WT and shank3 larvae were 10 μM Risperidone (Bruni et al., 2016; Hoffman et al., 2016), 5 mM LiCl and 200 μM CBZ, and 5 μM MPEP (Tucker et al., 2006). Genotype controls were exposed to DMSO (0.1%) in system water.

For PTZ trials, larvae were initially acclimated in 1 mL of system water at 28 °C in the Daniovision behavioral box for 30 minutes. Larvae were then recorded for 10 minutes to establish baseline behavior. Following a baseline recording, larvae were either exposed to 3 mM PTZ in 0.1% DMSO system water or 0.1% DMSO system water for ten minutes, before capturing ten minutes of behavior following drug exposure. Baseline and PTZ/DMSO data was then binned as total distance moved for 10 minutes pre and post PTZ exposure.

Statistics

Data were analyzed using PRISM 9 (graphpad, inc.); these same analyses could be conducted using R. Videos were manually screened before running data analyses, to determine that tracking software accurately captured individuals’ movements; if discrepancies between tracks and videos were noted, videos were retracked. No individuals or data points were excluded from behavioral analyses. Significance was assessed using the non-parametric Wilcoxon rank score test (Mann-Whitney rank scores). When there were more than two groups, a Kruskal-Wallis rank score test was first calculated and, if p<0.05, was followed by a Dunn’s multiple comparisons test to compare all treatments and genotypes. See Tables 2-41.